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The Role of TRIF and CXCL10 Signaling in the Murine Neonatal Response to Sepsis

Permanent Link: http://ufdc.ufl.edu/UFE0044079/00001

Material Information

Title: The Role of TRIF and CXCL10 Signaling in the Murine Neonatal Response to Sepsis
Physical Description: 1 online resource (112 p.)
Language: english
Creator: Cuenca, Alex Gervacio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: immunity -- innate -- neonatal -- sepsis
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neonatal sepsis and infection has an annual mortality in excess of 1 million. This number will more than likely continue to rise with the increasing numbers of premature babies that require prolonged intensive care unit stays. This is evident with shifts in the numbers of late onset neonatal sepsis which are more the result of nosocomial infection as opposed to early onset sepsis which is more related to maternal vertical transmission. Previous studies have suggested that neonates rely more on innate immunity for their antimicrobial response to bacterial infections than adaptive immunity. However, the innate immune response by neonates to bacterial infection remains poorly characterized. Here we demonstrate that TRIF-/-, but not MyD88-/-, neonates have impaired inflammatory cytokine/ chemokine production, in particular CXCL10, as well as decreased granulocyte phagocytic function following lipopolysaccharide 1 stimulation. We have also previously published that pretreatment of neonates with LPS is able to protect against polymicrobial sepsis. Here, we demonstrate that TRIF dependent inflammation is critical for this observed LPS adjuvant effect. In addition, we show that TRIF, but not MyD88, signaling is important for the neonatal innate immune response to gram negative E coli sepsis. This is important because we and other investigators have demonstrated that MyD88, but not TRIF, is important for survival to adult gram negative sepsis. This increased susceptibility in TRIF-/- neonates is associated with increased bacteremia in TRIF-/- neonates compared to MyD88-/- or WT neonates. We also demonstrate that despite increased numbers and percent phagocytic peritoneal granulocytes and macrophages, TRIF -/- neonates have significantly impaired reactive oxygen species production at 24 hours post E coli infection compared to WT animals. As CXCL10 production and the LPS adjuvant effect appeared to be decreased in TRIF-/- but not MyD88-/- neonates and CXCL10 has been shown to be important for adult granulocyte and macrophage function, we also wanted to explore the role of CXCL10 in the neonatal immune response to polymicrobial sepsis. We demonstrate that CXCL10 concentrations increase in the blood and peritoneum concomitantly with the peritoneal recruitment of granulocytes and macrophages following challenge with a murine model of neonatal sepsis. Blockade of CXCL10 with a polyclonal immunoglobulin not only decreases peritoneal macrophage and granulocyte numbers and function, but also worsens survival. Finally, we demonstrate that the protective effect of pretreatment by administration of a TLR4 agonist (lipopolysaccharide) to neonatal sepsis is dependent on an endogenous CXCL10 response, and that pretreatment of neonates with CXCL10 can also significantly improve macrophage and granulocyte function, and modestly improve outcome to polymicrobial sepsis. Together, these data suggest a critical role for both TRIF and CXCL10 signaling during neonatal sepsis. In addition, these data demonstrate a novel and crucial difference in the requirement for MyD88 and TRIF dependent signaling in the adult vs the neonate in response to gram negative infection. Importantly, these data may lead to the development of therapeutics that maximize TRIF dependent inflammation to improve innate immune responses to infection and outcomes in these particularly susceptible patients.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Alex Gervacio Cuenca.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Mathews, Clayton Elwood.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044079:00001

Permanent Link: http://ufdc.ufl.edu/UFE0044079/00001

Material Information

Title: The Role of TRIF and CXCL10 Signaling in the Murine Neonatal Response to Sepsis
Physical Description: 1 online resource (112 p.)
Language: english
Creator: Cuenca, Alex Gervacio
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2012

Subjects

Subjects / Keywords: immunity -- innate -- neonatal -- sepsis
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Neonatal sepsis and infection has an annual mortality in excess of 1 million. This number will more than likely continue to rise with the increasing numbers of premature babies that require prolonged intensive care unit stays. This is evident with shifts in the numbers of late onset neonatal sepsis which are more the result of nosocomial infection as opposed to early onset sepsis which is more related to maternal vertical transmission. Previous studies have suggested that neonates rely more on innate immunity for their antimicrobial response to bacterial infections than adaptive immunity. However, the innate immune response by neonates to bacterial infection remains poorly characterized. Here we demonstrate that TRIF-/-, but not MyD88-/-, neonates have impaired inflammatory cytokine/ chemokine production, in particular CXCL10, as well as decreased granulocyte phagocytic function following lipopolysaccharide 1 stimulation. We have also previously published that pretreatment of neonates with LPS is able to protect against polymicrobial sepsis. Here, we demonstrate that TRIF dependent inflammation is critical for this observed LPS adjuvant effect. In addition, we show that TRIF, but not MyD88, signaling is important for the neonatal innate immune response to gram negative E coli sepsis. This is important because we and other investigators have demonstrated that MyD88, but not TRIF, is important for survival to adult gram negative sepsis. This increased susceptibility in TRIF-/- neonates is associated with increased bacteremia in TRIF-/- neonates compared to MyD88-/- or WT neonates. We also demonstrate that despite increased numbers and percent phagocytic peritoneal granulocytes and macrophages, TRIF -/- neonates have significantly impaired reactive oxygen species production at 24 hours post E coli infection compared to WT animals. As CXCL10 production and the LPS adjuvant effect appeared to be decreased in TRIF-/- but not MyD88-/- neonates and CXCL10 has been shown to be important for adult granulocyte and macrophage function, we also wanted to explore the role of CXCL10 in the neonatal immune response to polymicrobial sepsis. We demonstrate that CXCL10 concentrations increase in the blood and peritoneum concomitantly with the peritoneal recruitment of granulocytes and macrophages following challenge with a murine model of neonatal sepsis. Blockade of CXCL10 with a polyclonal immunoglobulin not only decreases peritoneal macrophage and granulocyte numbers and function, but also worsens survival. Finally, we demonstrate that the protective effect of pretreatment by administration of a TLR4 agonist (lipopolysaccharide) to neonatal sepsis is dependent on an endogenous CXCL10 response, and that pretreatment of neonates with CXCL10 can also significantly improve macrophage and granulocyte function, and modestly improve outcome to polymicrobial sepsis. Together, these data suggest a critical role for both TRIF and CXCL10 signaling during neonatal sepsis. In addition, these data demonstrate a novel and crucial difference in the requirement for MyD88 and TRIF dependent signaling in the adult vs the neonate in response to gram negative infection. Importantly, these data may lead to the development of therapeutics that maximize TRIF dependent inflammation to improve innate immune responses to infection and outcomes in these particularly susceptible patients.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Alex Gervacio Cuenca.
Thesis: Thesis (Ph.D.)--University of Florida, 2012.
Local: Adviser: Mathews, Clayton Elwood.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2012
System ID: UFE0044079:00001


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1 THE ROLE OF TRIF AND CXCL10 SIGNALING IN THE MURINE NEONATAL RESPONSE TO SEPSIS By ALEX GERVACIO CUENCA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FO R THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2012

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2 2012 Alex Gervacio Cuenca

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3 For my mother, whose spirit moves me For my father, whose hands and heart live within me For my wife and her eternal support and love, despite my flaws And for my children, may they grow to love life a tenth as much as I love them

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4 ACKNOWLEDGMENTS This process was an undertaking. A four year clinical hiatus that has left me replete with the skills that I need to be a successful scientist and more i mportantly, a beautiful family. It has shown me that I am ready and willing to push my limits to not only be a constant self learner, but also a clinician thinking about the disease pathology of my patients and what I can do to improve their outcomes. Of c ourse, I would be lost without the help of several people that have helped shaped my goals and my dream to become a surgeon scientist. myself than anyone of my mentors in my entire life. His constant give and take, gentle and not so subtle nudging, and his unabashed curiosity for science to ask the questions that everyone wants to ask but lack the courage to ask have been immeasurably helpful for my scientific and academic growth. F or his way of turning my meandering mumbling into eloquent precise prose, his complete patience and sound advice for my paranoia and constant self doubt, despite the overwhelming responsibilities he has with his work and his family, and his absolute and un questionable understanding of translational our own accolade and by reflection, his own. Finally, and probably most importantly, for tempering all of that drive and am bition and showing me that being successful does not have to be at the expense of your family or friends. Also, my chairman, Dr. Kevin Behrns, deserves a tremendous amount of proud profession of intellectual and physical overachievers, refusing to be limited by a 24 hour day. Of course, this 4 year stint would not be possible without his

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5 understanding. A surgeon scientist himself, dedicated to his family, patient care, and his residents, he is the example of what I would like to become and a fantastic mentor and role model for me. To Dr. Clayton Mathews, I owe you a tremendous amount of thanks, for molding my grant writing ability and encouraging me to submit my proposa l to the NIH, when I subsequently funded. For the hours of bickering and banter about science and assays that I could and probably could/should run and for encouraging me to pursu e my dreams of becoming a surgeon scientist. To my other committee members, Dr. Shannon Wallet and Dr. Westley Reeves for pushing me and strengthening my science and for helping me, through this rigorous 4 year process. Thank you. To my fellow lab mates es pecially, Dallas Joiner, Ricky Ungaro, Dins Nacionales, Angela Avery, James L Wynn, Philip Scumpia, and Kindra Kelly Scumpia. To the National institutes of Health General Medical Sciences division. For funding my training grant and giving me a modicum of h ope that I might be able to succeed in science. my family has played in this. My wife, Angela, and my 2 children, Aidyn and Xander, I am so thankful that I have you to come home to. Without you, my career would be lackluster and my life without passion or direction. Thank you.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TA BLES ................................ ................................ ................................ ............ 9 LIST OF FIGURES ................................ ................................ ................................ ........ 10 LIST OF ABBREVIATIONS ................................ ................................ ........................... 12 ABSTRACT ................................ ................................ ................................ ................... 1 3 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ ........ 15 Neonatal sepsis ................................ ................................ ................................ ...... 15 Patient Population. ................................ ................................ ........................... 16 Epidemiology. ................................ ................................ ................................ ... 17 Early onset sepsis (EOS) ................................ ................................ ........... 17 Late onset sepsi s ................................ ................................ ....................... 18 Therapeutics in neonatal sepsis. ................................ ................................ ...... 20 Neonatal Innate immunity ................................ ................................ ................. 22 TLR signaling ................................ ................................ ................................ .......... 23 TLR4 and relevance to sepsis ................................ ................................ .......... 27 CXCL10/IP 10 ................................ ................................ ................................ ......... 29 Summary ................................ ................................ ................................ ................ 30 Overarching Hypothesis ................................ ................................ .......................... 33 2 TRIF DEPENDENT INFLAMMATION AND INNATE IMMUNE ACTIVATION IS CRITICAL FOR SURVIVAL T O GRAM NEGATIVE NEONATAL SEPSIS ............. 37 Background ................................ ................................ ................................ ............. 37 Methods ................................ ................................ ................................ .................. 39 Mice ................................ ................................ ................................ .................. 39 Cecal slurry (CS) model. ................................ ................................ .................. 39 Escherichia coli gram negative sepsis. ................................ ............................. 39 Pretreatment with a TLR4 agonist (bacterial LPS). ................................ .......... 40 Peritoneal, sera, and media cytokine concentrations. ................................ ...... 40 Flow cyto metry ................................ ................................ ................................ 41 Isolation of blood, bone marrow, and splenocytes for phenotypic analysis. ..... 41 Harvest of peritoneal washes. ................................ ................................ .......... 41 Isolation of splenic macrophages and neutrophils for functional analyses. ...... 42 In vitro stimulation with recombinant CXCL10. ................................ ................. 42

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7 Functional analysis of splenic macrophages and neutrophils or peritoneal macrophages or neutrophils. ................................ ................................ ......... 43 Statistical analyses. ................................ ................................ .......................... 43 Results ................................ ................................ ................................ .................... 43 TRIF / neonatal neutrophils have impaired phagocytic function response to LPS. ................................ ................................ ................................ .............. 43 TRIF / neonates have impaired cyto/chemokines production in response to LPS in vivo ................................ ................................ ................................ ... 44 CXCL10 partially reverses TRIF / defects in phagocytosis. ............................. 45 TRIF / neonates are not protected against polymicrobial sepsis in response to TLR4 adjuvant protection. ................................ ................................ ......... 45 TRIF / neonates are susceptible to gram negative sepsis. ............................... 46 TRIF / neonates produce decreased local inflammatory cytokines and chemokines in reponse to E coli gram negative sepsis. ................................ 47 Decreased early recruitment of peritoneal neutrophils and macrophages in but decreased late ROS production in TRIF / neonates in response to E coli neonatal sepsis. ................................ ................................ ...................... 47 Adult MyD88 / mice are susceptible to gram negative sepsis but not TRIF / or wild type adult mice ................................ ................................ .................. 48 Discussion ................................ ................................ ................................ .............. 48 3 SURVIVAL OF NEO NATES TO POLYMICROBIAL SEPSIS IS INDEPENDENT OF TLR SIGNALING ................................ ................................ ............................... 60 Background ................................ ................................ ................................ ............. 60 Materials and Methods ................................ ................................ ............................ 62 Mice ................................ ................................ ................................ .................. 62 Cecal slurry (CS) model ................................ ................................ ................... 62 Flow cytometry ................................ ................................ ................................ 62 Isolation of blood, bone marrow, and splenocytes for phenotypic analysis ...... 63 Harvest of peritoneal washes ................................ ................................ ........... 63 Isolation of splenic macrophages and neutrophils for functional analyses ....... 64 Functional analysis of splenic macrophages and neutrophils or peritoneal macrophages or neutrophils ................................ ................................ .......... 64 Statistical analyses ................................ ................................ ........................... 64 Results ................................ ................................ ................................ .................... 65 No differences in peritoneal recr uitment of neutrophils and macrophages at 24 hours following polymicrobial sepsis ................................ ........................ 65 Deletion of TRIF or MyD88 does not affect neutrophil reactive oxygen species production following phorbol e ster stimulation in neonates .............. 65 Survival of neonates to polymicrobial sepsis is independent of TLR signaling ................................ ................................ ................................ ........ 66 Discussion ................................ ................................ ................................ .............. 66 4 CRITICAL ROLE FOR CXCL10 (IP 10)/CXCR3 SIGNALING IN THE MURINE NEONATAL RESPONSE TO SEPSIS ................................ ................................ .... 73

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8 Background ................................ ................................ ................................ ............. 73 Materials and Methods ................................ ................................ ............................ 75 Mice ................................ ................................ ................................ .................. 75 Cecal slurry (CS) model ................................ ................................ ................... 75 Peritoneal and serum CXCL10 concentrations ................................ ................. 76 Flow cytometry ................................ ................................ ................................ 76 Isolation of blood, bone marrow, and splenocytes for phenotypic analysis ...... 76 Harvest of peritoneal washes ................................ ................................ ........... 77 In vitro CXCL10 chemotaxis assay ................................ ................................ ... 77 Blockade of CXCL10 ................................ ................................ ........................ 78 Peritoneal bacterial counts ................................ ................................ ............... 78 Isolation of spleni c macrophages and granulocytes for functional analyses ..... 78 In vitro stimulation with recombinant CXCL10 ................................ .................. 79 Functional analysis of splenic macrophages and granulocytes or peritoneal macrophages or granulocytes ................................ ................................ ....... 79 Pretreatment with a TLR4 agonist (bacterial LPS) ................................ ........... 80 In vivo administration of recombinant CXCL10 ................................ ................. 80 Statistical analyses ................................ ................................ ........................... 80 Results ................................ ................................ ................................ .................... 80 CXCL10 concentrations in the blood and peritoneum increase during polymicrobial sepsis ................................ ................................ ...................... 80 Peritoneal granulocyte and macrophage kinetics follow increases in CXCL10 pe ritoneal concentrations ................................ ................................ 81 CXCL10 induces the recruitment of both granulocytes and macrophages in vitro ................................ ................................ ................................ ............... 82 CXCL10 neutralization worsens survival to cecal slurry peritonitis and decreases recruitment of peritoneal macrophages and granulocytes ........... 82 CXCR3 expression increases on peritoneal granulocytes and macrophages duri ng septic challenge and deletion of CXCR3 decreases peritoneal granulocyte and macrophage numbers and worsens survival to neonatal sepsis ................................ ................................ ................................ ............ 83 In vitro stimulation with CXCL10 increases phagocyt ic ability of neonatal splenic macrophages and granulocytes ................................ ........................ 84 Blockade of CXCL10 worsens phagocytic ability ................................ .............. 85 Blockade of CXC1 0 inhibits adjuvant induced protection during sepsis and administration of CXCL10 improves outcome ................................ ............... 85 Discussion ................................ ................................ ................................ .............. 87 5 CONCLUSION AND FUTURE DIRECTIONS ................................ .......................... 101 LIST OF REFERENCES ................................ ................................ ............................. 103 BIOGRAPHICAL SKETCH ................................ ................................ .......................... 112

