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1 EXPRESSION AND EFFECTS OF INTERLEUKIN22 IN HUMAN SJ GRENS SYNDROME AND MOUSE MODELS By TEGAN N. LAVOIE A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE UNIVERSITY OF FLORIDA 2011
2 2011 Tegan N. Lavoie
3 I dedicate this to my mother and grandmother
4 ACKNOWLEDGMENTS I would like to thank Dr. Ammon Peck for giving me the opportunity to be a part of his laboratory and work on such an interesting project. Dr. Peck has demonstrated to me what proper leadership can accomplish and was always willing to help me develop my theories. Also I wish to thank Dr Cuong Nguyen for developing this project for me to work on and expand upon and for training me in laboratory techniques. He provided me with sufficient background information to begin my experiments and always encouraged critical thinking of complicated data. I also thank Dr. Carol Stewart for providing funding for my project and I would like to thank my committee members, Dr. Edward Chan and Dr. Naohiro Terada, for guiding me along the way. I would also like to thank the entire Peck lab for their guidanc e and support, specifically Byung Ha Lee and Ben Saylor. I also wish to thank Wendy Carcamo for helping me troubleshoot when problems arose and for being a great friend. Finally, I wish to thank my family who has never stopped believing in me. In particular, I would like to thank my mom, Lisa Graham, for always pushing me to be the best I can be and my grandma, Joan Elkins, for her helpful and humorous words of wisdom. I would like to thank my brother, Braden Lavoie, for always making me laugh, and my sister, Falen Graham, for just being her wonderful self. I would also like to thank my stepfather, Gregory Graham, for all of his support along the way. I am so grateful to have the unconditional love and support that they have provided me all of my life.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 LIST OF ABBREVIATI ONS ........................................................................................... 10 ABSTRACT ................................................................................................................... 11 CHAPTER 1 IN TRODUCTION .................................................................................................... 13 Sjgrens Syndrome ............................................................................................... 13 Sjgrens Syndrome Mouse Models ....................................................................... 15 The Nonobese Diabetic Mouse ....................................................................... 15 The C57BL/6.NOD Aec1Aec2 Mouse .............................................................. 16 The C57BL/6.NOD Aec1R1Aec2 Mouse.......................................................... 17 Interleukin22 .......................................................................................................... 18 The IL22 Encoding Gene ................................................................................ 19 Structure of Secretory IL 22 ............................................................................. 21 The IL22 Receptor .......................................................................................... 21 Sources of IL22 ............................................................................................... 24 Targets of IL22 ................................................................................................ 27 Biological Effects of IL22 ................................................................................. 28 2 MA TERIALS AND METHODS ................................................................................ 37 Human Subjects ..................................................................................................... 37 Sialometry ........................................................................................................ 38 Labial Salivary Gland Biopsy ............................................................................ 38 Mous e Samples ...................................................................................................... 39 Sialometry ........................................................................................................ 39 Organ Collection ............................................................................................... 39 Immunohistochemical Staining ............................................................................... 40 Quantification of Positive Staining .......................................................................... 41 RNA Isolation and Quantitative Real Time Polymerase Chain Reaction (PCR) ..... 41 Immunofluorescence .............................................................................................. 42 Determination of IL22 Levels in Sera and Saliva ................................................... 42 Luminex ............................................................................................................ 42 ELISA ............................................................................................................... 43 Flow Cytometry ....................................................................................................... 44 Mitochondrial Membrane Potential Assay ............................................................... 44 MTT Assay .............................................................................................................. 45 Cell Cycle Arrest Assay .......................................................................................... 45
6 Statistical Analyses ................................................................................................. 46 3 RESULTS ............................................................................................................... 47 Expression of IL22 and IL22R 1/2 in Labial Salivary Gland Tissues of pSjS Patients ................................................................................................................ 47 Expression of NKp46+, CD56+, and LTi like Cells in LSG Tissues of pSjS Patients ................................................................................................................ 48 Elevated Levels of Serum IL 22 and Signifciant Correlation with Saliva Flow in pSjS Patients ....................................................................................................... 49 IL 22 Levels in Sera Correlate with Major Parameters of pSjS ............................... 49 Expression of IL22 and IL22R 1 in Organs of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mice ............................................................................................... 50 Absent mRNA expression of IL22 in LSG of C57BL/6.NOD Aec1Aec2 and C57BL/6 Mice ...................................................................................................... 53 Expression of NKp46+ and LTi like Cells in LSG Tissues of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mice ......................................................................... 54 Lack of Saliva and Serum IL22 in C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mice ..................................................................................................................... 54 Analysis of IL 22 Producing Cell Populations within C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mice ............................................................................................... 55 IL 22 Induces Cell Cycle Arrest at the G2M Phase of the Cell Cycle .................... 60 4 DISCUSSION ......................................................................................................... 88 LIST OF REFERENCES ............................................................................................... 97 BIOGRAPHICAL SKETCH .......................................................................................... 111
7 LIST OF TABLES Table page 1 1 Important criterion for an ideal primary SjS mouse model. ................................. 33 1 2 Primary and secondary SjS m ouse m odels ........................................................ 34 1 3 Features of common spontaneous and transgenic mouse models for SjS ......... 35 1 4 Features of common knockout, immunization, infection, and transplantation chimera mouse models for SjS ........................................................................... 36 3 1 Basic laboratory tests, procedures, extraglandular manifestations, and oral findings. .............................................................................................................. 63
8 LIST OF FIGURES Figure page 3 1 Expression of IL22 and ILpatients.. ............................................................................................................. 64 3 2 Expression of NKp46, CD56, and subpopulations of NKp46 cells in labial sal ivary glands of pSjS patients. ........................................................................ 65 3 3 IL 22 levels in saliva and sera samples of pSjS patients .................................... 67 3 4 Correlation analysis of serum IL22 levels and saliva flow, focus score and disease duration.. ............................................................................................... 68 3 5 Correlation between serum IL22 levels and clinical dise ase parameters in pSjS patients ...................................................................................................... 69 3 6 Expression of IL22 and ILin the salivary glands of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. ....................................................................... 70 3 7 Expression of IL22 and ILof C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice.. ...................................................................... 71 3 8 Expression of IL22 and ILAec1R1Aec2 and C57 BL/6 mice ....................................................................... 72 3 9 Expression of IL22 and ILAec1R1Aec2 and C57BL/6 mice. ....................................................................... 73 3 10 Expression of IL22 and ILAec1R1Aec2 and C57BL/6 mice. ....................................................................... 74 3 11 Immunofluorescence staining reveals subpopulations of NKp46+ cells within the salivary glands of C57BL/6.NOD Aec1R1Aec2 mice.. ................................. 75 3 12 IL 22 levels in saliva and sera samples of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. .................................................................................................... 76 3 13 Flow cytometry gating examples.. ...................................................................... 77 3 14 NK cell populations present within the spleen of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. ............................................................................................. 80 3 15 NK cell populations present within the thymus of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. ............................................................................................. 81 3 16 NK cell populations present within the lymph nodes of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. ....................................................................... 82
9 3 17 NK cell populations present within peritoneal exudates cells in C57BL/6.NOD Aec1R 1Aec2 and C57BL/6 mice. ............................................... 83 3 18 LTi like cell populations present within the spleen, thymus, lymph nodes, and peritoneal exudates cells of C57BL/6.NODAec1R1Aec2 and C57BL/6 mice .... 84 3 19 IL 22 treatment of HSG cells results in halt in proliferation but not apopt osis.. ... 85 3 20 IL 22 treatment of HSG cells res ults in decreased proliferation .......................... 86 3 21 IL 22 induces cell cycle arrest. ........................................................................... 87
10 LIST OF ABBREVIATIONS IL Interleukin LF Lymphocytic foci LSG L abial salivary glands. LTi L ymphoid tissue inducer NK Natural killer p SjS Primary Sjgrens syndrome RARr elated orphan receptor gamma t SjS Sjgrens Syndrome sSjS Secondary Sjgrens Syndrome TH T Helper cell
11 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science EXPRESSION AND EFFECTS OF INTERLEUKIN22 IN HUMAN SJGRENS SYNDROME AND MOUSE MODELS By Tegan N. Lavoie May 2011 Chair: Ammon B. Peck Major: M edical Sciences Sjgrens syndrome (SjS ) is a complex autoimmune disease which targets the exocrine glands resulting in xerostomia/ keratoconjunctivitis sicca. Presently, we examined the presence, source and clinical correlations of IL22 in human SjS patients and in the C57BL/6.NOD Aec1R1Aec2 mouse model. The functional effects of IL22 were also studied using HSG cells as an in vitro model. Patients (n=31) who met the American European Consensus Group criteria for primary SjS (pSjS), together with normal controls (n=17), were randomly selected. Immunohistochemical staining revealed that IL22 i s expressed in the salivary glands most prominent in the leukocytic infiltrates, ductal epithelium, and myoepithelium. S everal unique cell populations expressing NKp46 involved in IL22 production were also found, including -CD3-NKp46+ NK, +CD3-NKp46+ LTi like, +CD3-NKp46LTi and +CD3+NKp46+ cells. IL 22 was detected at significantly higher levels in sera of SjS patients. The levels of IL22 present in sera showed statistically significant direct correlations with hyposalivation, anti SSB/La anti SSA /Ro /SSB/La combined, hypergammaglobulinemia and rheumatoid factor. IL 22 shows several direct
12 correlations with major clinical parameters. The data suggest that IL22 plays a critical role in the development of S j S and further study is needed to examine its function in human S j S. Interestingly, in the C57BL/6.NOD Aec1R1Aec2 mouse model ( currently considered one of the most phenotypically similar to the human disease) minimal to no IL 22 was present within the salivary or lacrimal glands, and levels present within the spleen, thymus, and lymph nodes were comparable to that of C57BL/6 controls. Both male (n=3) and female (n=3) mice were examined at time points 4, 8, 12, 16, 20, and 24 wks. The cytokine was entirely absent from saliva and sera and NK and LTi cell populations were present in miniscule amounts within the organs. Flow cytometry was used to further characteriz e these populations in mice. In order to better understand the in vivo function of IL22, a mouse model is needed which mimics similar overexpression patterns to human SjS patients. Mitochondrial membrane potential and MTT assays were performed to observe the in vitro effects of IL 22 treatment on HSG cells and r esults indicate that IL22 does not induce apoptosis in HSG cells, but instead halts the cell cycle at the G2M phase. The ability of IL22 to disrupt the cell cycle is likely a result of its ability to modulate common proproliferative mediators (ERK1/2, J NK). However, additional studies are required to understand the function of IL22 overex pression in human SjS patients and to determine if it acts in a protective or deleterious manner.
13 CHAPTER 1 IN TRODUCTION Sjgrens Syndrome Sjgrens syndrome (S j S) is a systemic chronic autoimmune disease that targets the exocrine glands, predominantly the salivary glands and lacrimal glands, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes) (1) The disease also presents with systemic manifestations involving the destruction of the thyroid gland (2) lungs (3) liver (4) and kidneys (5) The National Arthritis Data Workgroup using the Olmsted County, MN and 2005 US population prevalence estimates from the Census Bureau has estimated the prevalence of primary SjS (pS j S) in the USA approaches 1.3 million with a rang e of 0.43.1 million of the approximate 214.8 million population, with a femaleto male ratio of about 9:1, indicating a probable correlation between disease development and sex hormones (6) S j S can exist in one of two forms, either primary or secondary (7) p S j S affects salivary and/or lacrimal glands in the absence of other rheumatic diseases, while its more common secondary form occurs in the presence of other rheumatic diseases, such as systemic lupus erythematosus (SLE) (8) rheum atoid arthritis (RA) (9) scleroderma (10) and primary biliary cirrhosis (11) The degree of glandular destruction is related to the progressive development of lymphocytic infiltrations which are composed primarily of CD4+ and CD8+ T cells (12) B cells (13) macrophages and dendritic cells (14) According to the revised EuropeanAmeri can Consensus Group criteria, diagnosis of S j S includes th e signs of ocular and oral dryness, detection of infiltrating lymphocytes within minor salivary glands with quantification determined by histopathological evaluation, and the presence in serum of autoantibodies, specifically
14 anti SSA/Ro, antiSSB/La and antinuclear antibodies (ANA) (15) Recently, considerable interest has focused attention on serologic al evaluations showing the presence of rheumatoid factor (RF) elevated immunoglobulin levels (hypergammaglobulinemia), anti fodrin and the presence of antibodies to the muscarinic acetylcholine receptors, especially the type 3 receptor (M3R) which could impair secretory function (1624) The precise etiology of S j S remains elusive; however a number of possible theories have been postulated. Environmental triggers includ ing exposure to EpsteinBarr virus (25) hepatitis C virus (26) and retroviruses including both human Tcell ly mphocytic virus type I (HTLV 1) (27) and human endogenous retrovirus (HERV K113) (28) may initiate epithelial cell activation and a prolonged inflammatory response in genetically predisposed individuals, resulting in system ic autoimmunity. Other hypotheses, including epithelial/acinar cell apoptosis, emergence of autoreactive T cells, effect of autoantibodies and neurological dysfunction, could consequently contribute to various aspects of SjS pathogenesis (29) The challenge in attempting to understand the mechanism of human SjS pathogenesis is due to the difficult nature of detecting biological and immunological occurrence prior to overt clinical signs. Latestage disease is often the only parameter which is used to characterize the entire disease process. As a result, it remains difficult to grasp and understand the disease development. Therefore, animal models for S j S would permit the investigation of the full spectrum of possible etiologies from prior to during and post disease development Recently, we and others have reported the presence of CD4+TH17 lymphocytes within the leukocytic infiltrates observed in the salivary glands of SjS patients, as well as
15 animal models of SjS (30, 31) Although the focus of such studies has drawn attention to the possible role of IL17 in the development of disease, little attention has been placed on the role of IL22 produced by this same cell population. In the current study, we have examined the expression levels and clinical correlation between IL22 levels and multiple disease characteristics of S j S patients. Results indicate elevated levels of IL 22 in SjS patients and several IL22producing cell populations present within the salivary gland infiltrates of SjS pat ients. Furthermore, a direct association between the levels of detectable IL22 and the major criteria currently used in diagnosing S j S, leading us to propose that IL22 might play an important role in the pathogenesis of S j S. Sjgrens Syndrome Mouse Model s An ideal Sj S mouse model should fulfill a range of common characteristics present in human S j S, including etiological, clinical, histological, serological, and immunobiological features as detailed in Table 1 1 Furthermore, different models will represent SjS in either its primary or secondary form, as demonstrated in Table 1 2 which clarifies the relevance of each mouse model. For the purpose of this study, we have selected to use the C57BL/6.NOD Aec1Aec2 and strains due to their striking similar ities with the human disease. Tables 1 3 and 14 provide a brief overview of the most commonly used SjS mouse models with their characteristics. The Nonobese Diabetic Mouse The nonobese diabetic (NOD) inbred strain of mice were developed from a cataract prone subline (CTS) derived from outbred ICR mice (32) The NOD strain is not cataract prone, however, and is most commonly used as a model for human Type 1 insulin dependent diabetes mellitus (IDDM or T1D ) due to lymphocytic infiltrations (insulitis) wh ich cause the destruction of pancreatic islets. Onset of diabetes in highly
16 inbred NOD mice occurs between 90 and 120 days, with an incidence of 6080% in females and 2030% in males by 210 days (33) Spontaneous onset of diabetes in NOD mice presents with hyperglycemia, hypercholesterolemia, glycosuria, ketonuria, polyuria, polydipsia, and polyphagia, all common clinical features of human IDDM. While insulitis develops by 4 weeks (wks) of age, lymphocytic infiltrations in the salivary and lacrimal glands occur at approximately 1216 wks of age with corresponding loss of secretory function by 20 wks old (34, 35) At the onset of SjS like disease, various signature autoantibodies can also be detected, specifically, anti SSA/Ro, antiSSB/La and anti muscarinic receptor type III (M3R) which has been demonstrated to directly contribute to the secretory dysfunction in this animal model and SjS patients. The C 57BL/6.NOD Aec1Aec2 Mouse The NOD mouse model has provided important insight into the genetics of human SjS. The development of T1D in the NOD mouse is controlled by more than 18 chromosomal regions (36) Early studies involving replacement of individual insulin dependent diabetes ( idd) susceptibility intervals such as Idd3, Idd5, Idd13, Idd1 and Idd9 had minimal effect on the development of autoimmune exocrinopathy or SjS like disease. Both Idd3 and Idd5 are required for development of salivary and lacrimal dysfunction (37) When both NOD derived genetic regions were introduced to the SjS nonsusceptible C57BL/6 strain by crossing C57BL/6.NOD c3 mice carrying Idd3 ( Autoimmune exocrinopathy 1 ( Aec1 ) ) locus and C57BL/6.NOD c1t mice carrying Idd5 ( Aec2 ) locus, the C 57BL/6.NOD c3 .NOD c1t or C57BL/6.NOD Aec1Aec2 mouse strain was produced which is homozygous for both Idd3 and Idd5 chromosomal intervals (38) This double congenic strain fully recapitulated the SjS like disease process, exhibiting pathophysiological changes at early age, followed by lymphocytic infiltrations of the
17 salivary and lacrimal glands at 1216 wks of age, then accompanied by the production of autoantibodies to nuclear antigens (SSA/Ro, SSB/La) and M3R in the absence of T1D. The lymphocytic foci (LF) consis ted mainly of CD4+ and CD8+ T cells, as well as B lymphocytes with associated loss of saliva production by 20 wks of age. Due to the presence of T cells and sporadic numbers of dendritic cells and macrophages within infiltrates, an increase in the level s of proinflammatory cytokines such as i nterleukin17 (IL 17) and IL23 was also detected locally and systemically Similar observations are observed in human S j S patients (30) The C57BL/6.NOD Aec1R1Aec2 Mouse A recombinant inbred line, known as C57BL/6.NOD Aec1R1Aec2, was developed to define smaller genet ic regions that contain those genes necessary to induce autoimmune exocrinopathy by narrowing the Aec1 region (39) The genetic region of Aec 1 locus was shortened from a 48.5 cM segment to a centromeric piece spanning 19.2 cM. The resultant strain exhibited more rapid S jS like disease in males, with males developing salivary gland infiltrations at 10 wks of age compared to 19 wks in females. Females presented with more severe sialadenitis and larger infiltrations in the submandibular gland by 22 wks, however, they exhibited no dacryoadenitis whereas males exhibited significantly high levels of dacryoadenitis. Furthermore, a homogeneous nuclear ANA pattern was apparent in males as early as 5 wks of age but not until 10 wks in females. Both sexes demonstrated a significant loss of saliva flow rate (3540%) beginning at 5 wks of age, but only males displayed a loss of lacrimal gland secretory funct ion. The lack of lacrimal gland dysfunction in females may be attributed to the loss of a necessary gene on the shortened Aec1 locus which could regulate the sex dimorphism presented in SjS. For the purposes of this experiment, the
18 C57BL/6.NOD Aec1R1Aec2 m ouse model will be studied due to its high phenotypic homology with human SjS. Interestingly, the major histocompatibility complex (MHC) genes have little or no relation to the development of SjS in the NOD mouse. For example, the MHC class II region, when replaced from Ag7 to Ab locus in NOD mice, prevented the development of T1D, but the onset of SS l ike disease remained unaffected (40) Also, the NOD. H2h4 strain presents with exocrine gland infiltrations and compromised saliva flow without symptoms of T1D due to the replacement of the Ag7 allele with I Ak, but continues to develop spontaneous thyroiditis at a low occurrence (5%) (41, 42) The NOD.B10H2b strain also demonstrates a SjS like phenotype with inflammatory infiltrations in the exocrine glands without the occurrence of T1D due to the replacement of the diabetogenic MHC locus with the MHC locus of C57BL/10 strain that is nonsusceptible to T1D (43, 44) As a result, the NOD and NOD derived animal models have been critical in elucidating the genetic basis of Sj S development. Interleukin 22 Interleukin (IL) 22 is a cytokine belonging to the IL10 family which includes IL10, IL19, IL20, IL24, and IL26, IL 29 (45) IL 22 mediates host defenses against invading pathogens and r equires the presence of IL23 in order to induce its expression from immune cells (46) IL 22 is produced predominantly by CD4+TH17 cells, but its expression can also be found among other activated T cell subsets, natural killer (NK) cells, lymphoid tissue inducer (LTi) cells, and dendritic cells (DC), although at l ower levels (47) Its receptor complex is a heterodimeric molecule composed of IL22R 1 and IL10R2 (48) Upon interacting with its receptor, IL22 can transduce a signal through phosphorylation of tyrosine kinases Jak1 and Tyk2, followed
19 by the activation of STAT3, and to a lesser degree heterodimeric STAT1/3 during signaling cascade (49) IL 22 has also been reported to activate signal transductions via the MAPK pathways of ERK1/2, JNK, and p38 for induction of IL22 related genes (50) Since epithelial cells express high levels of IL 10R2 and IL22R, IL22 can initiate a strong response from epithelial cells which includes production of cytokines, chemokines, acute phase proteins, and a number of anti defensin, lipocalins, and S100 calcium binding protein (45) It is also involved in tissue repair following exacerbated immune responses and epithelial barrier functions against bacterial infections. (51) As CD4+ Th17 cells are the primary source of IL22, this cytokine h as been shown to be present at higher levels within T lymphocytedriven diseases being pathogenically associated with several autoimmune diseases including rheumatoid arthritis (52) and Crohns disease (53) as well as nonautoimmune diseases such as respiratory distress syndrome (54) and cystic fibrosis (55) At present, the role of IL 22 in SjS is unknown. The IL22 Encoding Gene In humans the IL22 encoding gene is found on the longer arm of chromosome 12, on 12q15, existing as a single copy gene (56) and 27 Kb from the IL 26 gene, indicating the occurrence of cytokine clustering on this particular genomic region which has been correlated with increased incidence of asthma and inflammatory bowl disease (5760) In mice, the IL 22 encoding gene is ; however, no evidence of a mouse hom olog of the IL 26 gene was apparent (56) The IL22 encoding gene exists as a single copy gene within the BALB/c and DBA/2 strains, however, it i s duplicated in the C57Bl/6, FVB and 129 strains. The duplicated copies
20 demonstrate 98% nucleotide similarity within the coding region and are distinguished as either IL. Both the human and mouse genomic sequences for IL22 can be found in the National Center of Biotechnology Information (NCBI) archives under the accession number NT_029419 (human) or NT_039500 (C57Bl/6J mouse). The mRNA encoding sections for humans is distributed from 30,785,331 to 30,790,587 bp in the human genome with a mR NA (NM_020525) sequence length of 1,147 bases according to the NCBI database as of February 2011. The mRNA is comprised of five exons following the form of other IL 10 cytokines. The first exon spans 239 bp in length with its first 53 bp containing the 5 untranslated region. The remaining 186 bp of exon 1, along with exon 2 (66 bp), exon 3 (144 bp), exon 4 (66 bp), and the first 79 bp of exon 5 encode IL22 protein and contain the stop codon. The final 554 bp of exon 5 encodes the 3 untranslated region, c ontaining six single and two duplicate copies of the ATTTA motif involved in control of mRNA degradation. Without including the stop codon, the total length of the open reading frame is 537 bp, therefore, the protein product should be approximately 179 aa in length. In mice, the genomic sections which encode the mRNA for IL 22 protein span from 59,973,407 to 59,978,490 bp. The mRNA ( NM_016971) has a length of 1088 bp and also consists of five exons, whose lengths are identical to that seen in humans. The fi rst 27 bp of exon 1 encode the 5 untranslated region. The remaining 186 bp of exon 1, along with exon 2 (66 bp), exon 3 (144 bp), exon 4 (66 bp), and the first 79 bp of exon 5 encode IL22 protein and contain the stop codon. The final 520 bp of exon 5 enc odes the 3 untranslated region Without including the stop codon, the total length of the open reading frame is 537 bp, therefore, the protein product should be approximately 179 aa in length.