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9 LIST OF TABLES Tabl e page 1 1 Therapeu tic Trials in neonatal sepsis ................................ ................................ 35

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10 LIST OF FIGURES Figure page 1 1 Incidence and mortality in neonatal sepsis. ................................ ........................ 34 1 2 TLR4 signaling. ................................ ................................ ................................ ... 36 2 1 TRIF / neonatal granulocytes have impair ed phagocytic function in response to LPS stimulation. ................................ ................................ .............................. 52 2 2 TRIF / neonates have decreased inflammatory cytokine/chemokines responses compared to MyD88 / and WT neonates ................................ .......... 53 2 3 TRIF / neonates are not protected against polymicrobial sepsis with TLR4 agonist pretreatment. ................................ ................................ .......................... 54 2 4 Partial reversal of TRIF / ne onatal granulocyte phagocytic dysfunction with CXCL10 replacement. ................................ ................................ ........................ 55 2 5 TRIF / neonates are more susceptible to gram negative infection compared to MyD88 / or WT neonates. ................................ ................................ .............. 56 2 6 TRIF / neonates produce decreased local inflammatory mediators c ompared to MyD88 or WT neonates ................................ ................................ .................. 57 2 7 Decrease innate immune effe ctor cell recruitment and ROS production in TRIF / post E coli infection ................................ ................................ ................. 58 2 8 MyD88 / adults are more susceptible to gram negative Escherichia coli sepsis compared to TRIF / and WT a dults ................................ .......................... 59 3 1 Recruitment of peritoneal macrophages and neutrophils following polymicrobial sepsis is similar between TRIF / MyD88 / and WT neonates .... 70 3 2 Similar reactive oxygen species production following phorbol ester stimulation in neutrophils from MyD88 / TRIF / and WT neonates is similar. ....... ................................ ................................ ................................ ....... 7 1 3 3 TLR signaling is dispensable for survival to neonatal polymicrobial sepsis .. ...... 72 4 1 Serum CXCL10 concentrations increase in neonatal sepsis. ............................ 90 4 2 Granulocyte and macrophage kinetics in neonatal sepsis. ................................ 91 4 3 Phenotypic characterization of granulocytes and macrophages ......................... 92

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11 4 4 Kinetics of Ly6g + /CD11b + and Ly6c + /CD11b + populations in neonatal sepsis.. ................................ ................................ ................................ ............... 93 4 5 Increased neonatal granulocytes and macrophages chemotaxis in response to a CXCL10 chemokine gradient ................................ ................................ ....... 94 4 6 CXCL10 blockade worsen s survival and cell recruitment ................................ ... 95 4 7 Deletion of CXCR3 significantly worsen s survival to neonatal sepsis ................. 96 4 8 Recombinant CXCL10 improves phagocytosis in granulocytes and macrophages. ................................ ................................ ................................ ..... 97 4 9 Inhibition of CXCL10 reduces phagocytic function of granulocytes and macrophages in vivo. ................................ ................................ .......................... 98 4 10 Inhibition of CXCL10 abolishes protective adjuvant effect of a TLR4 agonist in response to sepsis and CXCL10 administration modest ly improves survival to sepsis ................................ ................................ ............................... 99

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12 LIST OF ABBREVIATION S CD Cluster of differentiation marker CLP Cecal ligation and puncture CS Cecal slurry CXCL C X C chemokine EOS Early onset sepsis IFN Interferon IL Interleu kin IP Intraperitoneal IP 10/CXCL10 Interferon inducible protein 10 IV Intravenous IRF Interferon response factor MyD88 Myeloid differentiation primary response gene 88 NEC Necrotizing enterocolitis NF B Nuclear factor kappa B IKK I B kinase alpha sub u nit LBP LPS binding protein LOS Late onset se psis LPS Lipopolysaccharide RCT Randomized Control Trial SIRS Systemic Inflammatory Response Syndrome TNF Tumor necrosis factor TLR Toll like receptors TRAM TRIF related adaptor molecule TRIF TIR domain containing adapter inducing interferon

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13 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy THE ROLE OF TRIF AND CXCL10 SIGNALING IN THE MURINE NEONATAL RESPONSE TO SEPSIS By Alex Gervacio Cuenca May 2012 Chair: Clayton Mathews Major: Immunology and Microbiology Neonatal sepsis and infection has an annual global mortality in excess of 1 million. Previous studies have suggested that neonates rely more on innate immunity for their anti mic robial response to infection than adaptive immunity However the neonatal innate immune response remains poorly characterized. W e show that TIR domain containing adapter inducing interferon ( TRIF ) but not myeloid differentiation factor 88 ( MyD88 ) signaling is important for the neonatal innate immune response to gram negative E scherichia coli sepsis. This is important because we and other investigators have demonstrated that MyD88, but not TRIF, is important for survival to adult gram negative sepsi s. This increased susceptibility in TRIF / neonates to E coli is associated with increased bacteremia and poor i nflammatory mediator response compared to wild type neonates. We also demonstrate that despite increased numbers and percent phagocytic periton eal granulocytes and macrophages TRIF / neonates have significantly impaired reactive oxygen species production at 24 hours post E coli infection compared to wild type animals.

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14 As the chemokine, CXCL10 has been shown to be important for adult granuloc yte and macrophage function we also wanted to explore the role of CXCL10 in the neonatal immune response to polymicrobial sepsis. Blockade of CXCL10 not only decreases peritoneal macrophage and granulocyte numbers and function, but also worsens survival. Finally, we demonstrate that the protective effect of pretreatment by administration of a toll like receptor ( TLR ) 4 agonist to neonatal sepsis is dependent on an endogenous CXCL10 response, and that pretreatment of neonates with CXCL10 can also significa ntly improve macrophage and granulocyte function, and modestly improve outcome to polymicrobial sepsis. Together, these data suggest a critical role for both TRIF and CXCL10 signaling during neonatal sepsis. Also these data demonstrate a novel and cruci al difference in the requirement for MyD88 and TRIF dependent signaling in the adult vs the neonate in response to gram negative infection. Importantly, these data may lead to the development of therapeutics that maximize TRIF dependent inflammation to imp rove innate immune responses to infection and outcomes in these susceptible patients.

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15 CHAPTER 1 INTRODUCTION Neonatal sepsis Neonatal and infant infection and sepsis remain serious health care problems with an annual global mortality in excess of one m illion lives, and is the leading cause of mortality among infants in the first days of life 1 2 In addit ion, the incidence of infection and sepsis can vary widely depending on gestational age and time of onset, with severe infection or sepsis havin g higher incidence and death in very low birth weight (VLBW), less than 1500 g, and extremely low birth weight ( ELBW), less than 1000 g, premature neonates, within the first three to seven days of life 3 4 The economic burden of caring for and hospitalizing these infected infants is also not insignificant, and is estimated at approximately $700 million in the US alone 5 Infection and sepsis during the neonatal and infant period has also been recognized as an international issue, and in an attempt t o counter and improve health conditions for infants globally, the United Nations has outlined a series of eight goals known as the Millennium Development Goals to decrease by 2/3 the mortality of children under the age of five by 2015 5 Despite this, the mortality as a resu lt of neonatal sepsis has been largely unchanged over the last three decades (Figure 1 1) 6 As with most pediatric/neonatal medicine, baseline parameters for determi ning sepsis have been gleaned from the adult literature, and though parameters for sepsis have been somewhat accepted for the term infant, parameters for sepsis in the preterm infant are poorly defined 7 This creates delays in diagnosis and treatment and can subsequently adversely affect outcome. Another reason why the mortality rate as a result of neonatal sepsis ha s not

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16 changed is likely due to the inherent diffic ulty in overcoming the devel opmental state of the neonatal adaptive and innate immune response 8 9 For example, severa l groups have demonstrated distinct responses in neonat e compared to adult peripheral blood mononuclear cells (PBMCs) to inflammatory stimuli such as toll like receptor ( TLR ) ligands, including decreased c ytokine and chemokine production 8 10 12 Also, n eonates have poor responses to vaccines with a subsequent inab ility to maintain memory B cell immunity to polysaccharide antigens 13 15 As adaptive and, in particular, innate immunity are essential for the clearance of bacte rial infection s it is no surprise that these patients are so vulnerable to mortality in sepsis. Patient Population. As with adult population s where outcomes may differ based on the age of the patient, the care and outcomes of the neonate are also depende nt on the age and concordantly the weight of the baby. A term neonate is typically considered to be greater than or equal to 37 weeks gestational age, with prematurity being defined as less than 37 weeks of gestational age. As neonatal critical care has i mproved, clinical outcomes in preventing death of the premature neonate have dramatically improved with the youngest recorded survivors of prematurity being 21 23 weeks of gestation 16 17 As would be expected and further detailed below, these weights directly correlate with gestational age, and those babies with the VLBW or ELBW are at the highest risk for developme nt of and mortality related to neonatal sepsis 16 18

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17 Epidemiology. Despite advances in critical care and th erapeutics, the mortality to neonatal sepsis has remained largely unchanged over the past three decades (Figure 1 1) 3 6 19 Neonatal sepsis has been traditionally divided into two subtypes, early onset which occurs within the first three days of life usually from perinatal vertical transmission and late onset sepsis, which occurs between three to seven days of life which is usually nosocomial 20 In both ca ses, empiric antibiotics, such as ampicillin and gentamicin, are typically given early to cover broadly both gram negative and gram positive bacterial isolates, as signs of infection in the neonates may be and typically are more subtle than adults 20 The differences may seem insignificant but the predominant organisms in each o f these categories are dissimilar and therefore, strategies to prevent incidence may be different. In early onset sepsis, the predominant organisms from most to least prevalent are, group B streptococcus (GBS), Escher i chia coli (E coli), and Streptococcus viridians W hereas in late onset sepsis, Coagulase negative Staphylococcus or CoNS, Sta phylococcus aureus and E coli are the most prevalent 18 21 22 Regardless of category and pathogen, mortality and prevalence are the highest in premature infants, especially those babies that are categorized as very low birth weight (VLBW) or < 1500g 16 18 22 Early onset sepsis (EOS) Through the administration of perinatal/intrapartum antibiotics and screening, great strid es have been made in preventing the most common form of EOS, GBS. In fact, 22 To further suppor t these clinical efforts, there have been some studies investigating predictive models to determine the probability of EOS in an attempt to identify those neonates most at risk, such as a recent study that suggests that a model based on the GBS

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18 status, dur ation of ruptured membranes, gestational age, highest intrapartum temperature, and the administration of any form of intra partum antibiotic greater than four hours before birth was able to predict the onset of EOS relatively well (c statistic 1 = 0.800) 23 25 Despite these commonly implemented prophylactic measures and novel preventative strategies, a recent large prospective study of over 400,000 infants by Stoll and colleagues found that >30% of mothers who had babies that developed EO GBS sepsis were never appropriately screened and given antibio tics 22 While great stride s have been made in reducing the incidence of early onset GBS sepsis, gram negative sepsis is still associated with increase d mortality. The results of the current s tudy and other studies have demonstrated increased rates of early onset E coli septicemia e specially in the preterm infants (< 36 weeks of age) 22 26 As of yet, no preventative strategies have been deve loped to specifically address E coli and other gram negative bacteria related EOS aside from supportive care and antibiotics Late onset sepsis (LOS) The predominant organisms in LOS are CoNS and E coli and typically arise from nosocomial infections. As mentioned above, the risk factors for the development of LOS are different than EOS and are more related to neonatal intensive care than to maternal factors or delivery, such as chorioamnionitis 18 20 As low birth weight infants are at increased risk for the develop ment of LOS and have increased mortality to LOS compared to those low birth weight infants that were found to not be infected or have 1 C statistic is a statistical index to evaluate logistic regression and determine predictive ability of one or more variables for a given test with a value of 0.5 being equivalent to guessing and a value of 1.0 being perfectly predictive.

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19 sepsis 17 18 A large prospective trial by Perlman and colleagues found that total parenteral nutrition (TPN), and central venous catheters (CVCs) were risk factors for the development of catheter related LOS, whereas mechanical venti lation was an associated factor for non catheter related LOS 27 Samata et al. had confirmatory findings in a smaller case control study, that indeed TPN, mechanic al ventilation, and presence of central venous line were all important risk factors for the development of neonatal LOS 28 T his is not terribly surprising as the se are also risk factors for infection in the adult intensive care population 29 31 It is also typically within this late population that necrotizing enterocoliti s (NEC) occurs. NEC is a widespread abdominal inflammatory process that causes transmural bowel necrosis, perforation, and subsequent polymicrobial sepsis 32 As with other etiologies of neonatal sepsis, lower birth weight premature infants ELBW and VLBW, are at the highest ri sk for NEC development and mortality 16 Similar to EOS, aside from supportive care and antibiotics, few to no therapeutics exist for the treatment of LOS. As with EOS, LOS infants receive empiric antibiotics to broadly cover both gram positive and negative organisms, however since CoNS is one of the predominant organisms in LOS and is typically found to be resistant to many standard antibiotic regimens, such as amp icillin and gentamicin expanding coverage to include vancomycin is routine 33 However, a significant challenge has been to determine true infection from blood culture contaminant as over treating this organism and other nosocomial pathogen s simply leads to increased antibiotic resistance and gut colonization by potentially pathogenic enteric organisms 34 In both EOS and LOS settings, strategies to enhance neonatal immune function through the use of adjuvants

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20 or vaccines would obviously be highly advantageou s as one of the reasons why neonates are thought to be susceptible to infection is related to their distinct immunological state compared to adults. Therapeutics in neonatal sepsis. As with adult sepsis, despite the promise of preclinical and even sm aller clinical trials using immunomodulatory agents to improve outcome, when converted to more robust prospective randomized clinical studies, have either had limited efficacy or failed all together (Table 1 1) 34 Agents such as activated protein C or Xigris that were shown initially to have a small positive effect on sepsis mortality in adults, have been reported in neonatal sepsis case studies, but have never been examined in a robust clinical trial 35 Future studies with Xigris are not likely as it has been pulled from the market by the FDA due to concerns over safety. A meta analysis of another agent intravenous immun oglobulin (IVIg), suggested a positive effec t on neonatal sepsi s outcome but was recently shown in a larger prospective clinical trial to have no effect on outcome in neonatal sepsis 36 Other agents consider ed have included pentoxyfylline, glutamine supplementation, granulocyte colony stimulating factor, and granulocyte transfusion, all with the absence of significant benefit 34 Of the agents that have been attempted in large clinical trials, only lactoferrin for LOS neonatal sepsis and probiotics for NEC prevention have demonstrated any positive effect in terms of improvement in mortality to neonatal sepsis 34 37 38 Oral lactoferrin was studied in large trial o f 472 VLBW infants and demonstrated a relative risk (RR) 2 reduction of 0.34 for the development of LOS with lactoferrin alone or RR of 0.27 with 2 Relative risks that are less than one are p rotective.