21 Structure of Secretory IL22 The structure of IL22 consists of six helices, similar to that of other IL 10 family members. The six helices are denoted as helices A F and are situated in an antiparallel conformation which creates a monomeric, bundlelike protein, confirmed by X ra y diffraction, gel filtration chromatography, and dynamic light scattering studies (6163) Within its primary structure, IL 22 contains four cysteine residues which result in formation of two intramolecular disulfide bridge bonds The first disulfide bond occurs at Cys40 Cys132 and joins the N terminus to the DE loop and the second disulfide bond occurs at Cys89 Cys178 and links helices C and F together (62) Interaction of IL22 with its receptor subunit IL10R2 has been found to depend on the glycosylation on Asn54 in the IL22 sequence. IL22 has three potential N linked glycosylation sites and is glycosylated at each of these locations. These glycosylation sites include Asn54Arg55Thr56 in helix A, Asn68Asn69Thr70 in the AB loop, and Asn97Phe98 Thr99 in helix C (64) Howeve r, the tertiary structure of the protein remains unaltered whether glycosylated or not glycosylated. The IL22 Receptor In order for IL22 to induce cellular responses it must first bind to its specific cell surface receptors which belong to the class II c ytokine receptor family, similarly to all members of the IL 10 family (65) The IL22 receptor complex is composed of two distinct receptor chains, denoted as interleukin22 receptor alpha 1 (IL22R 1 ) (cytokine receptor family class 2 member 9, CRF29) and interleukin10 receptor 2 (IL10R2) (cytokine receptor family class 2 member 4, CRF24) (48, 49, 66) Both are structurally similar to IL 10R1 (67) Many receptors that bind IL10 family members exhib it crossreactivity with multiple cytokines from the IL 10 family, and IL22R 1 and IL10R2 are
22 no exception. When IL 22R 1 forms a heterodimer with IL20R2 (the second chain of the IL10 heterodimeric receptor), it is capable of binding interleukin20 (IL20) and interleukin24 (IL24) (68) Impressively, IL 10R2 can bind to many cytokines when existing as a heterodimer wit h either IL 10R1 (binds IL10), IL 20R1 (binds IL26) or IL 28R1 (binds IL28 IL 28, and IL29) (69) In a series of experiments conducted by the Walter group using surface plasmon resonance techniques, the kinetic binding data suggested that IL22 has a high affinity to the IL22R 1 subunit of the receptor complex but no affinity for the IL10R2 subunit (61, 64, 70) The IL10R2 subunit showed high affinity for the IL22:IL22R 1 complex. These findings were confirmed by the Fouser lab via ELISA which showed that biot inylated IL22 readily binds IL22R 1 Fc and not IL10R2Fc; however, IL10R2Fc did appear to bind with IL22:IL22R 1 Fc (71) Such studies support the theory that IL 22 initially binds IL22R 1 with high affinity and subsequently induces a conformational change in the cytokine to allow binding of IL10R2. The Sabat group used scans of overlapping peptides from the protein sequences of IL22 and IL10R2 to show that IL10R2 binding sites on IL22 likely include residues which are inaccessibl e on the surface of IL22 and only become accessible after conformational change is induced by IL22R 1 binding (72) The crystal structure of the IL22:IL22R 1 complex has been recently reported by two separate groups, suggesting that IL22 and IL22R form a 1:1 complex in which IL 22 residues located on helices A, F, and the AB loop interact with the ILL6 loops (70, 73) Site directed mutagenesis assays suggest that IL22 possesses a unique binding site for IL10R2, where it i nteracts with
23 five hot spot residues (Tyr51, Asn54, Arg55, Tyr114, and Glu117) on IL22 helices A and D (64) After IL 10R2 binds to the I L 22:IL the JAK/STAT signaling pathway leading to tyrosine phosphorylation of STAT3 in cells with endogenous receptor expression (69) Expression of STAT1 and STAT5 has been reported within tumor cell lines (49, 53, 74, 75) The Renauld group investigated the necessity of JAKs for IL 22induced STAT activation via Western blot analysis of c ell lysates and immunoprecipitation using anti JAK antibodies to demonstrate that IL22 transduces a signal through phosphorylation of tyrosine kinases Jak1 and Tyk2, but not Jak2 (50) This was confirmed by studies showing that in Jak1deficient cells there is no IL 22induced STATresponsive reporter gene activity present (50) Due to the typical association of Tyk2 with IL10R2, it is speculated that Jak1 would thus be the kinase associated with IL3, serine phosphorylation is vital for optimal IL22induced transactivation of the STATresponsive promoter and it was shown in H4IIE cells that IL 22 effectively induced the three major MAP kinase pathways ( extracellular signal related kinase 1/2 ( ERK1/2 ) c Jun N terminal kinase ( JNK) and p38 kinase for induction of IL22 related genes ) (50) Notably, the IL22R 10R2 complex is not the only receptor to which IL 22 binds. There is a nonmembrane bound singlechained IL22 binding protein (IL22BP) which binds IL 22 with high affinity and multiple studies have shown that binding of IL22BP to IL22 can inhibit the effects of cellular IL 22 by preventing binding to its membrane bound receptor complex (7681) In fact, the affinity of IL 22 to its binding protein is approximately 20or 1000fold greater than to its membrane bound receptor,
24 according to data reported by two separate research groups using the surface plasmon resonance technique with di ffering experimental conditions (70, 82) This difference in affinity is due to a significantly lower dissociation rate, indicating that IL22:IL22BP complexes are highly stable. Crystallization data indicates that IL22BP and ILshare overlapping IL22 binding surfaces, indicating that IL22 would be the only member of the IL10 family of cytokines to be under the influence of an inhibitory secreted receptor (83, 84) This innate soluble receptor is also known as IL 22R 2, which is expressed in CD4+ T cells, CD19+ B cells, and epithelium (81) Previous studies have shown that upon IL22 binding to IL22R 2, the soluble receptor can deactivate IL22 acti vity and prevent IL22 activat ion of STAT1, STAT3, and STAT5. As such, IL22 antagonist in the regulation of inflammatory responses. Sources of IL 22 The first report of IL 22 mRNA expression was in 2000 in murine models within T cell lines that had undergone IL 9 stimulation and in concanavalin A (ConA) activated spleen cells (76) In the same year, similar reports were published in the human system, describing IL22 mRNA expression in peripheral blood T cells that were activated with anti CD3 antibody or ConA (49) Subsequent experiments showed that IL22 expression can only be found in activated T cells and NK cells (85) IL 22 is not expressed in dendritic cells, monocyte derived macrophages, or nonhematopoietic cells (8688) In 2002, the Sabat group reported that IL22 expression was preferential to CD4+ memory cells, and high expression levels were apparent in the Th1 subset, with very low expression seen in the Th2 subset (85) In recent years, several additional Th cell subsets have been identified, of which the Th17 and Th22 subsets are also major
25 IL 22 producers (85, 8991) In fact, the frequencies of each of the three Th subsets among total IL22 producing T cells was determined by Duhen et al. to be from 3763% for Th22 cells, from 10 18% for Th17 cells, and about 35% for Th1 cells (89) The three IL22 producing subsets of T cells each possess their own unique characteristics. Th1 cells are characterized by their expression of the transcription fact or T bet as well as for their secretion of IFN (92) They are dependent on IL12 for generation and are typically CD3+CD4+CXCR5+CCR4-CCR6+/ -. Th17 cells are known for secretion of IL17A, IL17F, IL 6, and tumor necrosis factor alpha (TNF4 and IFN (91) Th22 cells are reported to have phenotype CD3+CD4+CCR10+CCR4+CCR6+ and to produce IL 22 but not IL17A or IFN (89, 90) cells is currently unknown as several groups have reported contradi ctory data as to its relevance. Dendritic cell interact ion with nave T cells is required to induce differentiation into Th1 or Th2 lineage, in either an IL12 or IL4 dependent manner, respectively. In humans, differentiation into a Th17 cell type is dependent on the combined presence of IL 23, TGF 6, and IL production of IL23R by approximately 50% (91) The products of Th1 cells (IL12, IFN 4) suppress the differentiation and development of Th17 cells (93, 94) It is unknown what effect TNFhas on Th17 development. Differentiation into Th22 cells was reported to be induced by plasmacytoid dendritic cells in a IL6 and TNF(89) Interestingly, in murine models only Th17 cells are producers of IL 22, not Th1 cells, and a Th22
26 subset of T cells has not yet been reported (46, 95, 96) Differentiation of mouse Th17 cells is thought to be induced by IL23, TGF 6, IL 21. In addition to IL22 production (9799) Upon IL23 stimulation, these cells secrete IL17, IL21, and IL22 and will also secrete ILabsence of stimulation (97, 99) like receptors (TLR) 1 and 2 and dectin1 (97) Production of IL22 has also been described in gut mucosa associated NK cells in b oth humans and mice (47) These cells are of phenotype CD3-NKp46++ and do not behave typically of NK cells Also reported are lymphoid tissue inducer ( LTi ) cells found within prenatal human lymphoid tissues and postnatal tonsils (47) LTi cells are vital in secondary lymphoid organ development and express CD4 and chemokine differentiation. They are likely responsible for IL22 expression in the intestine to promote the health of the mucosal lining. In addition, the presence of LTi cells can be analyzed by examining for its common markers, with phenotype of +CD3-CD56-NKp46-. Such human LTi cells can give rise to LTi like cells and NK cells which produce IL 17 and IL22 upon stimulation with IL23. If LTi cells are present within the lymphocytic infiltrates of the labial salivary glands of SjS patients, this finding would likely be critical in furthering our understanding of the pathogenesis of SjS in humans. Furthermore, IL22 expression has also been reported within NKT cells in murine models (100)
27 Targets of IL22 In order to determine the targets of IL22, the IL typically investigated within various tissues due to the ubiquitous nature of the IL10R2 subunit which comprises part of the receptor complex for multiple cytokines (88) Therefore, one can assume that ILa cell is a target of IL22 or not. Strikingly, IL22 does not target immune cells as demonstrated by reverse transcriptase polymerase chain reaction of bone marrow, blood mononuclear cells, thymus, and spleen for IL(88) No IL (monocytes, B cells, T cells, NK cells, macrophages, and immature and mature DCs) (8588) Confirming these findings, no STAT activation was apparent in blood immune cells that had been treated with IL22 (88) However, tissue cells located within the skin, kidneys, respiratory system (lung and trachea), and digestive system (liver, pancreas, intestine, and colon) are common targets of IL22 (88) Typically, tissues which express ILthe body and its surroundings and consist of epithelial cells. Within the skin, keratinocytes tend to express the highest levels of ILtimes lower levels seen in dermal fibroblasts and no expression seen in melanocytes, dermal microvascular endothelial cells, or subcutaneous adipocytes (88, 101) Cellular responsiveness of IL22 is highly influenced by the cytokines IFN TNF (87, 101, 102) They observed that expression of IL 10R2 in keratinocytes significantly increased when treated with IFN and timedependent, indicating that treated cells had heightened IL22 sensitivity under T1 and inflammatory conditions.