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21 lactoferrin with probiotics 38 39 Unfortunately, oral lactoferrin supplementation was not shown to decrease the incidence of NEC in VLBW. However, supplementation of premature neonates (<33 weeks gestational age) with prob iotics such as L actobacillus and B ifidobacterium was shown in a 2007 Lancet article by Deshpande et al. to be protective against NEC (RR 0.36, 95% CI 0.20 0.65) 40 The mechanisms behind these agents include both bacteriostatic and bactericidal protection, and are either through iron sequestration/bacterial membrane disruption (lactoferrin) or out competition of the enteropathogenic bacteria by probiotics 34 Impo rtantly, these agents do not directly address the neonatal immune system which is obviously a critical component of bacterial clearance. Preclinical investigations from our laboratory have demonstrated that by targeting the neonatal innate immune respon se with immunostimulatory adjuv ants, we can improve survival in a murine model of polymicrobial sepsis 8 Unfortunately, the adjuvant used in these studies that provide d the maximal amount of protection was lipopolysaccharide (LPS) otherwise known as endotoxin, which is a component of gram negative bacterial cell membranes 8 Though endotoxin or bacterial lipopolysaccharide is not a viable therapeutic adjuvant compound s based on immunostimulatory properties of LPS have been developed. One of t he best studied is 3 0 desac yl monophosphoryl lipid A ( MPL ) a modified lipid A variant o f Salmonella minnesota LPS. The MPL adjuvant has been combined with aluminum salts a traditional adjuvant compound use d in many vaccines in cludes both cancer and human papilloma virus vaccine preparations 41 42 Despite the abse nce of longitudinal evidence document ing long term complications of use, a large cohort of adolescent and adult patients, greater than

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22 60 ,000 individuals, have received the vaccine over the last 5 8 years with minimal side effects that were transient and ranged from pain at the injection site to headache to low grade rash/urticaria 41 In addition, studies to delineate the protective mechanisms of this endotoxin effect from its potentially deleterious effects are the part of the subject of this thesis and are described below. In addition, as many of these strategies may have some therapeutic synergism, a multiple armed approach to enhance neonatal innate immunity, as well as provide lactoferrin and probiotics, to directly target pathogenic bacteria, will certainly be of interest in future studies to further improve outcomes in neonatal sepsis. Neonatal Innate immunity Though for many years neonates were thought to have functionally impaired innate immune responses, a newly emerging hypothesis is that neonatal function is age appropriate, and develop s according to environmental exposures in an age specific manner as opposed to a linear progression toward adult immune function 43 44 To support this concept, many facets of neonatal immunity are similar to that of the fetus as opposed to an adult. For example, there is a striking absence of B cell germinal centers and large numbers of myeloid and stem progen itor cells in the neonatal spleen 45 46 As the spleen is one of the primary centers of hematopoiesis in the fetus, but in the adult is more of a clearance organ and as well as an important center for immunolo gical memory, neonates may have compromised pathogen defenses. Previously several studies have suggested that neonatal innate immune function may be geared towards extracellular pathogens which in turn may compromise inflammatory responses, and lead to a more suppressive immunological environment, as would be required in a growing fetus 43 47 48 Many reports have documented

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23 decreased Th1 type (cellular immunity/inflammatory) responses and increased Th2 (hum oral immunity/anti inflammatory) responses in neonates relative to adults 43 47 48 Despite the skewing towards humoral or antibody dependent immunity which in turn may be beneficial for opsonization of bacteria, several Th2 cytokines, such as IL 10, also tend to also suppress inflammatory responses, thereby de creasing innate immune activation and potentially clearance of infection. These findings are further compounded by reports that the baseline function of neonatal neutrophils and macrophages is somewhat decreased relative to adults. For instance, Ahmad and colleagues demonstrated that healthy neonates have decreased neutrophil reactive oxygen species (ROS) production and phagocytic function compared to adults 49 Yos t and colleagues have also documented decreased production of neutrophil extracellular traps or NETS by neonatal neutrophils compared to adults in response to lipopolysaccharide 50 Though many of these studies were descriptive rather than mechanistic studies, the sum of t hese findings demonstrates that neonatal immunity is distinc t from adults and further suggests that neonates are at an immunological disadvantage fo r clearing infections and preventing systemic dissemination of pathogens. However, strategies to mature neonatal immune responses may provide protection against infection and subsequent sepsis. TLR signaling Integral to innate immune recognition of pat hogens are the toll like receptors ( TLRs ) Initially described in early to mid 90s there are currently ten TLR s described in humans and 12 in mice 51 52 TLR 1, 2, 4, 5, and 6 are cell surface receptors and are typically thought to respond to pathogen associated molecular patterns (PAMPs) derived from bacteria or other microbial products 52 TLR 3, 7, 8 and 9 are intracellular

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24 receptors and recognize DNA/RNA from bacteria and viruses 52 These receptors are evolu tiona rily conserved and were first described in drosophila as the receptor toll 53 Since t hat first seminal discovery, TLRs have been found in many species and all serve a similar p urpose: pathogen surveillance and defense. In addition to PAMPs, TLRs have also been associated with the binding of danger a ssociated molecular patterns ( DAMPS ) / ala rmins. DAMPS are endogenous carbohydrates and/ or proteins that are usually found intracellularly but are released during tissue damage 54 They are also thought to be evolutionarily conserved as examp les of DAMPS are found in the plant kingdom as well 54 The bindi ng of these molecules activate innate immunity and are thought to be important for not only recruitment of innate immunity to the wound but also surveillance of the disrupted tissue for pathogen invasion and wound healing processes 54 Also, these molecules have been implicated in autoimmune processes 55 Probably the most controversial of these DAMPS is high mobility group box protein 1 or HMGB 1. Though initially purported to bind to T LR2 and/or 4, many of the initial studies used recombinant protein generated by E coli and demonstrated a robust immunologic response that may have been due to LPS contamination 56 More recently it was demonstrated that HMGB 1 isolated fr om cow thymus interacted or bound to recep tor for glycosylated end products (RAGE) which together was shown to stimulate TLR7 and 9 57 Though relatively new, other examples of DAMPS include mitochondrial a ssociated molecular patterns and IL 33 58 59 Of these receptors, TLR4 e arguably the best studied and described 51 Unlike other TLRs, activation of TLR4 requires the binding of LPS to the extracellular proteins, LPS binding protein (LBP), CD14, and MD 2 in order

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25 to effectively signal downstream 60 These prot eins facilitate binding of LPS and subsequent transfer of the oligosaccharide to either MD2 alone or TLR4 bound to MD2 60 The TLR4 bound MD2:endotoxin complex subsequently employ s two intracellular adaptor proteins, MyD88 and TRIF, to c arry out its downstream events (Figure 1 2 ) 51 61 It is the TLR4 LPS downstream signaling that is typically thought to be responsible for the systemic inflammatory response syndrome (SIRS) and septic shock co mponent of gram negative sepsis, as many studies from our laboratory and other investigators has demonstrated that a robust inflammatory response occurs following exposure of a given host to LPS 62 64 This inflammatory response typically leads to the elaboration of cytokines and chemokines such as tumor necrosis factor IL 1 CXCL10, and IL6, as well as the production of type 1 interferons, and 52 As indicated in Figure 1 2 the sum total of TLR4 signaling leads to both MyD88 and TRIF activation (Figure 1 2). Downstream MyD88 signaling through the IL 1 receptor associated kinase 4 (IRAK 4) initiates the phosphorylation of the I K NF B complex. This then subsequently leads to the release and translocation of NF B to the nucleus and production of inflammatory cytokines and chemokines 52 In addition, to NF B activation, p38 and mitogen activated protein kinase (MAPK) signaling is also activated through MyD88 to a TNF receptor associated factor 6 (TRAF6) /transforming growth factor activated kinase 1 pathway (Figure 1 2) 52 However, as the downstream consequence of p38/MAPK signaling is also thought to lead to the production of inflammatory cytokine/chemokines production, the thesis will focus on mainly NF B 52

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26 TLR4 signaling via TRIF typically leads to the phosphorylation and activation of interferon response factor 3 (IRF 3) 51 This pathway subsequently leads to the pr oduction of type 1 interferons Interestingly, downstream TRIF signaling also leads to the production of NF B associated inflammatory cytokines/chemokines. In addition, t hough both TLR3 and TLR4 utilize TRIF, only TLR4 uniquely uses a second adaptor protein, TRIF like adaptor molecule (TRAM), as an intermediate messenger for TRIF activation (see Fig ure 1 2 ) 65 66 TLRs 1,2, 5, 6, 7, 8, and 9 exclusively utilize MyD88 for downstream signaling, which in the case o f 7,8, and 9 leads to phosphorylation of IRF 5 and/or 7 and the production of Type 1 interferons, in addition to NF B dependent inflammatory mediators 51 52 67 Though MyD88 i s critical for the downstream signaling events of nearly every TLR, several investigators have demonstrated the poor expression of MyD88 in the neonate versus adult peripheral blood mononuclear cells ( PBMCs ) in response to TLR ligands 10 12 In addition, a study by Sadeghi et al. demonstrated that the expression of MyD88 varies in PBMCs depending on the gestat ional age of the neonate, with expression increasing as gestational age increases 12 These in vitro studies suggest that MyD88 dependent signaling pathways are no t as active in neonates as they are in adults; however, in vivo studies have not yet corroborated these data. No data exists for the expression or relative importance of TRIF signaling in neonatal sepsis. Though TLRs are expressed on both adaptive immune associated c ells, such as B and T lymphocytes we have found that this arm of immunity plays little to no protective role in the survival of neonates to sepsis 8 Ther efore the focus of this section will be on TLR activation of innate immune effector cells such as neutrophils and macrophages.

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27 TLR activation of neutrophils and macrophages is well known and contributes not only to the production of cytokines and chemokine s, but also to the exocytosis of bactericidal secretory granules. It also contributes to the activation of several members of the NADPH oxidase complex responsible for the oxidative burst, such as NOX1 and NOX2 as well as the generation of mitochon dri al as sociated reactive oxygen species 68 69 Less evidence exists for a direct TLR role in phagocytosis though an important role for MyD88 in macrophage phagocytosis of Listeria monocytogenes through phosphotidylinositol 3 kinase and Rac1 signaling has been reported 70 Also a recent report by Sotolongo and colleagues did suggest that TRIF dependent interferon production was critical for effective phagocytic uptake of gram negative organisms 71 In addition, it is clear that several cytokines and chemokines can augment the activation status of these innate immune effector cells to not only increase and improve effector functions, such as ph agocytosis and ROS production. TLR4 and relevance to sepsis As polymicrobial and gram negative sepsis are still critical and important causes of mortality in the adult population and LPS is a constituent of gram negative organism cell walls, the role of TLR4 signaling in sepsis has been frequently explored. However, an important distinction must be made between these investigations as some studies utilize highly lethal models (70 100% mortality) and other use less lethal models (0 30% mortality) of sepsi s. Importantly, the kinetics of the mortality in t hese models are different. In h igher letha lity models of sepsis, we observe mostly early death likely related to endotoxin induced SI RS or shock versus less lethal models where later deaths associated with a failure to control the inciting infection predominate For example, studies that have used higher lethality models have found that the deletion or

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28 blockade of TLR4 actually improves survival to septic insult 72 74 In contrast, deletion of TLR4 or one of its adaptor proteins, MyD88, in less severe models of sepsis appears to worsen survival 75 76 Despite the equivocal nature of these presented studies, the importance of balance in TLR4 dependent inflammation to recruit innate immune effector cells to the site of infection, but not cause overwhelming shock through exaggerated systemic inflammatory response syndrome ( SIRS ) responses is a recurrent theme. The outcomes in humans with deletions or mutations in TLR4 or MyD88 signaling are controversial, but are overall thoug ht to be deleterious. As the presence of these mutations is exceedingly rare, most study subject sizes are small and therefore conclusions may not be generalizable or valid. Despite some reports suggesting that there is no effect of TLR4 polymorphisms in s epsis, a recent report from Kumpf et al. of approximately 400 patients has suggested that polymorphisms in TLR4 signaling was associated with increased risk of severe infections (odds ratio 5.5 vs patients with a normal TLR4 gene) compared to normal patien ts 63 77 Rare deficiencies in other TLR related proteins, such as MyD88 or IRAK4, also do occur in humans Th ese humans are plagued by multiple bacterial infections, predominantly gram positive organisms such as S treptococcus pneumonia and S taphylococcus aureus In a study by Picard et al. most of patients studied had developed a noninvasive or invasive infec tion by age 2 (88% of patients studied) with a minority of patients (33%) developing their first infection during the neonatal period 78 79 As of yet, there is only one published report of patients with TRIF deletions or polymorphisms and their susceptibility to viral infections 80 However, patients with defects in NEMO or I K activators of NF B, would compromise the

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29 inflammatory pathway of both TRIF and MyD88 in TLR4 signaling 79 These patients present similarly as patients with dysfunctional MyD88 signaling and are prone to infection and subsequent sepsis 78 79 CXCL10/IP 10 As described above, TLR downs tream signaling leads to the production of many inflammatory cytokines and chemokines which function to recruit and activate innate immune effector cells during the course of infectio n 52 Unfortunately, many of these TLR ligands, in particular LPS, cannot be used as a therapeutic for obvious reasons, the most important of which is the fever, leukocytosis, my algia and hypotension associated with low doses to the massive SIRS response and life threatening shock that occur following increased administration 81 Of interest in 2002, Ness et al. found that blockade of one of the chemokines produced in response to LPS or during septic insult, CXCL 10 was found to increase the mortality associated with murine model of polymicrobial sepsis 82 The initial mechanism was thought to be related to immunomodulation of one of the chemokine r eceptors, CXCR2, whose ligands are IL 8, MIP2 and KC Gro, all of which are important for recruitment of both neutrophils and macrophages 82 However, this explanation may have been incomplete. CXCL10 belongs to the family of C X C chemokines that include monokine induced b y gamma interferon (MIG or CXCL 9) and interferon inducible T cell chemo attractant alpha (I TAC or CXCL 11). The receptor for all three chemokines is CXCR3. CXCL10 can be produced via either IRF3 dependent t ype 1 ( and and NF B dependent t ype 2 ( ) interferons 83 84 The CXCL 10/CXCR3 chemokine axis has been lin ked to several inflammatory processes 85 87 88 Although initially im plicated in T

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30 helper 1 (Th1) responses, CXCL10 /CXCR3 also has been found to be associated with innate immune function 86 88 For example, CXCL10 has also been shown to augment macrophage phagocytosis, parasite burden, and nitric oxide production during Leishmania infection 89 Also, during the course of this project, we have also found that type I interferon dependent CXCL10 production is important for survival in an adult model of polymicrobial sepsis 64 Aside from studies examining the role of CXCL10 as a biomarker of infection in neonates, n o studies to date have invest igated the function of CXCL 10 in neonates or in neonatal innate immunity 90 91 Summary It is clear that neonatal sepsis is still a critical problem globally. The issues are multifactorial but include 1) poor to subtle physiologic responses in neonates to infection which may lead to not only missed diagnoses but also overtreatment of patients 7 2) improvements in neonatal critical care that have led to increased survival of premature neonates, and therefore, increased ICU stay and exposure to nosocomial pathogens 7 27 and, 3) an immunologicall y distinct but functionally immature innate immune effector cells 8 43 Therefore, strategies are needed to prote ct infants from infection and the long term se quelae of neonatal sepsis 92 One possible route may be through the development of adjuvants that can ma tur e and/ or activat e neonatal inn ate immun ity As innate immune activation during infection is link ed to TLR signaling the subject of this thesis will focus mainly on u nderstanding the role of the TLR adaptor proteins, MyD88 and TRIF, and how they are immunologically distinct in neonate s relative to the adult in response to sepsis 52 This is important because many sepsis studies have been conducted in adult preclinical models and have focused on MyD88 signaling 75 76 93 94 These published data support a critical role for MyD88 i n the survival of adults to gram

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31 negative and polymicrobial sepsis and has been linked to the production of inflammatory mediators as well as innate immune function 75 76 93 94 In addition, a recent report has suggested that TRIF signaling in adults may play a n important role in the phagocytic ability of adult macrophages in response to Yersinia enterocolitica a gram negative organism 71 However given these adult data the role of MyD88 and TRIF in the innate immune response to ne onatal sepsis remains unknown. In contrast to the adult st udies described above, we demonstrate that TRIF but not MyD88 signaling is critical f or clearance and survival of neonates to E coli gram negative sepsis. Though no deficiencies in phagocytosis were observed as suggested by the Sotolongo et al. study, the production of reactive oxygen species was significantly decreased in TRIF / neonates relative to the wild type neonates 71 Importantly, as demonstrated in other studies investigating the role of MyD8 8 signaling in response to gram negative sepsis, MyD88 / but not wild type or TRIF / adults were shown to be more susceptible to E coli infection. These data suggest a novel critical role for TRIF signaling in the neonate, but not the adult, in respon se to gram negative infection. Another important goal of the thesis will be to identify soluble factors that are downstream of TLR signaling and can modulate neonatal innate immunity. Previous work from our laboratory as well as earlier work from Ness et al. has demonstrated that Type 1 interferon dependent CXCL10 can modulate adult innate immune function and is critical for survival to polymicrobial sepsis 64 82 Though we have previously shown that survival to neonatal sepsis was independent of type 1 interferon signaling, CXCL10