28 Additional studies demonstrated that epithelial cells in the intestines and lungs are also major targets of IL22. Notably, although fibroblasts tend to possess a weaker response to IL22 stimulation than do epithelial cells, this is likely due to their local cellular en vironment. For instance, IL22 was shown to have a significant effect on colonic subepithelial myofibroblasts and synovial fibroblasts in rheumatoid arthritis patients (52, 103) Hepatocytes and pancreatic acinar cells are also considerable target cells of IL 22 (66, 104) Biological Effects of IL22 At present, the precise mechanism of action of IL22 is obscure with respect to many diseases and immune processes. However, within the past decade several papers have helped to shed light on the elusive nature of IL22 allowing speculation of its many functions. For instance, we know that as a result of the ability of IL 22 to induce multiple anti microbial proteins ( AMPs defensins (BDs) in e pithelial cells, it suggests that IL22 may be a pivotal factor in epithelial host defense against pathogens in the lungs and the bowel (55, 88, 105) However, no current studies have been conducted in humans to determine the precise function of IL22 with respect to its antibacterial defense. Due to the preferential expression of IL22 by the Th1, Th17, and Th22 T cell subsets, it is likely that exacerbated lev els of the cytokine would be present in persistent, T cell mediated diseases such as rheumatoid arthritis (RA), inflammatory bowl disease (IBD), and psoriasis. In RA patients, it was found that IL22 expression was evident in the synovial tissues (both lining and sublining layers) and in mononuclear cells of the synovial fluid (52) T he number of CD4+IL 22+ T cells in the peripheral blood was increased when compared to healthy controls (106) Using synovial fibroblasts
29 extracted from RA patients, an in vitro study found that high levels of IL22 significantly increased CCL2 production and proliferation of the fibroblasts (52) An additional study reported the pathogenic nature of IL22 in a collageninduced arthritis animal model of RA, in which the less destructiv e course of arthritis was observed when IL22 was absent and was accompanied by increased CII specific and total IgG antibody production (107) This group also r eported that in vitro IL 22 induced osteoclastogenesis. In patients with IBD areas of inflammation within the gut are positive for CD4+IL 22+ T cells which are otherwise absent in gut tissues of healthy individuals (53, 103) In active ulcerative colitis these IL22 producing cells were localized to the lamina propria whereas in Crohns disease they were found throughout the submucosa (103) Leppkes et al. reported that ulcerative colitis was induced in RAG1deficient mice via adoptive transfer of IL 22deficient T cells, causing a severe disease phenotype indistinguishable from that observed by transferred wildtype cells (108) Strikingly, not only is IL 22 present within the blood of Crohns disease pat ients but the IL22 serum levels positively correlate to disease activity and severity (82) Other cytokines, including IL17, IFN of healthy controls. Remarkably, IL22 expression was entirely absent in the gut mucosa of indiv iduals with infectious colitis (103) With regards to both ulcerative colitis and Crohns disease, IL22 appears to be playing a protective role, locally or systemicall y, respectively (109, 110) P soriasis is perhaps the most widely studied disease with respect to IL22 influence. Within the lesional skin from psoriasis patients, high expression of IL22
30 mRNA was apparent compared with healthy controls where IL22 mRNA expression was either very low or absent (87, 88) Upregulation of IL22 was significantly greater than that of IL12, TNFlevels of IL22 were significantly correlated to IL20 levels, supporting the theory of IL22induced keratinocyte IL20 production (111) Furthermore, raised cutaneous levels of IL22 were positively correlated to increased IL22 levels in the blood, where IL17, and IFN remained unaffected and TNF(87, 111) Patients treated with antipsoriatic drugs presented with reduced levels of IL22, both in cutaneous IL22 mRNA levels and in IL22 blood levels (87, 112) Increased cutaneous levels of IL22 expression are also apparent in other T cell mediated skin diseases including atopic dermatitis (88) Upon careful examination of all currently available data concerning IL22 i n relation to psoriasis, it appears that the effector phase of psoriasis pathogenesis includes two distinct stages (69) The first stage, referred to as the proximal stage, is marked by immune cell infiltration of the skin. The second stage, or the distal stage, is when the typical keratinocyte alterations appear. During the proximal stage, induction of Th1, Th17, Th22, macrophage, DC, and neutrophilic granulocyteattracting chemokines allow TNFocess of leukocytic infiltration (101, 113) Also highly influential at this stage are IFN well as activation of antigenpresenting cells (APCs), and IL17 which may be responsible for the development of Munros microabscesses as it is a potent inductor of neutrophilic granulocyteattracting chemokines (101, 114) Pivotal players of the distal stage include IL22 and its downstream mediator IL20 which inhibit the keratinocyte cornification process. Ergo, IL 22 is directly responsible for producing the keratinocyte
31 alterations which lead to epider mal acanthosis, hypogranularity, parakeratosis, and hyperkeratosis (87, 101) However, IL 22 has no effect on the hyperproliferation of basal keratinocytes typical of psoriasis. Notably, TNFof IL 22 receptor pathway elements, to amplify effects of IL 22 (87, 88, 101, 102) In keratinocytes, the effects of IL 22 and IL17 can be additive, as is seen in the induction of antimicrobial proteins and CXCL8 (96, 101) Most effects are not exerted additively, however, as in the induction of psoriasis like keratinocyte changes (101) Therefore, since the IL22 system is responsible for the distal stage of psoriasis it would be an ideal target for future drug therapies. Interestingly, upon examining the role of IL22 in experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis in humans, it was found that IL22 was not required for disease development via use of IL22 knockout mice. However, use of IL22Ig gene therapy was proven to be effective in controlli ng EAE in rats, suggesting that in EAE IL22 serves a protective function in mediating the disease progress. Currently, the effect of IL 22 in SjS has yet to be defined. In order to determine if IL22 is influential in the development and progression of Sj S, the following must be established: Identify whether IL 22 is present within the infiltrating lymphocytic populations present within the salivary gland of human SjS patients Establish whether IL 22 effects are systemic or local via cytokine profiling of the sera and saliva Determine which IL22 secreting cell populations are present in the salivary glands Quantify whether or not IL22 expression correlates with common disease parameters (i.e., anti SSA/Ro /SSB/La antibodies, RF, saliva flow rate, etc.) Com pare human results with results observed in C57BL/6.NOD Aec1R1Aec2 SjS mouse model
32 Perform functional assays to observe effects of IL22 treatment in vitro
33 Table 11 Important criterion for an ideal primary SjS mouse model. Features Etiology Unknown (possible viral exposure) Clinical Xerostomia Keratoconjuctivitis sicca Histological Polyclonal lymphocytic infiltrations in the salivary and lacrimal glands Lymphocytic focus, >50 mononuclear cells/mm2 (CD4+ > CD8+) Monoclonal B cell proliferation Progressive destruction of the acinar and ductal cells Serological Hypergammaglobulinemia Anti SSA/Ro and anti SSB/La autoantibodies Anti fodrin autoantibody Rheumatoid factor Antinuclear antibodies Anti type 3 acetylcholine muscarinic receptor Additional o rgan i nvolvement Heart, blood vessels, lungs, liver, pancreas, stomach, kidneys, bladder, thyroid gland (secondary SjS) Immunobiology Diminished apoptosis of lymphocytes Abnormal MHC expression, H2+glandular ductal epithelium Epithelial cell expression of Fas/FasL Other 9:1 female:male ratio Disease presents in absence of other rheumatic diseases
34 Table 1 2 Primary and secondary SjS m ouse m odels Type of SjS Mouse m odel Secondary to Primary Aec1Aec2 NOD.B10 H2 b NFS/sld IQI/Jic CAII immunization PI3K K.O. ID3 K.O. Ar K.O. Ro immunization Secondary NOD Autoimmune diabetes NOD. H2 h4 Autoimmune thyroiditis MRL/lpr RA, SLE GVHR SLE BAFF Tg SLE IL 12 Tg SLE IL SLE MCMV SLE HTLV 1 tax Tg RA (115) TGF SLE (116) IL 6 Tg IL 10 Tg PBC (117) SLE/Neuropathy TSP 1 K.O. IBS K.O.: knockout; Tg: transgenic; SLE: systemic lupus erythematosus; RA: rheumatoid arthritis; PBC: primary biliary cirrhosis; IBS: inflammatory bowel disease
35 Table 1 3. Features of common spontaneous and transgenic mouse m odels for SjS Sjogrens syndrome patient Spontaneous mouse model Transgenic mouse model NOD Aec1 Aec2 NZB/ W F1 MRL/ lpr NFS/ sld IQI/ Jic Aly/ aly HTLV 1 BAFF IL 6 IL 10 IL 12 IL Autoantibodies Anti SSA/ Ro, Anti SSB/ La Yes Yes No No Yes Yes No No No Yes Yes Anti DNA (ANAs) Yes Yes Yes Yes Yes Yes Yes Yes Yes Anti fodrin Yes Yes Yes Yes Anti adrenergic receptor Yes Yes Yes Yes Yes Anti type3 muscarinic Ach receptor Yes Yes Yes Yes Yes Leukocytic infiltrate Time of onset (w ks) Yes Yes 12 Yes 10 Yes 16 Yes 8 Yes 16 Yes 8 Yes 24 Yes 24 Yes 52 Yes 2 Yes 8 Yes <16 Yes 48 Dacryoadenitis Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Sialadenitis Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Loss secretory function Time of onset (w ks) Target organ Yes Var 20 S, L Yes 19 S, L Var 24 L No Yes 72 S, L Yes 52 S Yes 8 S, L Var 16 S, L Var 12 S Pro inflammatory cytokine product Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Var: variable, m ale and female mice differ, : Not determi ned, S: Salivary glands, L: Lacrimal glands
36 Table 1 4. Features of common knockout, i mmunization, i nfection, and transplantation chimera mouse m odels for SjS Characteristic Sjgrens syndrome patient Knockout mouse model Immunization mouse model Infection mouse model Transplantation c himeras Id3 PI3K TGF TSP 1 Ar CAII Ro CMV GVHR Autoantibodies Anti SSA/ Ro, Anti SSB/ La Yes Yes Yes No Yes Yes Yes Anti DNA (ANAs) Yes Yes No Yes Anti fodrin Yes Yes Anti adrenergic receptor Yes Anti type3 muscarinic Ach receptor Yes Leukocytic infiltrate Time of onset (w ks) Yes Yes 8 Yes 8 Yes 1 Yes 24 Yes 48 Yes Yes 16 Yes 4 Yes 28 Dacryoadenitis Yes Yes Yes Yes Yes Sialadenitis Yes Yes Yes Yes Yes Yes Yes Yes Yes Loss secretory function Time of onset (w ks) Target organ Yes Yes 8 S, L Yes <8 S, L Yes >1 S Yes 24 L Yes 16 S Yes 4 S Pro inflammatory cytokine product Yes Yes Yes Yes Var: variable, m ale and female mice differ, : Not deter mined, S: Salivary glands, L: Lacrimal glands
37 CHAPTER 2 MATERIALS AND METHODS Human Subjects Participants were selected from patients seen at the Center for Autoimmune Disorders at the University of Florida. Initial evaluation of these patients was performed by the University of Florida/Shands department of Rheumatology. From a large cohort of patients who met the American European Consensus Group criteria for primary Sj S (24), a subset of 21 patients was selected randomly to include those with whole unstimulated salivary flow rates below the SjS criterion for hyposalivation (<0.1 m L / min ) and patients with flow rates above that level. All patients underwent extensive ser ologic evaluations, which included tests for the presence of antinuclear antibodies (ANAs), anti SSA/Ro, anti SSB/La, anticentromere antibodies, anti Scl70, anti Sm, anti RNP, anti doublestranded DNA, rheumatoid factor (RF), anticardiolipin antibodies, and lupus anticoagulant as well as levels of C3 and C4, level of C reactive protein, thyroid profile, liver function screen, renal screen, complete blood cell count, iron profile, and erythrocyte sedimentation rate. In addition, all patients underwent an ex tensive medical examination. Following the initial evaluation by a rheumatologist, each patient was referred to the Oral Medicine Clinic for a review of his or her medical history, an oral examination, an unstimulated whole salivary flow rate, and a labial salivary gland biopsy. Specimens from 17 subjects not known to have SjS were used as comparative controls. All procedures were reviewed and approved by the University of Florida Health Science Center Institutional Review Board and informed consent of all participants was obtained.