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32 can be produced via type 1 or 2 interferon pathways and may still be important for not only neonatal innate immune function but also survival to neonatal sepsis 8 The data presented below also identifies a n important role for the endogenous CXCL 10 response to neonatal polymicrobial sepsis In addition, CXCL10 pretreatment of innate immune effector cells is shown to improve chemotaxis and phagocytic ability of both neutrophils and macrophages. To further support the role of CXCL10 in neonatal inna te immune function, blockade of CXCL10 during polymicrobial sepsis was also demonstrated to decrease the phagocytic ability and the absolute numbers of peritoneal macrophages and neutrophils following sepsis. Importantly, blockade of CXCL10 also decreased bacterial clearance and increased mortality to polymicrobial neonatal sepsis. Finally we have been able to demonstrate that by pretreating neonatal mice with TLR ligands, in particular LPS, we are able to functionally improve innate immune function and subsequent survival to polymicrobial sepsis 8 Therefore, a nother component of the thesis will attempt to delineate which components and/or downstream products of neonatal sepsis. The da ta below suggests that TRIF, but not MyD88, signaling is critical for the TLR4 protective adjuvant effect in response to neonatal polymicrobial sepsis Also, we d emonstrate that blockade of CXCL10 completely abrogates this protection similar to the loss of TRIF. Though administration of CXCL10 only partially recapitulated the protective effect to polymicrobial sepsis, the data suggests that TRIF dependent inflamma tion, in particular CXCL10, is required for the observed adjuvant effect to neonatal sepsis. This is important because if novel therapeutics can be developed and combined to target both bacterial growth/activity, i.e. lactoferrin and probiotics, with

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33 strat egies to functionally improve neonatal innate immunity, we may be able to further augment outcome in this highly susceptible population. Overarching Hypothesis TRIF signaling leading to downstream production of CXCL10 is important for innate immune functio n and survival during neonatal sepsis and further, that modulation of these signaling pathways is responsible for the observed adjuvant induced protection to neonatal sepsis

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34 Figure 1 1. Incidence and mortality in neonatal sepsis. A. Incidence of ped iatric sepsis in the US 6 B. Mortality trends of early and late neonatal sepsis over two time periods: 1985 to 1991 and 1995 to 1998 3

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35 Table 1 1 Therapeutic Trials in neonatal sepsis (adapted from Tarnow Mordi et al.)

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36 Figure 1 2. TLR4 signaling. TLR 4 signaling proceeds through either MyD88 or TRIF adaptor proteins. Downstream MyD88 signaling leads to the production of inflammatory cytokines and chemokines whereas downstream TRIF signaling leads to both inflammatory cytokines/chemokines as well as Type 1 interferons.

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37 CHAPTER 2 TRIF DEPENDENT INFLAMMATION AND INNATE IMMUNE ACTIVATION IS CRITICAL FOR SURVIVAL TO GRAM NEGATIVE NEONATAL SEPSIS Background One million newborn children die each year from sepsis or severe infection 8 95 Mort ality rates among these babies range from 10 40% depending on birth weight and age at onset of sepsis, but are especially high in very low birth weight infants (VLBW) 6 19 In addition, despite progress in outcomes with post partu m group B streptococcus infections and sepsis, gram negative sepsis from Escherichia coli continues to be a significant problem in the neonatal intensive care units 18 22 It is now gen erally accepted that the functional status of neonatal innate and adaptive immunity may be one of the reasons why this population fares so poorly during infection 8 Numerous studies have examined the epidemiol ogy and the molecular markers associated with neonatal sepsis 3 6 19 21 but few studies have characterized t he underlying immunological resp onses and patho physiology Though many studies in adult models of infection and sepsis exist, examining the relative requirements of innate immunity and potential modulators of innate immune response, few studies have established these responses or mechani sms in the neonate and whether they are similar or different than in the adult. Of these modulators of innate immune activation, Toll like receptor (TLR) signaling is arguably one of the most important. TLR signaling proceeds through either myeloid differ entiation primary response gene 88 (MyD88) leading predominantly to the production of proinflammatory cytokines/chemokines or TIR domain containing adapter protein inducing interferon (TRIF) leading to the production of both Type I interferons, as well a s the production of proinflammatory cytokines and chemokines 52 Several adult

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38 studies have identified MyD88 signaling as critical for survival to gram negative infection or seps is, as well as important for the production of reactive oxygen species in neutrophils 76 93 96 98 The role of TRIF signaling in adult gram negative infection is somewhat controversial; the response to some gram negative infections such as Burholderia infection is thought to be MyD88 dependent, whereas TRIF signaling was recently shown to be critical for survival to Yersinia enterocolitica 71 93 The role of these TLR adapt or signaling proteins in the survival of the neonate to gram negative infection is unknown. We have previously shown that pretreatment of murine neonates with toll like receptor (TLR) 4 ligand, lipopolysaccharide (LPS) is able to significantly improve su rvival to polymicrobial sepsis 8 Although this effect was associated with increased activation/recruitment of innate immune effector cells, the mechanism was unknown. In this report, we demonstrate that TRIF, but not MyD88, is critical for the observed TLR4 adjuvant effect. In addition, since the TLR4 adjuvant effect was dependent on TRIF, we hypothesized that TRIF / but not MyD88 / neonates would be susceptible to gr am negative E coli sepsis. Indeed, TRIF / neonates were more susceptible to E coli sepsis compared to wild type (WT) and MyD88 / neonates. This was associated with decreased absolute numbers of peritoneal neutrophils and macrophages at 12 hours post E co li infection, decreased neutrophil and macrophage reactive oxygen species production, as well as increased peritoneal and blood bacterial counts in TRIF / compared to wild type neonates. Importantly, we also demonstrate that MyD88 / but not TRIF / you ng adults were more susceptible to E coli gram negative infection. This

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39 report presents novel findings which demonstrate that neonates are reliant on TRIF but not MyD88 signaling for survival to neonatal sepsis. Methods Mice Six to 8 week old mal e and female C57BL/6J (wild type (WT)) and TRIF / (C57Bl/6J Ticam1 Lps2 /J) mice were purchased from T he Jackson Laboratories (Bar Harbor, ME) and used for adult breeders and adult experiments MyD88 / ( B6.129P2(SJL) Myd88tm1.1Defr /J ) mice were obtained f rom S Akira, initially maintained at Rhode Island Hospital and Brown University, and transferred as a gift to UF for breeding and colony maintenance. Adult mice were mated in a harem schema with one male to two females. Neonatal mice were studied between f our and seven days of age. All experiments were approved by the Institutional Animal Care and Use Committee of the University of Florida, College of Medicine. Cecal slurry (CS) model Polymicrobial sepsis was induced as previously described 4 Briefly, cecal contents of adult C57BL/6 J mice were suspended in 5% dextrose solution (D5W) at a final concentration of 80 mg/mL and injected intraperitoneally (ip) into five to seven day old neonates. A n LD 70 (1.3 mg fecal contents/g body weight) o f cecal slurry, as determined from previous experiments, was used 4 Escherichia coli gram neg ative sepsis After institutional review board permission was obtained to review patient records, a clinical isolate of Escherchia coli was found from a blood culture drawn from a neonate (less than 30 days of age) with E coli urosepsis. The isolate was f urther characterized as Escherichia coli O11:H18. This isolate was then used in the murine

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40 studies described in this manuscript. E coli was grown in trypticase soy agar broth and plates (Fisher scientific). Neonatal C57BL/6 mice five to seven days old rec eived ip an LD 70 of E coli (2 x 10 3 CFUs) in 50 L of phosphate buffered saline Total volume of injected solution was less than 50 L. Neonates were either followed for survival for seven days or euthanized and tissues harvested at the time points indicat ed. Pretreatment with a TLR4 agonist (bacterial LPS). Neonatal C57BL/6 mice four to six days old received ip administration of lipopolysaccharide (1 g/g BW; ultrapure via ion exchange chromatography Escherichia coli O26:B6 (Sigma Aldrich St Louis, MO ) ) in physiologic saline 24 hours before challenge with cecal slurry. Control animals received saline only ip 24 hours before cecal slurry challenge. Total volume of injected solution was less than 50 L. Peritoneal, sera, and media cytokine concentrations Blood and peritoneal washes were harvested from neonates at the time points described following either LPS or E coli administration As reports have shown that heparin can bind to CXCL10 and possibl y reduce its detection in blood, serum was used for these assays 99 100 Blood was collected via intra cardiac puncture and centrifuged to collect the supernatant (serum). To collect peritoneal washes, the peritoneal cavity was instilled with 500 L of cold p hosphate buffered saline (PBS Cellgro, Manassas, VA). The peritoneal cavity was then opened over a sterile tissue culture dish and the peritoneal wash collected. Peritoneal washes were then centrifuged to remove debris and the supernatants were stored at 80 0 C. Media was collected from in vitro LPS stimulation experiments and centrifuged to remove cellular debris. The supernatant was then used for cytokine detection assays. Sera, peritoneal washes and media were then

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41 measured for IL 1 IL 6 IL 10, IL 12p70, KC, IFN TNF MCP 1, MIP 1 CXCL10 concentrations via Multiplex Luminex Assay (Miltenyi Biotech Auburn, CA ). Flow cytome try Single cell suspensions we re characterized using anti Ly6g APC, anti CD11b PECy7, and/or anti F4/80 APC Ale xf luor 750 (Ebioscience, San Diego, CA). All antibodies were purchased from Becton Dickinson (BD) (Franklin Lakes, NJ) unless otherwise indicated. Samples were acquired and analyzed on an LSR II flow cytometer reestar Inc, Ashland, OR) software. At least 1 x 10 4 live cells (SYTOX Blue ; Invitrogen, Carlsbad, CA) were collected for analysis. Isolation of blood, bone marrow, and splenocytes for phenotypic analysis. Blood was obtained via intracardiac puncture of isoflurane anesthetized neonatal mice into a heparinized syringe. Spleens were harvested from neonates and then dissociated through a 70 m sterile filter. To harvest bone marrow, both femurs and tibias from individual animals were collected and flushed with phosphate buffered saline (PBS) (Cellgro Manassas, VA ). Blood, splenocyte, and bone marrow suspensions were then subjected to erythro cyte lysis using ammonium chloride lysis solution. Cell suspensions were then stained for neutrophil ( Ly6g + /CD11b + ) or macrophage (F4/80 + /CD11b + ) cell surface markers and analyzed via flow cytometry Harvest of peritoneal washes. Peritoneal cell isolatio n was carried out in a similar manner as described previously 8 Due to the fact that peritoneal lavages from individual animals contained relatively few cells, perito neal washes from three to five animals were pooled for

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42 analysis and considered to be one sample. For each time point or condition analyzed, a total of three to four peritoneal samples were collected per experiment. To determine absolute numbers of innate i mmune effector cells, the percentage of macrophages or neutrophils within the total sample population was determined via flow cytometry, multiplied by the total sample cell number, and then divided by the total number of mice in the pooled sample. Absolute numbers represent total macrophages or neutrophils per mouse. Isolation of splenic macrophages and neutrophils for functional analyses. Spleens were disassociated through a 70 m sterile filter into RPMI 1640 media (Cellgro) with 10% heat inactivated l ow endotoxin fetal bovine serum and 0.5% penicillin/streptomycin (Cellgro). Suspensions were then overlayed on top of a Histopaque 1119 (Sigma Aldrich, St Louis, MO) density gradient and centrifuged. Mixed cell suspensions were then washed with media alon e, and individual cell populations were assayed for phagocytosis or reactive oxygen species production by flow cytometry. In vitro stimulation with recombinant CXCL10. Sple nocytes isolated by Histopaque 1119 (Sigma Aldrich) gradient were stimulated with or without recombinant murine CXCL10 at 100 ng/mL for four hours (R&D Systems Minneapolis, MN ). After incubation with CXCL10, cells were washed with PBS and subsequently assayed for phagocytic function or ROS production and phenotypically characterized v ia flow cytometry.

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43 Functional analysis of splenic macrophage s and neutrophils or peritoneal macrophages or neutrophils. For phagocytosis assays, 1 x 10 5 splenocytes isolated via density gradient or peritoneal cell suspensions were incubated with 1 x 10 8 fluorescent polystyrene microspheres (Fluospheres; Invitrogen, Carlsbad, CA) for 30 min at 37C. Cells were then stained and analyzed by flow cytometry, as described above. After cells were gated on either neutrophil ( Ly 6g + /CD11b + ) or macrophage (F4/80 + /C D11b + ) populations, FITC + cells were considered to be phagocytic cells. To assay for ROS production, 2 x 10 6 splenocytes isolated via density gradient were first stained for cell surface markers and lab eled with dihydrorhodamine 123 (Invitrogen). Cells we re then stimulated with 1 M of phorb ol 12 myristate 13 acetate (PMA Sigma Aldrich) at 37C and aliquots were evaluated by flow cytometry at various points over a 30 minute period using a LSR II flow cytometer (BD). A minimum of 1 x 10 4 live, non debris cells were collected for analysis. Statistical analyses. Continuous variables were tested for normality and equality of variances. test whereas differences among groups were evaluated by either on e post t test. Significance was determined at the 95% confidence level Results TRIF / neonatal neutrophils have impaired phagocytic function response to LPS Both the phagocytic function an d production of neutrophil extracellular traps have been demonstrated to be decreased or impaired in neonatal neutrophils relative to

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44 adults 8 49 50 To investigate if TLR signaling through TRIF or MyD88 dependent pathways played any role in the neonatal neutrophil function, splenocytes were harvested from neonates and stimulated with LPS (100ng/mL) for 24 hours. Following staining for neutrophil cell surface markers, cell suspensions were then either incubated with either FITC labeled polystyrene beads or evaluated for ROS production via phorbol ester stimulation. Thoug h no differences were noted between neutrophils from MyD88 / TRIF / and wild type neonates with regards to ROS respiratory burst following PMA stimulation (data not shown) neutrophils from TRIF / animals had significantly decreased phagocytic functio n relative to neutrophils from both MyD88 / and wild type animals in response to LPS (p<0.001) ( Figure 2 1 ). TRIF / neonates have impaired cyto/chemokines production in response to LPS in vivo One of the reasons why neutrophil phagocytic function may be impaired in TRIF / neonates may be due to poor inflammatory mediator production, which can have a paracrine effect on innate immune effector function 101 102 To assess if the inflammatory cyt okine production by immune cell populations was also impaired in TRIF / neonates, media supernatant from neonatal splenocytes stimulated with LPS (100 ng/mL) for 24 hou rs and assayed via luminex for the concentration of cyto/chemokines, including IL 1 TNF IFN and CXCL10. While both MyD88 / and TRIF / produced significantly less IL 6, TRIF / neonates produced significantly less IL 1 and CXCL10 ( Figure 2 2A ). These data suggest that deletion of TRIF, but not MyD88 in the neonate significantly i mpairs both neutrophil phagocytic function and cellular inflammatory responses to LPS. To determine if this is observed decrease in cytokine/chemokine production is recapitulated in vi vo, wild type MyD88 / and TRIF / neonates were injected with LPS at

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45 a dose of 1 g/g body weight (BW) and serum was harvested at 0, 6, and 24 hours post injection. Interestingly in contrast to the in vitro data, MyD88 / neonates produced significantly less IL 1 and TNF compared to wild type neonates, whereas TRIF / neonates produ ced significantly less TNF IL 6, and CXCL10 ( Figure 2 2B ). These results further support the data above and suggest that TRIF plays a critical role in the endotoxin mediated inflammatory responses in the neonate. CXCL10 partially reverses TRIF / defe cts in phagocytosis. We have previously demonstrated that CXCL10 is important for neonatal and adult neutrophil phagocytosis. As CXCL10 was significantly decreased following both in vitro stimulation and in vivo administration of LPS in TRIF / but not M yD88 / or wild type neonates, and TRIF / granulocytes had significantly decreased phagocytic function compared to wild type or MyD88 / neonates following LPS stimulation, we treated TRIF / and wild type neonatal splenocytes with or without 100 ng/mL o f recombinant murine CXCL10 in the presence of LPS. As demonstrated in Figure 2 3 incubation of TRIF / splenocytes with CXCL10 partially but significantly reversed the phagocytic function of TRIF / neutrophils, 84% with CXCL10 vs 70% without CXCL10, fo llowing LPS stimulation (p< 0.05). Though we were not are able to demonstrate complete reversal of the phagocytic defect in TRIF / mice via CXCL10, these data support the importance of TRIF signaling and the production of TRIF dependent inflammatory media tors in innate immune function. TRIF / neonates are not protected against polymicrobial sepsis in response to TLR4 adjuvant protection. We have previously shown that wild type neonates pretreated with LPS are protected against polymicrobial sepsis compa red to neonates pretreated with saline 8

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46 As the data above suggest that TRIF / neonates have impaired responses to LPS, including decreased phagocytic function and de creased inflammatory cytokine/chemokine production, we hypothesized that TRIF / neonates pretreated with LPS would not be protected against polymicrobial sepsis. To address this, wild type TRIF / and MyD88 / neonates were pretreated with LPS (1 g/g B W) 24 hours before polymicrobial sepsis challenge and subsequently followed for survival. As demonstrated in Figure 2 4 in contrast to both MyD88 / and wild type neonates, TRIF / neonates were not protected against cecal slurry (CS) induced polymicrobi al sepsis. These data suggest that the loss of TRIF, not only impacts the inflammatory responses and functional maturity of innate immune effectors cells recruited to the peritoneum, but also is critical for the TLR4 adjuvant protective effect that we have previously demonstrated 8 TRIF / neonates are susceptible to gram negative sepsis. Though the loss of MyD88 has been associated with susceptibility to gram negati ve infection in adults, the role of MyD88 in neonatal gram negative infection and sepsis is unclear 76 93 98 To explore if the decrease in endotoxin responses of TRIF / neonates demonstrated above extended also to gram negative infection, TRIF / MyD88 / and wild type neonates were challenged with 1 x 10 3 CFUs o f an Escherichia coli isolate from a neonate with urosepsis. As demonstrated in Figure 2 5A TRIF / neonates were more susceptible to E coli challenge compared to both MyD88 / and wild type neonates. This was associated with increased peritoneal and bloo d CFUs ( Figure 2 5B ).

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47 TRIF / neonates produce decreased local inflammatory cytokines and chemokines in response to E coli gram negative sepsis. As the downstream products of TLR signaling are inflammatory mediator s, we examined whether there were any diff erence s in the production of key cytokines or chemokines in TRIF / neonates compared to wild type or MyD88 / neonates that could potentially explain this increased susceptibility to gram negative sepsis. Peritoneal washes from TRIF / MyD88 / and wild type neonates were harvested at 0, 6, 12, and 24 hours post E coli administration and assayed for the concentration of IL 10, IL 12, IFN IL 1b, TNF IL 6, CXCL10, MCP 1, MIP 1 and keratinocyte chemo attractant ( KC ) via Luminex multiplex cytokine a rrays. There were significant differences in the peritonea l concentration of IL 6, KC, MIP 1 and CXCL10 in TRIF / neonates compared to MyD88 / or wild type neonates following E coli inoculation ( Figure 2 6 ). In addition, not surprisingly there were di fferences in TNF production between MyD88 / and TRIF / neonates compared to wild type neonates in response to E coli infection. No differences were noted in any of the other cytokines/chemokines( Figure 2 6 ). Decreased early peritonea l neutrophil and ma crophage recruitment and late ROS production in TRIF / neonates in response to E coli neonatal sepsis. To understand why TRIF / neonates exhibit susceptibility to gram negative sepsis compared to wild type neonates, we harvested peritoneal washes from TR IF / and wild type neonates 24 hours post E coli infection. TRIF / neonates had significantly fewer peritoneal neutrophils and macrophages compared to wild type neonates at 12 hours post E coli administration ( Figure 2 7A ). In terms of phagocytic functio n, though there were no differences in phagocytosis at 12 hours post E coli infection between TRIF / and wild type neonates ( data not shown ), TRIF / neonates had significantly more

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48 phagocytic peritoneal neutrophils compared to wild type animals ( Figure 2 7B ). Despite this increase in phagocytic neutrophils, TRIF / neonatal neutrophils and macrophages produced significantly less ROS than wild type neonates at 24 hours post E coli infection ( Figure 2 7C ). Together these data suggest that TRIF / neonates h ave impaired innate immune neutrophil and macrophage function compared to wild type neonates that subsequently impairs clearance of gram negative infection. Adult MyD88 / mice are susceptible to gram negative sepsis but not TRIF / or wild type adult mic e Infection and sepsis studies in adults have suggested that MyD88 signaling is more critical than TRIF signaling for survival to gram negative infection 76 93 98 To see if these observations held true in our model system, we administered 1 X 10 6 E Coli ip into adult MyD88 / TRIF / and wild type mice. Not surpri singly, MyD88 / adults were dramatically more susceptible to E coli challenge compared to TRIF / or wild type adults ( Figure 2 8A ). This increased susceptibility of MyD88 / adults to E coli was associated with increased bacteremia and peritoneal bacteri al counts (Figure 2 8B) These data suggest intrinsic differences in the TLR signaling adaptor protein pathways that are important for the clearance of gram negative infection between neonates and adults. Discussion Many studies have focused on the role of MyD88 in response to polymicrobial or gram negative sepsis in adults 75 76 93 94 96 98 Furthe r, these studies have suggested a critical role for TLR4 MyD88 signaling in the survival of adult mice to both polymicrobial MyD88 and TRIF signaling in the lethality of adults to endotoxemia, again the loss of MyD88 appears to offer more protection to lethal endotoxin challenge than th e loss of

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49 TRIF in these studies 52 103 TLR signaling in neonates, however, is not as well characterized. Our da ta suggest that TLR4 TRIF signaling in the neonate, in contrast to the young adult, is critical for respo nse to TLR4 agonists We demonstrate that the loss of TRIF in neonates impairs the production of several inflammatory cytokines and chemokines in respo nse to LPS stimulation. In addition, following in vitro stim ulation with LPS, our data show a reduced phagocytic function in TRIF / neutrophils compared to wild type or MyD88 / neutrophils. Previous work from our laboratory has also demonstrated a critic al role for CXCL10 in the phagocytosis and survival of neonates to polymicrobial challenge 101 In the current study, we show that the loss of TRIF in neonates impairs both in vitro and in vi vo CXCL10 production, as well as the phagocytic response of neutrophils to LPS. Further, in the current study, we also demonstrate that TRIF / neonates are not protected following LPS adjuvant pretreatment to polymicrobial sepsis. Similarly, our recent w ork shows that if the CXCL10 response to LPS is blocked, the protective adjuvant effect of LPS is lost in response to polymicrobial sepsis 101 These findings suggest a novel critical role for a TRIF dependent inflammation in response to LPS and gram negative infection. Importantly, we also demonstrate that TRIF signaling is not as critical in adults and suggest a reliance of neonates on TRIF signaling in contrast to adults who appear to be mor e dependent upon MyD88 for response and survival to gram negative or polymicrobial infection. Although a recent report by Sotolongo and colleagues has demonstrated that TRIF signaling is important for the adult survival to gram negative infection, it is i mportant to note that their model of infection utilized the gram negative enteropathogen Yersinia

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50 enterocolitica not Escherichia coli which may indeed have different immunopathological processes and requirements for clearance despite both being gram nega tive organisms 71 In addition, the susceptibility of MyD88 / adults to gram negative infection was not examined i n the report but was rather inferred from data investigating phagocytic function of MyD88 / TRIF / TLR4 / and wild type macrophages. Finally, Sotolongo et al. stressed and demonstrated the importance of TRIF dependent type I interferon production in the survival of adult mice to gram negative infection 71 In a previous report, we have demonstrated that loss of ty pe I Interferon signaling is not important for survival of neonates to gram negative infection 8 Though it was in a polymicrobial model of neonatal sepsis, most of the enteric organisms within the cecum that are administered through cecal slurry challenge are gram negative (> 99%). These data highlight important differences between the neonatal and adult responses to bacterial products and gram negative infection. Prev ious data have suggested that neonates rely more heavily on a functioning innate immune system for protective immunity than do young adults 8 This report moves that o bservation forward, and demonstrates for the first time that TRIF but not MyD88 signaling is essential for innate immune activation and is required for survival of neonates to gram negative infection. This is important, as much of the focus on early signal ing pathways of innate immunity to microbial infection has been on MyD88, as it is the primary adaptor protein responsible for downstream signaling for the majority of TLRs 52 In addition, these data suggest an ontological difference between adults and neonates that reflect differential requirement for TRIF dependent inflammation during the neonatal period and redirect

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51 the focus on therapeutics designed around TRIF dependent inf lammation and innate immune activation to improve outcome s in these particularly susceptible patients.

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52 Figure 2 1. TRIF / neonatal granulocytes have impaired phagocytic function in response to LPS stimulation. Phagocytic uptake of polystyrene FITC lab eled beads in WT, MyD88 / and TRIF / neutrophils following 24 hr stimulation with LPS (On e way ANOVA p <0.001; ** NS; *** p< 0.004) (n=4 5 animals per group).

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53 Figure 2 2. TRIF / neonates have decreased inflammatory cytokine/chemokines responses compared to MyD88 / an d WT neonates. A. Cytokine production from splenocytes isolated from WT, TRIF / and MyD88 / neonates following 24 hour stimulation with LPS (100ng/mL)(* p<0.05) (n=3 4 animals per group). B. Sera cytokine concentrations from WT, TRIF / and MyD88 / neonates at 0, 6, and 24 hours post LPS administration (One way ANOVA *p<0.05; **p<0.01; ***p<0.024) (n=4 5 animals per group).

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54 Figure 2 3. TRIF / neonates are not protected against polymicrobial sepsis with TLR4 agonist pretreatment. WT, TRIF / and MyD88 / neonates were treated with either LPS or saline 24 hours before CS challenge and followed for

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55 Figure 2 4. Partial reversal of TRIF / neonatal granulocyte phagocytic dysfunction with CXCL10 re placement. Splenocytes from WT or TRIF / neonates were stimulated with LPS (100ng/mL). TRIF / splenocytes were also either stimulated with or without CXCL10 (100ng/mL) (* p<0.01; ** p<0.05) (n=4 5 animals per group).

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56 Figure 2 5. TRIF / neonate s are more susceptible to gram negative infection compared to MyD88 / or WT neonates. A. WT, TRIF / and MyD88 / neonates were inoculated ip with E coli and subsequently followed for (C) were harvested 24 hours post E coli inoculation and plated on blood agar, and colony forming units (CFUs) counted (n=5 7 animals per group) (One way ANOVA p<0.05).

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57 Figure 2 6. TRIF / neonates produce decreased local inflammatory mediators co mpared to MyD88 or WT neonates. Peritoneal washes were obtained from TRIF / MyD88 / and WT neonates at 0, 6, 12, and 24 hours post E coli administration and assayed for concentration (n=5 animals per time p<0.05 ** p<0.001) (n=4 5 animals per group).

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58 Figure 2 7. Decrease innate immune effector cell recruitment and ROS production in TRIF / post E coli infection A. Periton eal washes were harvest from WT and TRIF / neonates at 12 and 24 hours post E coli inoculation and assayed for the presence of neutrophils and p<0.04 p<0.01 ** p<0.001 ). B. Peritoneal washes from WT, TRIF / and MyD88 / neonates 24 hours post E coli were incubated with FITC l abeled polystyrene beads and phenotyped for percent phagocytic neutrophils or macrophages via flow cytometry ( washes from WT, TRIF / and MyD88 / neonates 24 hours post E coli were phenotyped for the presence o f neutrophils and macrophages and subsequently stained for DHR 12 test *p=0.016 ** p<0.001 ) (n=4 5 animals per group ).

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59 Figure 2 8. MyD88 / adults are more susceptible to gram negative Escherichia coli seps is compared to TRIF / and WT adults. A. Six to twelve week old WT, TRIF / and MyD88 / were inoculated ip with E coli and followed for collected from MyD88 / TRIF / and W T adults 24 hours post E coli infection (One way ANOVA p<0.009 ** p<0.001) (n=5 animals per group).

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60 CHA PTER 3 SURVIVAL OF NEONATES TO POLYMICROBIAL SEPSIS IS INDEPENDENT OF TLR SIGNALING Background Toll like receptors (TLRs) are a well describe d and conserved set of pathogen recognition receptors, and are thought to be critical to host surveillance and recognition of pathogen invasion 51 52 Downstream TLR signaling either proceeds through one of three different pathways MyD88, TRIF, or in the unique case of TLR4, both MyD88 and TRIF 52 Elaboration of MyD88 signaling through IRAK4 leads to the phosphorylation of IkK and the early activation/translocation of NF B to the nucleus, as well as p38 MAPK activation, and the production of inflammatory cytokines and growth factors 52 TRIF signaling typically leads to the production of IRF 3 dependent type 1 interferons, as well as late activation of NF B and the production of inflammatory cytokines 52 Many studies in adult models of infection and sepsis have suggested that TLR signaling is critical for pathogen surveillance, activation of innate immunity, and subsequent clearance of infection 75 76 93 104 105 In fact, loss of MyD88, which is utilized by the majority of TLRs, has been shown to worsen survival to a cecal ligatio n and puncture, the gold standard murine model for the study of polymicrobial sepsis, as well as increase the susceptibility of adult mice to gram negative infection 75 93 In addition, loss of MyD88 has been linked to decreased innate immune function, specifically phagocytosis and reactive oxygen species production 70 94 Despite the vast number of studies exploring the role of these proteins in adult infection and sepsis, the role of these proteins in neonatal sepsis is unclear. Several in vitro investigations have demonstrated decreased expression of MyD88 and

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61 expression/production of inflammatory cytokines following stimulation with the TLR4 agonist, lipopolysaccharide 11 12 Also, a longitudinal study by Corbett et al. examined the cytokine production of peripheral nuclear cells in response to TLR ligands of children from neon ate to age 2, and suggested that the development of TLR responses is not necessarily impaired in neonates and young children, but may just be specific for the developmental stage of immunity 44 In addition, there have been epidemiological studies that have documented the presence of mutations in TLRs, MyD88, and other downstream TLR associated kinases in humans, who, not surprisingly, have an increased incidence o f viral and bacterial infection during the neonatal and early childhood period 78 80 However, these studies are largely non mechanistic and do not specifically a ddress or document the role of MyD88 and TRIF in abdominal polymicrobial sepsis, of which necrotizing enterocolitis is a major contributor during the neonatal, and in particular, the premature neonatal population. Using a model of abdominal polymicrobial neonatal sepsis model d eveloped in our laboratory 4 we demonstrate that despite the loss of TRIF or MyD88, neonates are still able to recruit neutrophils and macropha ges to the peritoneum following a polymicrobial challenge 4 In addition, we demonstrate that innate immune effector cells from wild type, TRIF / and MyD88 / neonates are still able to produce equivalent amounts of reactive oxygen species in response to the phorbol ester, phorbol 12 myristate 13 acetate. Importantly, wild type, TRIF / and MyD88 / mice also have similar survival to polymicrobial sepsis either at an LD 4 0 or an LD 70 dose and further, the simultaneous deletion of both TRIF and MyD88 does not increase the mortality of neonates to an LD 4 0 dose of polymicrobial sepsis.