38 Sialometry Unstimulated whole saliva was collected by the drooling method during the late afternoon, between 2:30 PM and 4:30 PM. Study participants were asked to refrain from oral hygiene procedures, smoking, eating, and drinking for at least 2 hours prior to the test session. They were seated comfortably in an upright position and instructed to allow their saliva to flow into a preweighed vessel for a period of 15 min. Sealed containers were then reweighed to determine the weight of saliva expectorated. The unstimulated salivary flow rate was determined by gravitation, using a scale accurate to 0.01 gm. On the assumption that 1 gm of saliva is equivalent to 1 m L the measured volume was expressed as flow rate in mL/ min (27,28) The saliva samples were immediately frozen and stored at 80C. Labial Salivary Gland Biopsy Labial salivary gland biopsies were performed on SjS patients in the Oral Medicine Clinic within 2 weeks of the initial Oral Medicine Clinic visit. A local anest hetic was injected into the lower lip followed by a small incision to the right or left of the lip midline. Five or 6 minor salivary gland lobules were carefully harvested and placed into formalin fixative. Resorbable sutures were placed. Standard paraffin preparations were prepared, sectioned at 5m thickness, and stained with hematoxylin and eosin (H&E). The slides were examined for the presence of lymphocytic infiltrates and/or foci by 3 boardcertified oral and maxillofacial pathologists using standardized criteria. A focus was defined as an aggregate of score was reported as the number of foci per 4 mm2 of tissue, up to a maximum of 12 foci (29,30).
39 Mouse Samples Strains of mice used in this study were C57BL/6J, C57BL/6.NOD Aec1Aec2, and C57BL/6.NOD Aec1R1Aec2. A total of three male and three female mice were used for each time point. For flow cytometry, immunohistochemical staining, and immunofluorescence studies, time points for C57BL/6J mice were 4, 8, and 20 wk and for C57BL/6.NOD Aec1R1Aec2 were 4, 8, 12, 16, 20, and 24 wk. Saliva and sera profiling was performed in both C57BL/6J and C57BL/6.NOD Aec1R1Aec2 mice at 4, 8, 12, 16, and 20 wk. Real time PCR studies used C57BL/6J and C57BL/6.NOD Aec1A ec2 mice at 4, 8, 12, 16, and 20 wk time points. These mice were bred and maintained under specific pathogenfree conditions in the mouse facility of the Department of Pathology, Immunology, and Laboratory Medicine within the Health Science Center at the U niversity of Florida, Gainesville. All mice received acidified water and food ad libitum and were maintained on a 12 hr light dark schedule. Studies described were approved by the University of Florida Institutional Animal Care and Use Committee. Sialometr y Saliva was stimulated via intraperitoneal injection of 100L of phosphatebuffered saline (PBS) containing isoproterenol (0.02mg/m L ) and pilocarpine (0.05mg/m L ). Saliva was collected from the oral cavity of mice at the abovementioned time points starting one min after injection of the secretagogue. Saliva samples were stored at 80C until needed. Organ Collection At each previously mentioned time point, mice were euthanized by cervical dislocation after deep anesthetization with isoflurane. Peripheral bl ood, salivary glands,
40 lacrimal glands, spleen, thymus, lymph nodes, and peritoneal exudate cells were harvested for analyses. Immunohistochemical Staining Labial salivary gland biopsied tissues from SjS patients were surgically removed and placed into 10% phosphate buffered formalin for 24 hours. Tissues were embedded in paraffin and sectioned to a thickness of 5Deparaffinization of paraffinembedded slides was performed by xylene immersion and subsequent dehydration in ethanol. Antigenretrieval was performed in 25 m M Tris/EDTA buffer, pH 9.1 followed by incubation with CytoQ Background Buster (Innovex Biosciences, Richman, CA). After sections were blocked with normal goat serum (S 1000, Vector Labs, Burlingame, CA) and avidin/biotin blocker (Vector Labs, Burlingame, CA), they were incubated overnight at 4C with either anti IL 22 (1/500 dilution, ab18499), anti IL 22R 1 (cross reacts with IL 22R 2, 1/100 dilution, ab5984 ) (Abcam, Cambridge, MA) anti NKp46 (1/100 dilution, MAB1850, R&D Systems) or anti CD56 (1/100 dilution, #3576, Cell Signaling Technology) Isotype controls were performed using rabbit IgG. Slides were washed for 5 minutes (min) and incubated for 30 min in biotinylated goat anti rabbit IgG followed by incubation with biotinylated horseradish peroxidase using Vectastain ABC kit ( SC2023, Vector Labs, Burlingame, CA). The staining was developed by addition of diaminobenzidine (DAB) substrate (Vector Labs, Burlingame, CA) and counterstained with hematoxylin. To ensure that positive inf iltrate staining for IL 22R 1 was not due to high background staining, an additional run was performed using 10 m M citrate buffer for the antigenretrieval step with all other procedures unchanged. Images were taken using the Zeiss Axiovert 200M microscope with the AxioCam MRc 5 camera (Carl Zeiss Microimaging, Inc., Thornwood, NY).
41 Q uantification of Positive Staining Immunohistochemistry was used to examine the staining patterns of IL22 and IL 22R 1 positive cells within lymphocytic foci of 11 SjS patient s and of NKp46 and CD56 positive cells within lymphocytic foci of 3 SjS patients. Similar staining patterns were observed, and for purposes of quantification, SjS Patient #5 was used to represent IL 22 and IL22R 1 staining, and SjS Patient #S086405 was us ed to represent NKp46 and CD56 staining. Five representative lymphocytic foci were chosen from each representative patient. Both total cell count s and positive cell counts were enumerated visually by three individuals blinded to the coded sections. Positiv e cell counts were compared to confirm similar values. RNA Isolation and Quantitative Real Time Polymerase Chain Reaction (PCR) In mice, total RNA was isolated from whole salivary gland s of 3 male C57Bl/6J and C57Bl/6.NOD Aec1Aec2 mice from time points 4, 8, 12, 16, and 20 wk using RNeasy Mini Kits (Qiagen, Valencia, CA) Complementary DNA (cDNA) was obtained through reverse transcription using MuLV Reverse Transcriptase according to manufacturers instructions ( M0253L, New Englan d BioLabs, Inc ., Ipswich, MA ) Each PCR was performed in triplicates on the StepOne Plus Real Time PCR System (Applied Biosystems, Carlsbad, CA ). The reaction mixture contained 5 L of TaqMan Fast Universal PCR Master Mix ( # 4352042) 0.5 L of IL 22 primer mixture ( # Mm00444241_m1, Applied Biosystems Carlsbad, CA ) 0.2L of 18S primer and 2 of cDNA, and was brought to a total volume of 1 L with nucleasefree H2O. The thermal cycling conditions involved an initial denaturation step at 95C for 20 seconds followed by 40 cycles of 95C for 1 second and 60C for 2 0 seconds.
42 Immunofluorescence As previously mentioned, both human and mouse t issues were embedded in paraffin and sectioned to a thickness of 5embedded sl ides was performed by xylene immersion and subsequent dehydration in ethanol. Antigenretrieval was performed in 25 m M Tris/EDTA buffer, pH 9.1 After sections were blocked with donkey serum, they were incubated overnight at 4C with rabbit anti mouse ROR yt (1/100 dilution, ab78007, Abcam, Cambridge, MA), goat anti mouse sc1127, Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and rat anti mouse NKp46 (1/100 dilution, #137602, Biolegend, San Diego, CA) in Dako A ntibody D iluent (#S0809, DAKO Corporation, Carpinteria, CA) Isotype controls were performed equal volume of antibody diluent only. Secondary antibodies were added one at a time and incubated for 1 hr, with 3X5 min washes in between: Alexa Fluor 488 donkey anti rabbit IgG (1/100 dilution, A 21206, Molecular Probes Eugene, OR ), Alexa Fluor 594 donkey anti rat IgG (1/100 dilution A 21209, Molecular Probes Eugene, OR ), and Cy 5 conjugated Affinity Pure rabbit anti goat IgG (1/100 dilution, 305175003, Jackson ImmunoResearch Laboratories, Inc. Westgrove, PA ) One drop of Vectashield Mounting Medium for fluorescence with DAPI ( H 1200, Vector Laboratories, Inc., Burlingame, CA ) was added to each slide before cover slip was added. Images were taken using the Zeiss Axiovert 200M microscope with the AxioCam MR camera (Carl Zeiss Microimaging, Inc., Thornwood, NY). Determination of IL22 Levels in Sera and Saliva Luminex For human samples, m easurements of IL22 in serum and saliva samples were performed using the human IL22 LE GENDplex custom cytokine panel (BioLegend,
43 San Diego, CA). All procedures were performed according to the manufacturers instructions. Briefly, cytokine standards and samples were mixed in appropriate buffer and added to each well of a 96well filter plat e. Sonicated antibody conjugated beads were added to each well and incubated overnight in the dark at 4 C. Following 2 washes with wash buffer, 25 L of detection antibody was added to each well and incubated for 60 min at room temperature, followed by inc ubation with 25 L of Streptavidin Phycoerythrin solution for 30 min at room temperature. All readings were carried out using the Luminex 200 System (Luminex Austin, TX). Data was analyzed using MILLIPLEX Analyst software (Millipore, Billerica, MA) with standard curve generated from 0.64 to 2000 pg/m L ELISA For mouse samples, measurements of IL22 in serum and saliva were obtained using the Mouse IL22 ELISA Ready SET Go! reagent set (eBioscience, San Diego, CA). All procedures were performed according to the manufacturers instructions. ELISA plate was coated with capture antibody and incubated overnight in the dark at 4C. After washing five times with wash buffer (1X PBS, 0.05% Tween20) wells were blocked with 1X Assay diluents and incubated at room temperature for 1 hr. Following five washes, standards and samples were added to each well and allowed to incubate overnight at 4C. Wells were washed 5 times with wash buffer and detection antibody was added and allowed to incubate at room temperat ure for 1 hr. After 5 washes, Avidin HRP was added and wells were incubated at room temperature for 30 min. Wells were then washed 7 times and substrate solution was added followed by 15 min incubation. Stop solution (1 M H3PO4) was added and plate was read in ELISA plate
44 reader at 450 nm with values at 570 nm subtracted. Standard curve was generated from 8 to 1000 pg/mL. Flow Cytometry Flow cytometric analysis was performed on harvested spleen, thymus, lymph node, and peritoneal exudate cells from C57Bl/6J and C57Bl/6.NOD Aec1R1Aec2 mice at the previously mentioned time points. Spleen, thymus, and lymph node cells were obtained by extraction through wire mesh and were washed in 1% PBS. Spleen cells were treated with ammonium chloride to remove red blood cel l contamination. Cells from each organ were stained for three sets of cell surface markers: FITC rat anti mouse NKp46 ( #560756), PE hamster anti mouse CD3), PECy7 rat anti mouse CD11b ( #552850), and APC hamster anti mouse CD27 ( #560691), or; FITC rat anti mouse NKp46, PE hamster anti Cy7 rat anti mouse B220 ( #552772), and APC rat anti mouse CD4 (#553051), or; FITC rat anti mouse NKp46, PE hamster anti PE Cy7 rat anti mouse CD11b, and APC rat antimouse CD4 (BD Bios ciences, San Jose, CA) Positive color controls were created by staining spleen cells in separate wells with anti mouse CD4 in FITC, PE, PECy7, and APC. Negative color control was created using unstained cells of each cell type. Flow cytometric data acqui stition was performed on the Accuri C6 Flow Cytometer (Accuri Cytometers, Inc., Ann Arbor, MI) and data analysis was performed using FlowJo Flow Cytometry Analysis Software (Tree Star, Inc., Ashland, OR) Mitochondrial Membrane Potential Assay Human saliv ary gland cancer (HSG) cells were plated at a concentration of 100,000 cells/m L in 500 l of Dulbeccos Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), and 1% Penicillin/Streptomycin. HSG cells were then subject to
45 treatment with IL 22 cytokine (#782IL/CF, R&D Systems, Minneapolis, MN) at a concentration of either 0, 1, 3, 20, 30 or 100 ng/m L Time points under observation were 2, 6, 24, and 48 hr. Using the Mitochondrial Membrane Potential Kit (#28000251, Agilent Technologies, Inc., Santa Clara, CA), a t each time point, 1X JC 1 was added and incubated for 15 min at 37C. JC1 was removed, and cells were washed with 1X Assay Buffer. After removal of buffer, PBS was added and cells were exami ned via fluorescence microscopy using the Zeiss Axiovert 200M microscope with the AxioCam MR camera (Carl Zeiss Microimaging, Inc., Thornwood, NY). MTT Assay Human salivary gland cancer (HSG) cells were plated at a c oncentration of 100,000 cells/mL in 500 L of Dulbeccos Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), and 1% Penicillin/Streptomycin. HSG cells were then subject to treatment with IL 22 cytokine (#782IL/CF, R&D Systems, Minneapolis, MN) at a concentration of either 0, 10, 50, 100 or 500 ng/m L with three wells for each time poi nt (6, 24, and 48 hr). After 2 hr, 20 of MTT solution (M 0283, Sigma) was added. After 4 hr, 100 L of supernatant was discarded from each well and 150 L MTT solvent (M 0408, Sigma) was added to dissolve purple MTT crystals. Contents from each well wer e transferred completely to an ELISA plate and absorbance values were read using an ELISA plate reader at wavelength 570 nm, with background absorbance at 655 nm subtracted. Cell Cycle Arrest Assay Human salivary gland cancer (HSG) cells were plated at a c oncentration of 100,000 cells/m L in 500 L of Dulbeccos Modified Eagle Medium (DMEM), 10% fetal bovine serum (FBS), and 1% Penicillin/ Streptomycin HSG cells were either left
46 untreated or were treated with IL22 (#782IL/CF, R&D Systems, Minneapolis, MN ) at a concentration of 100 ng/ L Cells were allowed to proliferate for 72 hr at 37C with 5% CO2 and were then treated with Vybrant DyeCycle Ruby stain according to manufacturers instructions (V10273, Invitrogen, Carlsbad, CA) adding 1 L of Vybrant Dy eCycle Ruby stain to HSG cells at a concentration of 5 x 105 cells/m L at a volume of 0.5 m L HSG cells were incubated with stain for 30 min at 37C protected from light. Cells were analyzed on Accuri C6 Flow Cytometer (Accuri Cytometers Inc., Ann Arbor, MI) and data analysis was performed using FlowJo Flow Cytometry Analysis Software (Tree Star, Inc., Ashland, OR). Statistical A nalyses Statistical evaluations were determined using the MannWhitney U test Linear regression analysis was performed for linear correlation between IL22 levels and saliva flow, focus score, or disease duration using Spearman r test to obtain the r and p values. The onetailed p value <0.05 was considered significant. Data are presented as mean SEM. Statisti cal analyses and graphs were generated by the GraphPad InStat and Prism software s, respectively ( GraphPad Software, La Jolla, CA) For murine studies, all statistics were performed using the student unpaired twotailed t test. The two tailed p value<0.05 w as considered significant.
47 CHAPTER 3 RESULTS Expression of IL 22 and IL22R 1/2 in Labial Salivary Gland Tissues of pSjS Patients A major hallmark of pSjS is the presence of lymphocytic foci (LF) in minor labial salivary gland (LSG) biopsies (118) Occassionally, these LF organize into germinal center (GC)like structures composed predominatly of CD4+T cells and CD20+ B cells, but can also contain plasma cells, dendritic cells (DC s), natural killer (NK) cells and/or macrophages (119, 120) Recent findings have identified the presence of TH17 cells which, on activation by DCs, subsequently secrete their signiture cytokine, IL17A (30, 31) However, TH17 cells also produce IL22. Interestingly, to date, neither IL22 nor its receptor, IL 22R 1 have been characterized in salivary g lands even though IL22 receptors are expressed on many epithelial cell populations To determine if IL22 and/or IL22 receptors could be detected in LSG tissues of pSjS patients, an immunohistochemical analysis was carried out on histological sections of 11 patients. It should be pointed out that LSG biopsies were acquired only from patients who were not receiving corticosteroids, as such therapy could affect levels of inflammation and alter patient profiles. Thus, the number of biopsies tested were fewer than the number of patients participating in the overall study. As shown in Figure 31A positive staining for IL 22 was confirmed within the infiltrating leukocytic populations, as well as on ductal cells. Weak positive staining was also observed within the myoepithelial cells surrounding the acinar cells, while acinar cells per se appeared to show minimal, if any, staining. A pproximately 45% of the inflammatory cells within the LF proved positive for IL22 (Figure 31B ) Similarly, staining for the IL 2 2 receptor revealed that ILwas also highly expressed
48 unexpectedly within the leukocytic infiltrations of the LSG tissues of pSjS patients, but also on ductal and myoepithelial cells (Figure 31A ). Quantitatively, approximately 56% of the infiltrating cells appeared to express IL22R (Figure 31C) These data indicate that infiltrating lymphocytic cells in LSG in pS j S are significantly positive for IL22 and IL2 2R Expression of NKp46+, CD56+, and LTi like Ce lls in LSG T issues of pS j S Patients The source of IL22 production is generally thought to be TH17 cells, however IL22 can also originate from a subset of NK cells and LTi cells (47, 121, 122) To determine if NK cells were present within the lymphocytic foci of SjS patients, immunohistochemis try was used, staining for two common NK cell markers, NKp46 and CD56. CD56 positive cells include activated T cells and NK cells, while NKp46 positive cells include resting and activated NK cells, but not T or B cells. NKp46 is one of the natural cytotoxi city receptors which recognize tumor targets and specific types of virus (123, 124) It is expressed in all NK cells of various mouse strains, but only expres sed by a certain subset of NK cells in humans. Precursors and progenies of NKp46+ cells are thought to contribute significantly to the production of IL22 (51) Therefore, to determine if NKp46 was present in the LSG of pSS patients, immunohistochemical analysis was conducted. As shown in Figure 3 8A NKp46 is predominantly expressed by infiltrating leukocytes in the LSG, regardless of the size of the LF, with an average of approximately 8.7% of total infiltrates (quantified in Figure 38B) Also shown in Figure 3 8A CD56 is also mainly expressed by infiltrating leukocy tes in the LSG, regardless of the size of the LF, with an average of approximately 6.3% of total infiltrates (quantified in Figure 38B) Further analysis revealed that NKp46+ cells were also positive for with CD3 marker ( Figure 8C ). As a
49 result, several unique cell populations have been identified in the LSG of pS j S patients +CD3-NKp46++CD3+NKp46++CD3-NKp46-. Elevated L evels of S erum IL22 and S ignifciant C orrelation with S aliva F low in pS j S Patients To determine the systemic levels of IL22 present in saliva and sera of pS j S patients, saliva and sera samples from 31 pS j S patients and 17 heal thy control volunteers were each assayed for IL22 leve ls using the Luminex platfor m. As presented in Figure 3 10A l evels of IL22 in salivas of pS j S patients and control subjects did not differ significantly, with most salivas containing level s below detection of the assay. In Figure 310B, however, IL 22 levels in sera of patients proved to be significantly higher than those of control subjects (18. 402.24 pg/m L vs. 12.852. 76 pg/m L p=0.0389). More importantly, signficiant correlation was found between IL22 levels in sera with each participants corresponding saliva flow, most specif ically in patients with lower saliva flow tending to have higher levels of serum IL22 (Figure 311A) No statistically significant correlation was obtained between IL22 levels and focus scores or estimated disease durations of the patients (Figure 3 11 B,C). IL 22 Levels in S era C orrelate with M ajor P arameters of pS j S Patients participating in this study have to date been evaluated for as many as 34 different clinical parameters associated with pS j S in addition t o saliva flow (Table 3 1). To determine if any correlations exist between clinical parameters and IL22 levels, patients were separated into negative and positive groups for each specific clinical examination parameter. As presented in Figure 312A E, comparing the levels of serum IL 22 within t he two groups for eachclinical parameter revealed dramatically higher levels of IL22 in positive groups and statistically significant correlations between the