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62 Materials and Methods Mice Six to 8 week old male and female C57BL/6J (wild type), MyD8 8 / (strain) and TRIF / (C57Bl/6J Ticam1 Lps2 /J) mice were purchased from T he Jackson Laboratories (Bar Harbor, ME) and used for adult breeders and adult experiments MyD88 / ( B6.129P2(SJL) Myd88tm1.1Defr /J ) mice were obtained from S Akira and were maint ained at Rhode Island Hospital and Brown University. These mice were then transferred to the University of Florida (UF) and used for adult breeders and adult experiments. TRIF / /MyD88 / double knockout mice were generated at UF via breeding of TRIF / an d MyD88 / mice. Adult mice were mated in a harem schema with one male to two females. Neonata l mice were studied between five and seven days of age. All experiments were approved by the Institutional Animal Care and Use Committee at the University of Flor ida, College of Medicine. Cecal slurry (CS) model Polymicrobial sepsis was induced as previously described 4 Briefly, cecal contents of adult C57BL/6 J mice were susp ended in 5% dextrose solution (D5W) at a final concentration of 80 mg/mL and injected intraperitoneally (ip) into five to seven day old neonates. A n LD 4 0 (1.0 mg fecal contents/g body weight) or L D 70 (1.3 mg fecal contents/g body weight) of cecal slurry, as determined from previous experiments, was used 4 Flow cytometry Single cell suspensions we r e characterized using anti Ly6g APC, anti CD11b PECy7, and/or anti F4/80 APC Alexfluor 750 (Ebioscience, San Diego, CA). All

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63 antibodies were purcha sed from Becton Dickinson (BD Franklin Lakes, NJ) unless otherwise indicated. Samples were acquired and analyzed on an LSR II flow cytometer software. At least 1 x 10 4 live cells (SYTOX Blue ; Invitrogen, Carlsbad, CA) were collected for analysis. Isolation of blood, bone marrow, and splenocytes for phenotypic analysis Blood was obtained via intracardiac punctu re of isoflurane anesthetized neonatal mice into a heparinized syringe. Spleens were harvested from neonates and then dissociated through a 70 m sterile filter. To harvest bone marrow, both femurs and tibias from individual animals were collected and flus hed with p hosphate buffered saline (PBS Cellgro). Blood, splenocyte, and bone marrow suspensions were then subjected to erythrocyte lysis using ammonium chloride lysis solution. Cell suspensions were then stained for neutrophil ( Ly6g + CD11b + ) or macrophage ( Ly6g F4/80 + CD11b + ) cell surface markers and analyzed via flow cytometry Harvest of peritoneal washes Peritoneal cell isolation was carried out in a similar manner as described previously 8 Due to the fact that peritoneal lavages from individual animals contained relatively few cells, peritoneal washes from three to five animals were pooled for analysis and considered to be one sample. For each time point or condition analyzed, a total of three to four peritoneal samples were collected per experiment. To determine absolute numbers of innate immune effector cells, the percentage of macrophages or neutrophils within the total sample population was determined via flow cyto metry, multiplied by the total sample cell number, and then divided by the total number of mice

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64 in the pooled sample. Absolute numbers represent total macrophages or neutrophils per mouse. Isolation of splenic macrophages and neutrophils for functional a nalyses Spleens were disassociated through a 70 m sterile filter into RPMI 1640 media (Cellgro) with 10% heat inactivated low endotoxin fetal bovine serum 0.5% and penicillin/streptomycin (Cellgro). Suspensions were then overlayed on top of a Histopaque 1119 (Sigma Aldrich, St Louis, MO) density grad ient and centrifuged. Mixed cell suspensions were then washed with media alone, and individual cell populations were assayed for phagocytosis or reactive oxygen species (ROS) production by flow cytometry. Functional analysis of splenic macrophage s and neut rophils or peritoneal macrophages or neutrophils To assay for ROS production, 2 x 10 6 splenocytes isolated via density gradient were first stained for cell surface markers and la beled with dihydrorhodamine 12 3 (Invitrogen). Cells were then stimulated wit h 1 M of phorb ol 12 myristate 13 acetate (PMA Sigma Aldrich) at 37C and aliquots were evaluated by flow cytometry at various points over a 30 minute period using a LSR II flow cytometer (BD). A minimum of 1 x 10 4 live, non debris cells were collected f or analysis. Statistical analyses Continuous variables were tested for normality and equality of variances. test whereas differences among groups were evaluated by either one way ANOVA with either post t test. Significance was determined at the 95% confidence level

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65 Results No differences in peritoneal recruitment of neutrophils and macrophages at 24 hours following polymicrobial sepsis The downstream conse quence of TLR signaling leads to the production of many inflammatory cytokines and chemokines, which serve to recruit and activate innate immune effector cells to localize and clear infection. In addition, the loss of MyD88 has been associated with decreas ed reactive oxygen species production in response to a pure TLR agonist, as well as increased susceptibility of adults to gram negative, gram positive, and polymicrobial infections 75 76 93 94 The role of TRIF or MyD88 in the neonatal innate immune function/re sponse to polymicrobial sepsis is unknown. To address this, peritoneal washes from wild type, TRIF / or MyD88 / neonates (5 7 days old) that were given an LD 4 0 dose of cecal slurry were harvested at 24 hours post sepsis challenge and phenotyped via flow cytometry for t he presence of neutrophils (Ly6g + CD11b + ) or macrophages (Ly6g F4/80 + CD11b + ). Surprisingly, there were no significant differences in the recruitment of neutrophils or macrophages to the peritoneum following polymicrobial challenge (Figure 3 1). This data suggests that despite t he loss of either MyD88 or TRIF neonates are still able to mount a sufficient inflammatory response to recruit innate immune effector cells to the site of infection. Deletion of TRIF or MyD88 does not affect neutroph il reactive oxygen species production following phorbol ester stimulation in neonates Though there were no differences in the recruitment of innate immune effector cells, specifically macrophages and neutrophils, there may be specific defects in innate imm une function. To investigate this, peritoneal washes from wild type, TRIF / or MyD88 / neonates 24 hours post polymicrobial sepsis challenge were stained for neutrophil cell surface markers and evaluated for reactive oxygen species production.

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66 Interesti ngly, despite the loss of TRIF or MyD88, PMA stimulated neonatal neutrophils were still a ble to produce reactive oxygen species (Figure 3 2). Survival of neonates to polymicrobial sepsis is independent of TLR signaling Despite these similarities in recruitment and innate immune function between TRIF / MyD88 / and wild type neonates in response to polymicrobial infection, the loss of TRIF and/or MyD88 in the neonate may still impact survival to polymicrobial sepsis. To address this TRIF / MyD88 / and wild type neonates were challenged with either an LD 4 0 or LD 70 challenge of CS and followed for survival for 7 days. Surprisingly, there were no differences noted in the survival between groups with either the LD 4 0 or the more lethal LD 70 polymicro bial challenge (Figure 3 3A and B). To address if the complete loss of TLR signaling impacted the survival of neonates to polymicrobial sepsis, TRIF / /MyD88 / or wild type neonates were challenged with an LD 4 0 dose of CS and followed for survival. Again, there were no significant differences in survival between TRIF / /MyD88 / or wild type neonates in response to cecal slurry (Figure 3 3C). This data further supports the role of other pathogen recognition receptors in the absence of TLR signaling to prov ide protection during a polymicrobial insult. Discussion Since the discovery of toll like receptors in the mid 1990s, a number of pathogen recognition receptors have been reported 52 106 All of these receptors have a central theme in common which is that they recognize conserved sequences, carbohydrate, protein, and/or nucleic acid of pathogens and signaling down stream to promote inflammation and host recognition of invasion. Of these, the TLRs have received the most focus as they were the first to be discovered and are arguably the best characterized 52 However, the majority of the investigations have been focused on adult

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67 preclinical studies and the role of these pathogen recognition receptors in neonatal sepsis, let alone polymicrobial abdominal sepsis, is unknown. We demonstrate tha t despite the loss of key TLR signaling proteins, MyD88 or TRIF, neonates are able to recruit innate immune effector cells and survive two different lethalities of polymicrobial sepsis. This is important as the loss of MyD88 in adults has been associated w ith increased mortality in adult preclinical murine models of sepsis 75 In addition, we demonstrate that the loss of both TRIF and MyD88 proteins does not impact survival to polymicrobial sepsis. This is surprising as TLR signaling is thought to be critical for immunity to infection, and would certainly be thought to have a role in the survival of neonates to infection and sepsis 51 52 61 However, given these results, the question of how these murine neonates are responding and subsequently surviving polymicrobial sepsis is remains unknown. It is likely that more than just the TLR system is affected and activated following polymicrobial challenge, due to the magnitude of the challenge and the presence of a variety of bacterial products in the cecal slurry model. Certainly, nucleotide oligomerization domain (NOD) like receptors are also involved in the response, as these receptors recognize a variety of bacterial products such as muramyl dipeptide and bacterial CpG (nonmethylated CpG repeats), and are responsible for the inflammasome/caspase 1 mediated IL 1 IL 18, and IL 33 production 106 In addition, though less is known about the ro le of RIG I receptors, they have been shown to be stimulated by gram negative bacterial RNA to produce a type 1 interferon response, and also have been shown to have a role in the phagocytic function of macrophages during gram negative infection 107 108 Therefore, though TLRs are thought to be critical for the

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68 response to many pathogens, there is redundancy with in the pathogen recognition receptors that may allow for the loss of one system, so there is some mechanism to provide protection to the host in the absence of one set of receptors. In addition, this may be reflective of a difference in the role of adaptiv e immunity in the neonate and adult in response to polymicrobial sepsis. We have previously shown that the adaptive immune response does not play a role or provide protection during polymicrobial sepsis 8 However, we have also recently shown that B cells are critical for survival to adult polymicrobial sepsis and provide inflammatory cross talk with the innate immune response during CLP 109 Is this observation a reflection of the fact that TLR signaling is critical for the adaptive immune response, and specifically the B cell response, during adult sepsis, but is dispens able for neonatal polymicrobial sepsis? More studies are needed to clarify this issue. In sum, we present the novel observation that in the absence of TLR signaling, neonates are able to survive polymicrobial sepsis. This was associated with a similar innate immune response to polymicrobial challenge as well as similar production of innate immune reactive oxygen species production to a phorbol ester. Future studies are needed to clarify the role of TLR signaling in the cecal slurry, polymicrobial sepsi s model in the adult, to recapitulate previous findings by the Hotchkiss lab in the CLP model 75 and show that these observations are not simply an artifact of the preclinical model in neonates 75 In addition, the role of the other pathogen recognition receptors systems in the response t o neonatal polymicrobial sepsis will need to be explored to identify whether or not there is a neonatal reliance on one pathogen recognition receptor system vs another in response to polymicrobial sepsis. Regardless, these data

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69 add new insight into the rol e of TLR signaling in neonates during abdominal polymicrobial sepsis and reveal a difference in the adult vs neonatal response to infection and sepsis.

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70 F igure 3 1. Recruitment of peritoneal macrophages and neutrophils following polymicrobial sepsis i s similar between TRIF / MyD88 / and WT neonates. Peritoneal washes from TRIF / MyD88 / and WT neonates were harvested 24 hours following polymicrobial challenge and phenotyped via flow cytometry for the presence of macrophages and neutrophils (n=4 5 animals per group).

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71 F igure 3 2 Similar reactive oxygen species production following phorbol ester stimulation in neutrophils from MyD88 / TRIF / and WT neonates is similar. Splenocytes from neonatal mice were stained for neutrophil c ell surface markers and labeled with DHR 12 3. Cell suspensions were then stimulated with PMA and read at baseline and 10 minute intervals to 30 minutes (n=3 4 animals per group; data are representative of 2 experiments).

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72 F igure 3 3 TLR signaling is dispensable for survival to neonatal polymicrobial sepsis. Five to seven day old TRIF / MyD88 / and WT animals were subjected to either an LD 4 0 (A) or LD 70 (B) dose of polymicrobial sepsis and followed for survival (Data represent the summary of 3 exp eriments). C. WT or MyD88 / /TRIF / neonates were subjected to an LD 4 0 challenge of polymicrobial sepsis and followed for survival for 7 days.

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73 CHAPTER 4 CRITICAL ROLE FOR CXCL10 (IP 10)/CXCR3 SIGNALING IN THE MURINE NEONATAL RESPONSE TO SEPSIS Background Death associated with neonatal sepsis remains largely unchanged over the last two decades, and is responsible for more than 1 million deaths per year worldwide 3 9 Mortality rates are particularly elevated in very low birth weight infants (<1500 g at birth) and have been reported as high as 50% 6 19 In human neonates who survive severe sepsis, developmental morbidity also has long term social and economic consequences 16 92 110 Although these stagnant outcomes closely mirror results in the adult population, ther e are distinct immunological differences between the adult and neonatal response to sepsis. For example, adult mice rely on both adaptive and innate immunity for survival 111 while neonatal mice rely more heavily on their innate immune responses 8 111 Neutrophils fro m neonates exhibit impaired production of neutrophil extracellular traps (NETS), and have reduced phagocytic function and reactive oxygen species productio n (ROS) when compared to adults 8 50 These findings highlight the increased importance of the innate immune response during sepsis in the neonate, and the need to delineate mediators of neonatal innate immune modula tion. The ultimate goal is to not only understand how neonates respond to severe infection, but also to identify potential therapeutic targets. We have previously shown that pretreatment of neonatal mice with a TLR4 or TLR7/8 agonist could dramatically im prove outcome to subsequent polymicrobial sepsis Published in Infection and Immunity 2011 Jul;79 (7):2746 54

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74 through the improvement of innate immune function, whereas adaptive immunotherapy with a glucocorticoid induced TNF receptor (GITR) agonistic antibody that improves outcome in adult polymicrobial sepsis fa iled to improve outcome in neonates 8 These results highlight the need for different immunologic approaches for the treatment of adult versus neonatal sepsis. It is well known that TLR stimulation induces the production of multiple pro inflammatory cytokines and chemokines, as well as type I ( and ) interferons 8 51 61 These mediators serve to recruit and modulate early immunological responses to protect the host against infection. Interferon inducible protein 10 (IP 10) or CXCL10 is a CXC chemokine that has been shown to be produced in response to both type I ( and ) and the pro inflammatory type I I interferons ( ) 51 66 Many studies have linked CXCL10 to productive TH 1 responses, but CXCL10 has also been more recently associated with neutrophil infiltration and function 64 85 86 Increased CXCL10 production has been described in multiple adult infection models, and increased plasma concentratio ns have been associated with bacterial infection in neonates 91 Additionally, blockade of CXCL10 has been demonstrated to worsen survival in an adult murine model of experime ntal sepsis 82 The present study was undertaken to examine the physiologic and pharmacologic role of CXCL10 in innate immune modulation during neonatal sepsis. We d emonstrate that the influx of granulocytes and macrophages into the peritoneal cavity during polymicrobial sepsis is temporally associated with increases in peritoneal CXCL10 concentrations. Blockade of CXCL10 significantly reduces the influx and phagocyt ic activity of peritoneal granulocytes during polymicrobial sepsis, and worsens survival. In

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75 addition to providing evidence that CXCL10 is critical for the effective recruitment of innate immune effector cells during neonatal sepsis, we demonstrate that e xogenous CXCL10 can directly stimulate granulocyte and macrophage phagocytic function in vitro. Finally, to investigate the potential therapeutic role of CXCL10 in neonatal sepsis, we show that blockade of CXCL10 prevents the protective effect of pretreat ment with a TLR4 agonist to polymicrobial sepsis, and that pretreatment with CXCL10 prior to polymicrobial sepsis is able to modestly augment survival. These data suggest that production of CXCL10 during polymicrobial sepsis is important for the efficient recruitment and function of neonatal innate immunity, and may also be a critical component of therapies designed to improve outcomes in neonatal sepsis. Materials and Methods Mice All experiments were approved by the Institutional Animal Care and Use Com mittee at the University of Florida. Six to eight week old male and female wild type or CXCR3 / C5 7BL/6 mice were purchased from T he Jackson Laboratories (Bar Harbor, ME), and used for adult breeders. Adult mice were mated in a harem schema with one mal e to two females. Neonatal mice were studied between four and seven days of age. Cecal slurry (CS) model Polymicrobial sepsis was induced as previously described 4 Br iefly, cecal contents of adult C57BL/6 mice were suspended in 5% dextrose solution at a final concentration of 80 mg/mL and injected intraperitoneally (ip) into five to seven day old neonates. Either an LD 40 (1 mg fecal contents/g body weight) or an LD 70 (1.3 mg fecal contents/g body weight) of cecal slurry, as determined from previous experiments, was used 4

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76 Peritoneal and serum CXCL10 concentrations Blood and perito neal washes were harvested from neonates at the time points described following induction of polymicrobial sepsis. As reports have shown that heparin can bind to CXCL10 and possibly reduce its detection in blood, serum was used for these assays 99 100 To collect peritoneal washes, the peritoneal cavity was instilled with 500 L of cold phosphate buffered saline (PBS) (Cellgro, Manassas, VA). The peritoneal cavity was then opened over a sterile tissue culture dish and the peritoneal wash collected. Peritoneal washes were then centrifuged to remove debris and the supernatants we re stored at 80 0 C. Serum and peritoneal washes were then measured for CXCL10 concentrations via ELISA (R&D Systems, Minneapolis, MN). Flow cytometry Single cell suspensions we re characterized using anti Ly6g APC, anti Ly6c PerCPCy5.5, anti Gr 1 APC, anti CD11b PECy7, anti CXCR3 PE, and/or anti F4/80 APCeFlour 780 (Ebioscience, San Diego, CA). All antibodies were purchased from Becton Dickinson (BD) (Franklin Lakes, NJ) unless otherwise indicated. Samples were acquired and analyzed on an LSR II flow cytom 4 live cells (SYTOX Blue ; Invitrogen, Carlsbad, CA) were collected for analysis. Sorting was conducted on a FACSAria TM (BD) and cells were sorted to > 95% purity. Cytospins were prepared from sorted cell populations and Wright Giemsa stain ed (Sigma Aldrich, St. Louis, MO ). Isolation of blood, bone marrow, and splenocytes for phenotypic analysis Blood was obtained via intracardiac puncture of isoflurane anesthetized neonatal mice into a heparinized syringe. Spleens were harvested from neonates and then dissociated through a 70 m sterile filter. To harvest bone marrow, both femurs and

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77 tibias from individual animals were collected and flushed with PBS. Blood, splenocyte, and bone marrow suspensions were then subjected to erythrocyte lysis using ammonium chloride lysis solution. C ell suspensions were then stained for granulocyte (Gr 1 + or Ly6g + CD11b + ), monocyte (Ly6c + CD11b + ) or macrophage (F4/80 + CD11b + ) cell surface markers and analyzed via flow cytometry Harvest of peritoneal washes Peritoneal cell isolation was carried out in a similar manner as described previously 8 Due to the fact that peritoneal lavage from individual animals contained relatively few cells, peritoneal washes from three t o five animals were pooled for analysis and considered to be one sample. For each time point or condition analyzed, a total of three to four peritoneal samples were collected per experiment. To determine absolute numbers of innate immune effector cells, th e percentage of macrophages or granulocytes within the total sample population was determined via flow cytometry, multiplied by the total sample cell number, and then divided by the total number of mice in the pooled sample. Absolute numbers represent tota l macrophages or granulocytes per mouse. In vitro CXCL10 chemotaxis assay Splenocytes were harvested from neonates, overlayed on top of Histopaque 1119 (Sigma Aldrich St Louis, MO), and centrifuged. Splenocytes were then washed in PBS, resuspended in R PMI 1640 and 10% fetal bovine serum (FBS), and placed in the top well o f an 8 micron BD Falcon FluoroBlo k TM Multiwell insert (BD). Inserts with splenocyte suspensions were then placed on top of RPMI 1640 and 10% FBS media containing 0, 100, or 200 ng/mL of recombinant CXCL10 (R&D systems) to establish a gradient and placed at 37 o C. After 2 and 6 hours of culture, bottom wells and the bottom

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78 of the insert were washed with ice cold PBS twice and cells scraped. Cell suspensions were counted and assayed via flo w cytometry for the presence of Gr 1 + CD11b + cells (granulocytes) and F4/80 + CD11b + cells (macrophages). Blockade of CXCL10 Neonates were injected ip with 0.5 mg of either rabbit polyclonal anti CXCL10 IgG (gift from SL Kunkel, Ann Arbor, MI) or control r abbit IgG (Fisher Scientific, Pittsburgh, PA). Specificity and activity of polyclonal rabbit anti CXCL10 has been verified in previous studies 112 Due to th e fact that ages of mice could vary by as much as two days, individual litters were split into two cohorts, those that received anti CXCL10 versus those that received control rabbit IgG to eliminate or reduce the effect of age and/or size variability. IgG was purified from either polyclonal anti CXCL10 antisera or control rabbit sera using protein A columns (Pierce, Rockford, IL), and then dialyzed against PBS (Cellgro) before use. Peritoneal bacterial counts Animals were administered eith er control IgG or anti CXCL10 two hours before cecal slurry challenge. Twelve hours following septic challenge, neonates were euthanized and injected with 500 L of sterile PBS. Peritoneal washes were then collected, serially diluted, and cultured on blood agar plates under aerobic conditions at 37 o C. Colonies were counted 18 24 hours following culture. Isolation of splenic macrophages and granulocytes for func tional analyses Spleens were dissociated through a 70 m sterile filter into RPMI 1640 medium (Cellgro) with 10% heat inactivated, low endotoxin FBS, and 0.5% penicillin/ streptomycin (Cellgro). Suspensions were then overlayed on a Histopaque 1119

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79 (Sigma Aldrich) density gradient and centrifuged. Mixed cell suspensions were then washed with medium alone, and individual cell populations were assayed for phagocytosis or reactive oxygen species (ROS) produ ction by flow cytometry. In vitro stimulation with recombinant CXCL10 Isolated splenocytes were stimulated with or without recombinant murine CXCL10 at 100 ng/mL for four hours (R&D Systems). After incubation with CXCL10, cells were washed with PBS and su bsequently assayed for phagocytic function or ROS production and phenotypically characterized via flow cytometry. Functional analysis of splenic macrophages and granulocytes or peritoneal macrophages or granulocytes For phagocytosis assays, 1 X 10 5 spleno cytes isolated via density gradient or peritoneal cell suspensions were incubated with 1 X 10 8 fluorescent polystyrene microspheres (Fluospheres; Invitrogen, Carlsbad, CA) for 30 min at 37 C. Cells were then stained and analyzed by flow cytometry, as descr ibed above. After cells were gated on either granulocyte (Gr 1 + CD11b + ) or macrophage (F4/80 + CD11b + ) populations, FITC + cells were considered to be phagocytic. To assay for ROS production, 2 X 10 6 splenocytes isolated via density gradient were first staine d for cell surface markers and la beled with dihydrorhodamine 12 3 (Invitrogen). Cells were then stimulated with 1 M of phorbol 12 myristate 13 acetate (PMA) (Sigma Aldrich) at 37 C and aliquots were evaluated by flow cytometry at various points over a 30 minute period using the LSR II flow cytometer (BD). A minimum of 1 X 10 4 live, non debris cells were collected for analysis.

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80 Pretreatment with a TLR4 agonist (bacterial LPS) Neonatal C57BL/6 mice four to six days old received LPS (1 g/g BW ip; ultrapure via ion exchange chromatography Escherichia coli O26:B6 (Sigma Aldrich)) in physiologic saline 24 hours before challenge with cecal slurry. Control animals received saline only ip 24 hours before cecal slurry challenge. Total volume of injected solution was less than 50 L. In vivo administration of recombinant CXCL10 R ecombinant murine CXCL10 (R&D) (20ng) in physiological saline or physiological saline alone was administered ip to neonatal C57BL/6 mice 5 7 days old. Total volume administered was 50 L per mouse. Statist ical analyses Continuous variables were tested for normality and equality of variances. test whereas differences among groups were evaluated by either one pos t t test. Significance was determined at the 95% confidence level using a two tailed test. Results CXCL10 concentrations in the blood and peritoneum increase during polymicrobial sepsis Peritoneal washes and sera were harvested f rom neonates at multiple time points during polymicrobial sepsis, and CXCL10 concentrations were determined. As demonstrated in Figure 4 1A and B, CXCL10 serum (A) and peritoneal co ncentrations (B) increase at 6 and 12 hours after induction of sepsis, resp ectively, but subsequently decrease by 24 hours.

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81 Peritoneal granulocyte and macrophage kinetics follow increases in CXCL10 peritoneal concentrations The recruitment of innate immune effector cells is important for the containment and control of bacteria l infection, as well as abscess formation and localization 113 The characteristics of the adult response to sepsis have been well described, but have not been fully delineated in the neonate 64 114 115 Blood, spleen, bone marrow, and peritoneal washes were collected at multip le time points from neonates that were exposed to an LD 40 polymicrobial sepsis. Cells were then stained for granulocyte (Gr 1 + CD11b + ) or macrophage (F4/80 + CD11b + ) cell surface markers. As expected, granulocyte percentages increase in the blood two hours fo llowing sepsis, whereas macrophage percentages slowly increase over the next 24 hours (Figure 4 2A and B). Both granulocyte and macrophage numbers increase significantly in the peritoneum within 12 and 24 hours of polymicrobial sepsis, respectively (Figure 4 2C and D). This correlates with a decrease in absolute numbers of granulocyte and macrophages in the bone marrow and spleen (Figure 4 2C and D). As the murine marker Gr 1 is comprised of two epitopes, one more specif ic for mature granulocytes (Ly6g ) a nd the other more representati ve of monocyte populations (Ly6c ) including myeloid precursors, it was unclear as to what the relative contribution of monocytes and/or mature granulocytes is to the sepsis elicited peritoneal exudate. To address this concern, the kine tics of mature granulocyte (Ly6g + CD11b + ) and monocyte (Ly6g Ly6c + CD11b + ) populations in blood, bone marrow, spleen, and peritoneum during neonatal sepsis were further determined. Though some heterogeneity is evident between the cellular morphology of Gr 1 + CD11b + versus Ly6g + CD11b + versus Ly6g Ly6C + CD11b + cells ( Figure 4 3 ), mature granulocytes comprise the majority of both Gr

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82 1 + CD11b + versus Ly6g + CD11b + sorted populations. In addition, the kinetics of Ly6g + CD11b + (granulocytes), but not Ly6g Ly6c + C D11b + (immature/mature monocytes), following cecal slurry are nearly identical to the Gr 1 + CD11b + population ( Figure 4 4 ). Therefore, although th e Gr 1 marker can identify both granulocyte and monocyte populations, these data indicate that the majority of infiltrating cells to the peritoneum are indeed mature granulocytes and Gr 1 + CD11b + cells will be referred to as granulocytes for the remainder of this report. CXCL10 induces the recruitment of both granulocytes and macrophages in vitro Though the data ab ove suggest that granulocyte and macrophage recruitment to the peritoneum following sepsis is associated with increases in peritoneal CXCL10 concentration, there is little evidence to suggest that CXCL10 may directly recruit these cells following septic in sult. To address this concern, splenocyte suspensions were cult ured in the top well of a FluoroBlo k TM 8 micron cell culture insert and placed on top of increasing concentrations of CXC L10. As demonstrated in Figure 4 5 A an d B, both granulocyte and macroph age numbers were significantly increased in the bottom wells of cultures containing 100 ng/mL and 200 ng/mL of recombinant CXCL10 compared to those wells without CXCL10 after 6 hours. This suggests that CXCL10 can recruit both granulocytes and macrophages directly. CXCL10 neutralization worsens survival to cecal slurry peritonitis and decreases recruitment of peritoneal macrophages and granulocytes The data shown above suggest that CXCL10 may be important for the recruitment of granulocytes and macro phages to the peritoneum during polymicrobial sepsis. To further explore the requirement for CXCL10 production during neonatal sepsis, we investigated whether blockade of CXCL10 would affect the recruitment of peritoneal

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83 macrophages or granulocytes. Perit oneal exudates were examined from anti CXCL10 antibody treated animals 12 hours following induction of polymicrobial sepsis. CXCL10 blockade significantly decreases both peritoneal macrophage and g ranulocyte recruitme nt (Figure 4 6A and B). To examine th e overall importance of CXCL10 to sepsis response, survival of neonates to septic challenge was examined. Neonates were administered 0.5 mg of control IgG or anti CXCL10 IgG ip two hours before polymicrob ial sepsis. As shown in Figure 4 6 C, blockade of CXC L10 significantly decreases survival to sepsis compared to controls. This was also associated with increased peritoneal bacterial counts at 12 hours following sepsis in those animals administered anti CXCL10 compared to those administered control IgG. Thes e data suggest that CXCL10 production is critical for the appropriate recruitment of innate immune effector cells and subsequent survival to polymicrobial sepsis. CXCR3 expression increases on peritoneal granulocytes and macrophages during septic challenge and deletion of CXCR3 decreases peritoneal granulocyte and macrophage numbers and worsens survival to neonatal sepsis Recruitment of innate immune effector cells to the site of infection is thought to require a chemokine gradient 116 To determine if changes in the expression of the receptor for CXCL10, CXCR3, mirror the changes in CXCL10 concentrations, peritoneal washes from neonates at zero, six, and 24 hours during polymicrobial sepsis were collec ted. As demonstrated in Figure 4 7A and B, peritoneal granulocyte and macrophage CXCR3 expression increases by 24 hours post induction of sepsis. Peritoneal granulocytes (Figu re 4 7A) and macrophages (Figur e 4 7 B) from healthy mice have little to no CXCR3 expression, and CXCR3 expression increases with time

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84 after induction of sepsis, although delayed in time relative to peritoneal CXCL10 concentrations. Since blockade of CXCL10 was demonstrated to worsen sur vival to cecal slurry peritonitis, we next evaluated the requirement of CXCR3 expression for the recruitment of peritoneal granulocytes and macrophages, as well as the survival of neonates to sepsis. CXCR3 / or wild type mice were subjected to an LD 40 sep tic challenge and either sacrificed at 12 hours post septic challenge and analyzed for the presence and number of peritoneal granulocytes and macrophages or followed for survival for 7 days. Though there were no differences in the relative percentages of p eritoneal granulocytes or macrophages, there were significantly more peritoneal macrophages and neutrophils in wild type neonates compared to CXCR3 / neonates f ollowing sepsis (Figure 4 7C and D). In addition, the absence of CXCR3 significantly worse ned s urvival to sepsi s (Figure 4 7 E). These data further support a critical role for CXCL10/CXCR3 signaling in the survival of neonates to septic challenge. In vitro stimulation with CXCL10 increases phagocytic ability of neonatal splenic macrophages and gran ulocytes CXCL10 has been demonstrated to augment the phagocytic function of adult murine and human neutrophils 64 Therefore in addition to recruitment, CXCL10 may play a critical role in the modulation of neonatal innate immune phagocytic function and/or reactive oxygen species production. To address this question, neonatal splenocytes from healthy neonatal mice were isolated and incubated with or without recombinant CXCL10 in vitro for four hours. Althou gh CXCL10 pretreatment had no effect on ROS production ( data not shown ), CXCL10 pretreatment significantly augmented the phagocytic function of both neonatal granul ocytes and macrophages (Figure 4 8A and B).