50 positive and negative groups for anti SSB/La (11. 3753. 149 vs 21. 6833. 722, p=0. 0175), anti SSA/ Ro /SSB/La combined (8 8151. 874 vs 22. 5964. 370, p=0. 0367), rheumatoid factor (RF) (9. 2572. 033 vs 22. 1914. 049, p=0. 0225), and hypergammaglobulinemia (13. 3502. 547 vs 26. 0685. 954, p=0. 0333), but not with anti SSA/Ro alone. These correlation analyses clearly support a close association between the levels of IL22 and pS j S patients who are positive or negative for anti SSB/La, both anti SSA/Ro /SSB/La combined, RF, and hypergammaglobulinemia in the diagnosis of pS j S. Ex pression of IL 22 and IL22R 1 in Organs of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mice Upon determining the presence of IL22 within the salivary glands of human SjS patients, it became necessary to establish whether or not the cytokine was upregulated within SjS mouse models. The C57BL/6.NOD Aec1R1Aec2 strain was chosen for said examination due to its high phenotypic homology to the human disease, as it could be argued that this model is the most representative model to date for human SjS. Therefore, it is im perative to our understanding of SjS disease development and progression with respect to IL22 to have a mouse model which exhibits similar expression patterns of the cytokine. C57BL/6 mice were used as comparative normal controls. To determine if IL22 and/or IL 22 receptors could be detected in salivary gland tissues of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice, an immunohistochemical analysis was carried out on histological sections of 3 males and 3 females at each of the abovementioned timepoints Negative isotype controls were performed using rabbit IgG (Figure 36A). As shown in Figure 36B at all time points for both C57BL/6.NOD -
51 Aec1R1Aec2 and C57BL/6 mice there is weak to no positive staining for IL22 within the infiltrating leukocytic populat ions. P ositive staining for IL 22 was apparent on ductal cells and w eak positive staining was observed within the myoepithelial cells surrounding the acinar cells, with minimal to no staining on acinar cells S taining for the IL2 2 receptor revealed that I L corrected here to not cross react with IL) was also not expressed within the leukocytic infiltrations of the salivary glands but was highly positive on ductal and myoepithelial cells (Figure 31A ). These data indicate that infiltrating lymp hocytic cells in salivary glands of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice are negative for IL 22 and IL2 2R does not represent the human condition. To determine whether IL22 or IL rgans of the SjS mouse model, we examine the lacrimal glands, spleen, thymus, and lymph nodes. Similar observations were made in the lacrimal glands of both C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Again, negative isotype controls were performed using rabbit IgG (Figure 37A). As shown in Figure 37B at all time points there is weak to no positive staining for IL 22 within the infiltrating leukocytic populations for both strains of mice. P ositive staining for IL 22 was apparent on ductal cells and w eak pos itive staining was observed within the myoepithelial cells surrounding the acinar cells, with minimal to no staining on acinar cells S taining for IL showed no expression within the leu kocytic infiltrations but was highly positive on ductal and myoepithelial cells (Figure 3 7B ). Therefore, infiltrating lymphocytic cells in the lacrimal glands of both C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice are negative for IL 22 and its receptor
52 Upon examining the spleen for IL22 (isotype controls shown in Figu re 38A), strong positive staining was revealed for the cytokine within the white pulp in the periarteriolar lymphoid shield (PALS), composed of lymphocytes and concentric layers of reticular fibers and flattened reticular cells (Figure 38B) This finding is understandable as the majority of cells within the PALS are CD4+, indicating a possible Th17 or Th22 phenotype and IL22 secretion. No significant staining was observed in the red pulp, follicles or marginal zone. Very weak to no staining for ILwas apparent in both strains at all time points regardless of gender, with no changes seen over time or between genders or strains (Figure 38C) Again, staining was visible within the PALS region however, due to the very minimal staining and the known c omposition of PALS to be T cells and macrophages, it is highly unlikely that the IL22 receptor could be present within this region of the spleen. Therefore, this staining is likely representative of high background. The thymus was then examined for the presence of IL22 and ILNegative isotype controls are shown (Figure 39A). Cells positive for IL22 were found scattered within both the medulla and the cortex of the organ (Figure 39B) As observed in the spleen, staining within the thymus was again apparent in both strains at all time points regardless of gender, with no changes seen over time or between genders or strains. Minimal to no staining was observed for ILfor both gender s and strains (Figure 39C) Lastly, the presence of IL22 and ILwas examined within the cervical lymph nodes of both the SjS mouse model and the normal controls. Again, negative isotype controls are provided (Figure 310A). With a similar staining pattern to that seen in the thymus, cells positive for IL22 were found
53 scattered within both the medulla and the cortex of the organ (Figure 310B) Staining within the lymph node was again apparent in both strains at all time points regardless of gender, with no changes seen over time or between genders or strains. Minimal to no staining was observed for IL(Figure 310C) These findings suggest that while IL22 is not present within the salivary glands (as observed in human SjS patients) or the lacrimal glands, it is present within other lymphoid organs such as the spleen, thymus, and cervical lymph nodes. However, due to the presence of IL22 in both the SjS mouse model and the normal control mice, it is unlikely that this cytokine plays a role in the disease process observed in the murine model. Absent mRNA expression of IL 22 in LSG of C57BL/6.NOD Aec1Aec2 and C57BL/6 Mice Upon performing real time PCR analysis within the whole salivary glands of C57 BL/6.NOD Aec1Aec2 and C57BL/6 mice for IL22 expression, it was apparent that no IL22 was present in the glands of either strain in both genders at all time points (data not shown). This again contradicts the human condition in which PCR analysis revealed upregulated IL22 expression in SjS patients when compared to controls. The real time PCR reaction was repeated for 40 cycles, at which point none of the samples produced CT values. Each sample had an 18S internal control which produced expected CT values (between 89), eliminating the possibility of cDNA levels being too low for sufficient analysis. The results confirm previous histology findings, indicating that IL22 is absent from the salivary glands of this SjS mouse model.
54 Expression of NKp46+ and LTi like Cells in LSG T issues of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mice In human SjS patients, the presence of NKp46+ subpopulations with phenotype s +CD3-NKp46++CD3+NKp46++CD3-NKp46was confirmed within the salivary gland tissues. To determine if these subpopulations were present in the C57BL/6.NOD Aec1R1Aec2 mouse model, similar immunofluorescence stainings C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice are shown (Figure 311). It was found that in C57BL/6.NOD Aec1R1Aec2 mice at 8 wks of age, +CD3+NKp46+ cells were present, albeit in low quantities (approximately 10 per section). At 20 wks of age, a small subset of +CD3-NKp46+ cells was apparent within LF. Neither population +NKp46+ IL 22producing subsets in mouse salivary glands was significantly lower than that observed within human SjS patients, p roviding a possible explanation as to why the mouse model does not exhibit overexpression of IL22. Lack of Saliva and S erum IL22 in C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mice To determine the systemic levels of IL22 present in saliva and sera of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice, saliva and sera samples from 3 males and 3 females from time points 4, 8, 12, 16, and 20 wks were each assayed for IL22 levels by ELISA Due to values being identical between genders, male and female data was pooled to give n=6 for each time point. As presented in Figure 3 1 2 A l evels of IL22 in salivas of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 were below detection level in all cases Futhermore, the absorbance readings for all samples were comparable to the absorbance reading obtained for a blank well, indicating that no IL22 was present
55 within the saliva and not the possibility of a low cytokine level not detected by the assay. In Figure 31 2 B the same observation is made for the sera of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice, with IL 22 being completely absent from the sera of both disease and control models. This data supports previous findings of IL22 being absent from the salivary glands of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. It also further dichotomizes the C57BL/6.NOD Aec1R1Aec2 SjS mouse model from the human disease, as human SjS patients present with increased levels of serum IL22 when compared to healthy controls. Analysis of IL 22 Producing Cell Populations within C57BL/6.NOD Aec1R1Aec2 and C57BL/6 Mi ce Flow cytometric analysis was performed to determine the presence of lesser known IL22producing cell populations such as NKp46+CD3-CD11bhigh/lowCD27high/low NK cells and CD4+CD3-NKp46-CD11bLTi cells. Using 3 males and 3 females from both C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20, 24 wk) and normal control C57BL/6 (4, 8, 20 wk) mice, lymphocyte populations from spleen, thymus, cervical lymph nodes, and peritoneal exudate cells were analyzed for these markers. An example of gating for each cell populations within each organ is provided in Figure 313A H. Analysis of splenocyte population for NKp46+CD3-CD11bhigh/lowCD27high/low NK cells shows several significant differences between SjS mouse models and controls, as well as between males and females. Figure 314A provides an overall comparison of the total population of NKp46+CD3NK cells within the spleen, regardless of their maturity. In Figure 314B, each population of NKp46+CD3NK cells in the spleen is broken down according to their expression of CD11b and CD27, where from most mature to least mature: CD11bhighCD27low, CD11bhighCD27high, CD11blowCD27high, CD11blowCD27low. It was
56 found that in C57BL/6.NOD Aec1R1Aec2 male mice, there is significant decrease in total NKp46+CD3NK cells from 4 to 24 wks of age (p= 0.0003), with a significant decline in the number of CD11b+CD27+ NK cells (p= 0.0042). Also, a significant drop in total NK cells is seen from 12 to 16 wks of age (p= 0.0012) with again a significant decli ne in the number of CD11b+CD27+ NK cells (p= .0012). Female C57BL/6.NOD Aec1R1Aec2 mice do not observe a significant difference in total NK cells over time, however, from 4 to 24 wks there is a statistically significant decline in the population of CD11b+CD 27+ NK cells (p= 0.0008). Male C57BL/6 controls noticed a significant increase in total NK cells from 4 to 8 wks (p= 0.0080) and a decline from 8 to 20 wks (p= 0.0207), with respective rise in CD11b+CD27+ NK subpopulations from 4 to 8 wks (p= 0.0095) and drop from 8 to 20 wks (p= 0.0026). Among female C57BL/6 mice, no significant changes in NK cell populations were observed over time. Upon comparison of male C57BL/6.NOD Aec1R1Aec2 to C57BL/6 mice, male controls exhibited a significantly higher level of CD11b+CD2 7NK subpopulations (p=0.0180) at 4 wks Subsequent comparison of female C57BL/6.NOD Aec1R1Aec2 to C57BL/6 mice revealed no significant differences between strains. Finally, performing statistical analyses between genders of same strain mice showed that in C57BL/6.NOD Aec1R1Aec2 mice males exhibited higher total NK cell percentages at 4 wks (p=0.0225) and 8 wks (p=0.0099), as well as with significantly higher levels of CD11b+CD27NK subsets at 8 wks (p=0.0249) when compared to females. In C57BL/6 mice, males presented with higher total NK cell percentages at 4 wks (p=0.0220) and 8 wks (p=0.0016), with significantly higher levels of CD11b+CD27NK subsets at 4 wks (p=0.0155) and 8 wks (p=0.0077) when compared to females.