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85 Blockade of CXCL10 worsens phagocytic ability To further support this proposed role in vivo neonates were administered 0.5 mg of anti CXCL10 or control rabbit IgG ip two hours prior to sepsis. Peritoneal washes were then collected from these animals 14 hours later and assayed for phagocytic uptake of FITC labe led beads. As shown in Figure 4 9 blockade of CXCL10 significantly worsens phagocytic function by both granulocytes (Figu re 4 9B) and macrophages (Figure 4 9 C). These data further support the hypothesis that CXCL10 not only functions to promote recruitment of inflammatory cells (granulocytes and macrophages) to the peritoneum during sepsis, but also serves to modulate phagocytic function for efficient clearance of infection. Blockade of CXC10 inhibits adjuvant induced protection during sepsis an d administration of CXCL10 improves outcome We have previously demonstrated that 24 hour pretreatment of neonates with TLR agonists confers protection to sepsis 8 Th is effect was associated with increased neutrophil recruitment to the peritoneum. Based on these observations and the data presented above, we examined whether CXCL10 was required for the observed adjuvant induced protective effect. First, we characterize d the serum and intraperitoneal CXCL10 concentrations in response to LPS pretreatment (1 g/g BW) followed by an LD 70 induction of sepsis. As demonstrated in Figure 4 10A and B, CXCL10 concentrations peaked within four hours after LPS administration, decr eased to basal levels within 24 hours, and then subsequently increased again in response to sepsis. To determine whether these increases in CXCL10 concentrations were associated with increases in peritoneal granulocytes and macrophages following adjuvant

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86 administration, neonates were injected with either 0.5 mg of control rabbit IgG or rabbit anti CXCL10 IgG two hours prior to the LPS pretreatment. Peritoneal washes were then harvested 24 hours following LPS admi nistration. B lockade of CXCL10 before admini stration of LPS is associated with decreased recruitment of both macrophages and granulocytes to the peritoneum (Figure 4 10C and D) To address whether this decrease in macrophage and granulocyte recruitment reduced or inhibited the adjuvant induced prote ctive effect to sepsis, 24 hours following LPS or saline pretreatment, sepsis was induced and survival evaluated. Surprisingly, blockade of CXCL10 two hours before LPS pretreatment completely prevents the protective effe ct of LPS pretreatment (Figure 4 10 E). Further the effect of CXCL10 blockade appeared to be specific to LPS pretreatment and not sepsis induced CXCL10, as blockade of CXCL10 either two or 22 hours after LPS pretreatment did not block the protective adjuvant effect of LPS ( data not shown ). T hese data suggest that CXCL10 production is critical for the recruitment of innate immune effector cells and subsequent protective adjuvant effect of a TLR4 agonist pretreatment. If CXCL10 is important for the recruitment and phagocytic function of granu locytes and macrophages to the peritoneum during neonatal sepsis, and LPS pretreatment improves outcome in a CXCL10 dependent manner, then pretreatment with CXCL10 may serve to improve survival to sepsis. To address this, neonates were either administered recombinant murine CXCL10 (20 ng) or saline 12 hours prior to induction of neonatal sepsis, and then followed for survi val. As demonstrated in Figure 4 10 F, administration of CXCL10 modestly improves survival of neonates in response to CS challenge from 44 % to 70% (p=0.066). Though not statistically significant, these data

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87 suggest that CXCL10 may be one mediator that contributes to the improved outcome to sepsis seen with a TLR adjuvant pretreatment schema. Discussion Sepsis in both neonates and adults is a complex disorder that despite advances in novel therapeutics and critical care management, is still incompletely understood. Due to the complexity of the host response to sepsis, identifying mediators that modulate innate immunity would be invaluable in the neonatal setting as the recruitment of functional innate immune effector cells to the site of infection appears to be critical for survival to polymicrobial sepsis 8 To this end, we have identified a role for CXCL10/CXCR3 signaling during neonatal sepsis. We have demonstrated that the production of CXCL10 during neonatal sepsis not only participates in the recruitment of granulocytes and macrophages into the perit oneum (Figure 4 4 A and B), but also modulates their phagocytic function (Figure 4 8 ). Additionally, we showed that ex vivo treatment of granulocytes and macrophages with CXCL10 significantly improv ed phagocytic function (Figure 4 8 ), a finding consistent with what we saw with adult murine and human neutrophils 64 Though pre vious work from our laboratory has demonstrated that type I interferon dependent production of CXCL10 may be important for the function of adult neutrophils and survival to adult sepsis, type I interferons do not appear to play a significant role in the survival of neonates during polymicrobial sepsis 8 64 Wynn et al. demonstrated that type I interferon receptor knockout mice have similar survival to neonatal polymicrobial sepsis compared to wild type mice 8 As CXCL10 can be induced by either type I or II interferons, these data suggest that though adults may rely more heavily on type I

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88 interferon dependent CXCL10 production for survival to sepsis, neonates do not require type I interferons for survival and are clearly different in this regard. Few studies have clearly linked CXCL10 production to either outcome from sepsis in neonates, or their neutrophil and/or macrophage function. Vasquez and Soong have demonstrated an improvement in macrophage phagocytic function and a decrease in Leishmania parasite burden mediated by CXCL10 stimulation 89 Additionally, elevat ed plasma concentrations of CXCL10 have been associated with severe infection in human neonates 90 91 However, no c lear link between neonatal innate immune function and the role of CXCL10 has ever been described. This report provides a clear and novel mechanistic role for CXCL10 in the modulation of both granulocytes and macrophages during neonatal sepsis. The immuno modulatory effects of CXCL10 on neonatal innate immunity suggest that therapeutics targeted to increase the production of CXCL10 may be of utility as a preventative strategy in neonates to septic insult. To support this, we have also shown that CXCL10 is a critical component of the observed benefit to survival seen with TLR4 adjuvant induced protection to neonatal sepsis 8 Blockade with a CXCL10 antibody prevented LPS p retreatment from protecting neonatal mice from sepsis induced lethality. However, blockade of CXCL10 as little as two hours after LPS administration or two hours prior to sepsis did not block the protective adjuvant effect of LPS ( data not shown ). Due to the fact that CXCL10 concentrations rise within two hours post LPS administration (Figure 4 10 A and B), the abolition of the protective effect of TLR agonist administration appears to be due to CXCL10 blockade in response to the LPS ( data not shown ). Howev er, although CXCL10 appears to be critical for this protective response,

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89 it is clear that it is more than likely working in concert, and not singularly, with other cytokines/chemokines to improve survival to sepsis. This is evidenced by the rather mode st i mprovements in survival (25 %) noted in those animals pretreated with CXCL10 and then subjected to polymicrobial sepsis (Figure 4 10 E). Production of CXCL10 in the neonate during infection and sepsis appears to serve a vital role in the recruitment and pha gocytic function of both granulocytes and macrophages. Further, as CXCL10 has been shown to be a critical part of LPS adjuvant induced protection and CXCL10 pretreatment of neonates has been demonstrated to modestly improve survival to sepsis, therapeutics designed to promote or augment CXCL10 production may be efficacious in preventing mortality during neonatal sepsis.

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90 Figure 4 1. Serum CXCL10 concentrations increase in neonatal sepsis. Blood and peritoneal washes were collected from neonates (n=4 ) at multiple time points post sepsis (LD 40 ). Sera (A) and peritoneal washes (B) were then assayed for CXCL10 concentration (Values represent mean SD).

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91 Figure 4 2. Granulocyte and macrophage kinetics in neonatal sepsis. Spleen, blood, bone m arrow, and peritoneal exudates were collected from neonates at different time points after sepsis (LD 40 ). Tissue cell suspensions were then stained for granulocytes (A and C) (Gr 1 + CD11b + ) and macrophages (B and D) (F4/80 + CD11b + ) and analyzed via flow c ytometry. Panels A and B represent the percentage of total live cells in the blood, while panels C and D show total cell number in the spleen, bone marrow, and peritoneum. (n=4 per group per time point; Values represent the mean y ANOVA p<0.01)

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92 Figure 4 3. Phenotypic characterization of granulocytes and macrophages. Splenocytes were harvested from neonates and then stained for Gr 1, Ly6g, Ly6c or CD11b. Splenocyte suspension were then sorted via flow cytometry to ~95 % purity. Cytospins and Wright Giemsa staining were done on sorted Gr 1 + CD11b + Ly6g + CD11b+, or Ly6c + CD11b + populations. Photographs were taken at 100 X under oil immersion.

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93 Figure 4 4. Kin etics of Ly6g + /CD11b + and Ly6c + /CD11b + populations in neonatal sepsis. Spleen, bone marrow, and peritoneal washes were taken at 0, 2, 6, 12, and 24 hours following septi c challenge and stained for Ly6g, Ly6c and CD11b. Granulocytes (Ly6g + /CD11 b + )(A), immature monocytes (Ly6g Ly6c hi CD11b + )(B), and mature monocytes (Ly6g Ly6c mid CD11b + )(C), are reported as total live cells per mouse. Values represent the mean SD of two experiments (n=6).

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94 Figure 4 5. Increased neonatal granulocytes and m acrophages chemotaxis in response to a CXCL10 chemokine gradient. Splenocytes were harvested and cultured in the to p well of an 8 micron FluoroBlo k TM insert and placed on top of increasing concentrations of CXCL10. After 6 hours, the bottom of the inse rt and bottom well were washed and analyzed for the presence of granulocytes and macrophages via flow cytometry. (n=6 per p<0.001).

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95 Figure 4 6. CXCL10 blockade worsens survival and cell recruitment. Two hours prior to sepsis (LD 40 ), neonates were administered either anti CXCL10 IgG (squares and solid line) or control IgG (diamonds and hashed line) (0.5 mg). Peritoneal washes were harvested from neonates administered anti CXCL10 or control IgG 12 hours post sepsis. Cells were subsequently stained for granulocytes (A) (Gr 1 + CD11b + ) and macrophages (B) (F4/80 + CD11b + ), and analyzed by flow cytometry. Figure represents the summary of two experiments. Values represent the mean SD (n=6 each, test p<0.05; error bars indicate SD). (C) Another cohort of animals were then monitored for survival for seven days. Figure p< 0.003) (D) Peritoneal bact erial counts were determined following sepsis in neonates that were administered either control IgG or anti CXCL10 test p =0.026)

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96 Figure 4 7. Deletion of CXCR3 significantly worsens surv ival to neonatal sepsis. Peritoneal granulocytes (Gr 1 + CD11b + ) and macrophages (F4/80 + CD11b + ) were collected from neonates at zero, six, and 24 hours post sepsis (LD 40 ) and stained for CXCR3 expression. Isotype controls are represented as dashed lines and CXCR3 expression is in solid lines. Mean fluorescence intensity (MFI) of CXCR3 expression in granulocytes (A) and macrophages (B) were measured by flow cytometry (Mean SD). Figur e represents the summary of two experiments (n=6)). Peritoneal washes from wild type (WT) or CXCR3 / neonates 12 hours following an LD 40 CS challenge were harvested and analyzed via flow cytometry for the presence of granulocytes (C) or macrophages (D) (n=4 per group; test p<0.05) (E) WT or CXCR3 / neonates were subjected an LD 40 septic challenge and then followed for survival for 7 days. Figure represents the summary of two exact test p<0.002).

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97 Figure 4 8. Recombinant CXCL10 improves phagocytosis in granulocytes and macrophages. Neonatal splenocytes isolated via density gradient were incubated with or without murine CXCL10 (100 ng/mL) for four hours. Cells were then wa shed and incubated with FITC labeled beads and stained for granulocytes (A) (Gr 1 + CD11b + ) and macrophage (B) (F4/80 + CD11b + ) cell surface markers. Cells were first gated on granulocyte or macrophage populations and then FITC+ cells were considered phago cytic. Figure represents the summary of three experiments and histograms are representative samples from one of the test p< 0.05)

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98 Figure 4 9. Inhibition of CXCL10 reduces phagocytic function of granulocytes and macrophages in vivo. Two hours prior to sepsis (LD 40 ), neonates were administered either anti CXCL10 or control IgG (0.5 mg) ip. Twelve hours following sepsis (LD 40 ), peritoneal washes were harvested and incubated with FITC labeled latex beads and subsequently stained for granulocytes (Gr 1 + CD11b + ) and macrophage (F4/80 + CD11b + ) cell surface markers. Cells were first gated on granulocyte or macrophage populations and then FITC+ cells were considered phagocytic. Histograms (A) of r epresentative samples and the summary of two experiments for both granulocytes (B) and macrophages (C) are presented (n=6 per group; Values represent the test p< 0.05)

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99 Figure 4 10. Inhibition of CXCL10 abolishes protec tive adjuvant effect of a TLR4 agonist in response to sepsis and CXCL10 administration modestly improves survival to sepsis. Sera (A) or peritoneal washes (B) were collected from neonates at multiple time points post sepsis and assayed via ELISA for co ncentration of CXCL10. Dashed line represents the concentrations of CXCL10 from nave animals following septic challenge. (C, D, and E) Two hours prior to LPS or saline administration, animals were administered either anti CXCL10 (squares) or control (d iamonds and triangles) IgG (0.5 mg). Twenty four hours following LPS administration, peritoneal washes were harvested and cells were stained for granulocytes

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100 (C) (Gr 1 + CD11b + ) and macrophages (D) (F4/80 + CD11b + ) and analyzed by flow cytometry. Figure r epresents the summary of three experiments (* test p<0.05; error bars indicate SD). (E) Twenty four hours following LPS or saline pretreatment, neonates were challenged with an LD 70 sepsis and then followed for survival. Figure represents th e LPS and anti CXCL10 + LPS p<0.001). (F) Twelve hours prior to sepsis, neonates were administered either recombinant murine CXCL10 (20 ng) or physiologic saline. Following sepsis animals were then followed for survival for seven days. Figure represents the summary of three

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101 CHAPTER 5 CONCLUSION AND FUTURE DIRECTIONS Despite our success in critical and supportive ICU care, we are still struggling to improve outcomes in neonatal sepsis. Several therapeutics such as probiotics and/or lactoferrin have been shown recently in large randomized clinical trials to improve outcome by targeting pathogenic bacteria 34 37 39 117 This however, leaves an avenue open for the development of immunothera peutics to target neonatal innate immune responses. Herein, we demonstrate a critical role for both TRIF signaling and CXCL10 in the innate immune response to neonatal sepsis. We show that TRIF signaling is crucial for effective neonatal innate immune func tion and survival during gram negative E coli sepsis. Also, we demonstrate that CXCL10 is an important chemokine for granulocyte/macrophage chemotaxis to sites of inflammation, as well as phagocytic function. In addition, TRIF or CXCL10 signaling deficien t neonates are shown to be impaired in their ability to clear gram negative and polymicrobial sepsis, respectively, and as such succumb to these infections more so than their wild type counterparts. In sum, these data suggest that TRIF dependent inflammati on, including CXCL10 production, contribute to and are critical for effective innate immune function during infection and neonatal sepsis. In addition, we also explored the role of TLR signaling during neonatal polymicrobial sepsis and showed that survival to neonatal polymicrobial sepsis is independent of TLR signaling. We demonstrate that despite the loss of MyD88 or TRIF, murine neonates are able to recruit innate immune effector cells in response to polymicrobial sepsis. In addition, we also show that d eletion of TRIF or MyD88 does not

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102 affect the production of neutrophil ROS in response to a phorbol ester. Finally, we demonstrate that TRIF / MyD88 / or TRIF / MyD88 / murine neonates have similar survival to polymicrobial sepsis compared to wild type controls. As many of the pathogen recognition receptor systems recognize conserved viral and bacterial products and lead to the production of inflammatory mediators, these data suggest a redundancy in neonates that leads to innate immune activation and su bsequent protection during polymicrobial infection. Future studies will be conducted to determine which if any PRR systems are required for survival to polymicrobial sepsis. Also, we show that TRIF and CXCL10 are critical for the observed TLR4 adjuvant e ffect that we have shown in a prior report 8 Together these data suggest that the development of therapeutics designed to maximize and improve upon TRIF dependent infl ammation and CXCL10 signaling may be a useful adjunct to clinical trial proven therapeutics such as, probiotics and lactoferrin. Importantly, utilization of these strategies may be key in the development of vaccines to prevent disseminated infection and su bsequent sepsis in neonates. Combining these efforts into a multimodal therapy will likely be important, as one of the most important lessons learned from the adult preclinical sepsis trials and their subsequent translation to the clinic has been that sing le focused therapeutics are simply not effective 118 119 As such, further investigations to determine which asp ects of TRIF dependent inflammation in the innate immune response, in addition to CXCL10, will be important to hone in on for future therapeutics to treat and/or prevent infection in the neonatal population.

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112 BIOGRAPHICAL SKETCH Alex Gervacio Cuenca was born in Kingston, New York in 1976. In 1997, he graduated from New College of Florida with a Bachelors of Arts degree in biology. His first exposure to science occurred during his undergraduate education when he worked on the development of Geminivirus resistant transgenic tom atoes at a research agricultural station. He subsequently relocated to Tampa to further explore his interest in basic science. After 3 years of working in a tumor immunology lab under the mentorship of Dr. Eduardo Sotomayor, he realized his love for medic ine and immunology and subsequently matriculated into medical school at the University of Florida (UF) in 2002. After completing his MD in 2006, he started his general surgery residency at the UF. Under the mentorship of Dr. Lyle Moldawer, he matriculated into the PhD program at UF in the concentration of Immunology and Microbiology during his clinical hiatus in 2008. Due to his interest in pediatric surgery, he decided to investigate the role of toll like receptor signaling in the innate immune response d uring neonatal sepsis. One of the novel findings presented in his thesis was the dependence of neonates, but not adults, on the TLR adaptor protein, TRIF, in response to gram negative sepsis. Together with his work examining the role of CXCL10 in neonatal sepsis, a downstream consequence of TLR signaling, his findings suggest that enhancement of TRIF associated inflammation in neonates can improve outcome in neonatal sepsis. He will return to his clinical duties in July of 2012 under the guidance of Dr. Kev in Behrns with the hope of entering a pediatric surgery fellowship in 2015. He hopes to pursue a career as a pediatric surgeon and clinical investigator pursing his love for both immunology and the surgical care of children.