57 Analysis of thymocyte population f or NKp46+CD3-CD11bhigh/lowCD27high/low NK cells showed minimal differences between SjS mouse models and controls, as well as between males and females. Figure 315A provides an overall comparison of the total population of NKp46+CD3NK cells within the th ymus regardless of their maturity Interestingly, the thymus contained the lowest percentage of total NK cells compared with other organs analyzed. In Figure 315B, each population of NKp46+CD3NK cells in the thymus is separated based on their expression of CD11b and CD27. It was found that in C57BL/6.NOD Aec1R1Aec2 male and female mice, there is no significant difference in total NKp46+CD3NK cells over time and the subpopulations of NK cells also do not differ significantly with age. Male and female C 57BL/6 controls also showed no statistically significant differences in total NK cells or NK cell subpopulations at different time points. Comparison of male C57BL/6.NOD Aec1R1Aec2 to C57BL/6 mice gave no significant differences, however, comparison of fem ale C57BL/6.NOD Aec1R1Aec2 to C57BL/6 mice revealed a significantly higher number of NKp46+CD3NK cells at 20 wks in the SjS model (p= 0.0335). Finally, performing statistical analyses between genders of same strain mice showed that in both C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice no significant differences were apparent. Cell surface marker staining of cervical lymph node lymphocyte population for NKp46+CD3-CD11bhigh/lowCD27high/low NK cells shows several significant differences between SjS mouse models and controls, as well as between males and females. Figure 316A shows an overall comparison of the total population of NKp46+CD3NK cells within the lymph nodes. I n Figure 316 B, four subpopulations of NKp46+CD3NK cells in the lymph nodes are shown bas ed upon maturity level. In both C57BL/6.NOD -
58 Aec1R1Aec2 and C57BL/6 male and female mice, there is no significant change in total NKp46+CD3NK cells between time points. Upon comparison of both male and female C57BL/6.NOD Aec1R1Aec2 to male and female C57B L/6 mice, respectively no differences were observed. Finally, performing statistical analyses between genders of same strain mice showed that in C57BL/6.NOD Aec1R1Aec2 mice, males exhibited higher total NK cell percentages at 8 wks (p=0.0.0215) and had si gnificantly higher levels of CD11b+CD27NK subsets at 8 wks (p=0.0254) when compared to females. In C57BL/6 mice, males presented with higher total NK cell percentages at 20 wks (p=0.0424), with significantly higher levels of CD11b+CD27NK subsets at 8 w ks (p=0.0123) and 20 wks (p=0.0424) when compared to females. Cell marker analysis of lymphocytes within peritoneal exudate for NKp46+CD3-CD11bhigh/lowCD27high/low NK cells shows no significant differences between SjS mouse models and controls, but signifi cant differences between males and females. Figure 317A provides an overall comparison of the total population of NKp46+CD3NK cells within the peritoneal exudates and Figure 314B shows the subpopulations of NKp46+CD3NK cells according to their expres sion of CD11b and CD27 to determine maturity It is interesting to note that total NK cell percentages were the highest in the peritoneal exudate cells when compared with the spleen, thymus, or lymph nodes It was found that in C57BL/6.NOD Aec1R1Aec2 male mice, there is no significant decrease in total NKp46+CD3NK cells from 4 to 24 wks of age but there is a significant decline in the number of CD11b+CD27+ NK cells (p= 0.0175). Also, a significant drop in the percentage of CD11b+CD27+ NK cells occurred from 12 to 16 wks (p= 0.0454). Female C57BL/6.NOD Aec1R1Aec2 mice do not observe a significant difference in total
59 NK cells over time. Male C57BL/6 controls noticed a significant decrease in total NK cells from 8 to 20 wks (p= 0.0079) while in female C57BL/6 mice, no significant changes in NK cell populations were observed over time. No difference was observed between disease mouse models and normal controls for either males or females. Finally, performing statistical analyses between genders of same strain m ice showed that in C57BL/6.NOD Aec1R1Aec2 mice males had a significantly higher percentage of total NK cells at 4 wks when compared to females (p= 0.0125) and in C57BL/6 mice, males showed significantly higher levels of total NK cells at 8 wks when compared to females (p= 0.0026). Lymphocytes from each tissue were then analyzed for CD4+CD3-NKp46-CD11bLTi cells (Figure 318) Analysis of splenocyte population for CD4+CD3-NKp46-CD11bLTi cells revealed significant differences between SjS mouse models and controls, as well as between males and females. In male C57BL/6.NOD Aec1R1Aec2 mice, a statistically significant decrease in CD4+CD3-NKp46-CD11bLTi cells occurs from 4 to 20 wks (p= 0.0079), 4 to 24 wks (p= 0.0460), and 12 to 16 wks (p= 0.0218) while an increase occurs from 20 to 24 wks (p= 0.0315). In female C57BL/6.NOD Aec1R1Aec2 mice a decline in LTi cells is noted from 4 to 8 wks (p= 0.0184), 4 to 20 wks (p= 0.0242), and 12 to 16 wks (p= 0.0414), while a marked increase is seen from 8 to 12 wks (p= 0.0188). Male C57BL/6.NOD Aec1R1Aec2 mice demonstrate a significantly higher level of LTi cells within the spleen when compared to females at 8 wks (p= 0.0041) as do male C57BL/6 mice when compared with females (p= 0.0150). Staining for CD4+CD3-NKp46-CD11bLTi cells in the thymus also revealed significant differences between SjS mouse models and controls, as well as between
60 males and females. In C57BL/6.NOD Aec1R1Aec2 mice, a statistically significant decrease in CD4+CD3-NKp46-CD11bLTi cells occurs from 12 to 16 wks in males (p= 0.0033) and in females (p= 0.0017). Male C57BL/6.NOD Aec1R1Aec2 mice demonstrate a significantly higher level of LTi cells within the thymus when compared to C57BL/6 males at 4 wks (p= 0.0134). Also, male C57BL/6.NOD Aec1R1Aec2 mice show significantly lower percentages of LTi cells at 8 (p= 0.0237) and 16 wks (p= 0.0293) when compared to females. Lymphocyte analysis for CD4+CD3-NKp46-CD11bLTi cells in the lymph nodes also gave significant differences between SjS mouse models and controls, as well as between males and females. In C57BL/6.NOD Aec1R1Aec2 mice, a statistically significant decrease in CD4+CD3-NKp46-CD11bLTi cells occurs from 4 to 8 wks (p= 0.0134) in and 4 to 20 wks (p= 0.0357) in females. Male C57BL/6.NOD Aec1R1Aec2 mice demons trate a significantly lower level of LTi cells within the lymph nodes when compared to females at 24 wks (p= 0.0339). Also, at 4 wks female C57BL/6.NOD Aec1R1Aec2 mice show significantly higher percentages of LTi cells when compared to C57BL/6 controls at 4 wks (p= 0.0255) Analysis of peritoneal exudate cells showed no significant differences in LTi population percentages among disease models and normal controls or between sexes. IL 22 Induces Cell Cycle Arrest at the G2 M Phase of the Cell Cycle To investigate the functional effects of IL22, an in vitro model using human salivary gland cancer (HSG) cells was employed. First, the mitochondrial membrane potential assay (MMP) was conducted to observe if IL22 induced apoptosis in HSG cells (Figure 319A) HSG cells were treated with either 0, 1, 3, 10, 30, or 100 ng/mL of IL 22 and cells were examined via fluorescence microscopy after 2, 6, 24, and 48 hrs.
61 Green cells represent apoptotic cells while red cells or red and green cells represent live cel ls. Upon examination, it was revealed that minimal to no apoptotic cells were visible and therefore IL22 was not inducing apoptosis in HSG cells. However, as IL22 concentration increased beyond 30 ng/mL the number of cells in each well was comparable to that seen at time point 0 hr (image not shown), indicating instead that a halt in cell proliferation was occuring in wells with IL22 concentrations of 30 and 100 ng/mL starting at the 6 hr time point. This experiment was repeated using flow cytometry with FL1H detecting green apoptotic cells and FL2H detecting red living cells (Figure 319B). HSG cells were treated with higher concentrations of IL22 (0, 10, 50, 100, and 500 ng/mL) and analyzed after 24 and 48 hrs. Percentages of green, apoptotic cells r emained constant throughout all IL 22 concentrations at both time points, which confirms the previous visual MMP assay data, indicating that IL22 does not induce apoptosis within HSG cells. To observe the effects of IL22 on proliferation of HSG cells, the MTT proliferation assay was performed (Figure 320). HSG cells were treated with IL22 at concentrations of 0, 10, 50, 100, or 500 ng/mL and absorbance measurements were taken at 6, 24, and 48 hrs after treatment. Higher absorbance values indicate a higher number of proliferating cells. Results show that after IL22 treatment for 6 hrs, no significant difference in the number of proliferating cells was observed. The same observation is true at 24 hrs of IL22 treatment. However, after 48 hr significant di fferences are seen in proliferation of HSG cells that were untreated when compared to HSG cells treated with IL 22 at 10 (p= 0.0138), 50 (p= 0.0167), 100 (p= 0.0247), and 500 (p= 0.0092) ng/mL. These results indicate that not only is the concentration of IL22 relevant to decreased
62 proliferation of HSG cells, but the length of time that treatment is provided is also crucial. To determine at which phase the cell cycle is arrested in HSG cells treated with IL22, a cell cycle arrest assay was performed using Vybrant DyeCycle Ruby stain. HSG cells were either treated with 100 ng/mL of IL22 for 72 hrs or left untreated and then analyzed via flow cytometry. Analysis revealed that 19.7% of untreated HSG cells were in the G2M phase of the cell cycle compared to 27.4% of IL 22 treated HSG cells. These results indicate that treatment of HSG cells with IL22 causes a halt in cell proliferation which occurs at the G2M phase of the cell cycle.
63 Table 3 1. Basic laboratory tests, procedures, extraglandular manifestations, and oral findings Participants Age (yr) Sex Race Duration (yr) Clinical tests & procedures pS j S (n=31)* 2479 F** (n=31) Caucasian (n=29) Asian (n=1) Hispanic (n=1) 4 27 ANA profiles: ANA, ANA titer, antiSSA/Ro60, antiSSB/La, anti dsDNA, RF, anti centromere, anti CCP, antiRNP, antithyroid, as appropriate, and CBC, ESR, and Schirmer's test. Manifestations: Peripheral /Cranial Neuropathy, Chronic Bronchitis, Lymphoma, Interstitial nephritis, Hypergammaglobulinemia, Autoimmune Thyroiditis, Interstitial cystitis, RT acidosis, Joint inflammation, Pleural Involvement, Liver Disease, GERD, Fibromyalgia, Cognitive impairment. Oral: Salivary gland enlargement, Unstimulated whole sialometry, Labial salivary gland biopsy and focus score, tobacco his tory. Controls (n=17)** 22 67 F (n=16) M (n=1) Caucasian (n=11) Hispanic (n=4) African American (n=2) N/A N/A Not all pSjS patients were evaluated with the same panel of clinical tests. **Healthy volunteer controls who were randomly selected. ***Abbreviations: F: female, M: male, yr: year, ANA: antinuclear antibody, SSA/ Ro : SSA antigen/ Ro SSB/ La: SSB antigen/ La, RF: rheumatoid factor, CCP: cyclic citrullinated peptide, CBC: complete blood count, RNP: ribonucleoprotein, ESR : erythrocyte sedimentation rate, GERD: gastroesophageal reflux disease, N/A: not applicable.
64 A B C Figure 3 1: Expression of IL22 and ILpatients. Explanted labial salivary gland tissues from pSjS patients (n=11) were fixed and embedded in paraffin. Sections were cut to 5 m and stained using either anti IL 22 or anti IL or both antibodies were performed using rabbit IgG. Staining was developed by using DAB substrate and counterstaining was done with hematoxylin. Representative stained sections from patients ID#5 and ID#7 are shown with original magnification at 200X with insets showing the closeup of the square boxes (A). Average percentage of IL22 positive cells in LF (n=5) (B) and ILpositive cells (n=5) (C) are determined by manual counting.
65 A B C Figure 32. Expression of NKp46, CD56, and subpopulations of NKp46 cells in labial salivary glands of pSjS patients. Immunohistochemical analysis was performed on LSG biopsies of pS j S patients (n=11). Tissues were fixed and kness, and stained using anti -
66 NKp46 or anti CD56 Staining was developed using DAB substrate with hematoxylin counterstain. Isotype control was performed using rabbit IgG A) Representative sections from one S j S patient is provided at 200X magnification wi th insets showing the closeups of square boxes. B) Average percent of enumerated NKp46and CD56positive cells are presented. C) Immunofluorescence staining was performed on LSG biopsy from a pSjS patient (n=11). LSG biopsied sections from pSS patient stained with DAPI (blue), anti CD3 (purple), and anti NKp46 (green) is shown at 400X magnification. White arrow: RO+CD3-NKp46cells, red arrow: +CD3+NKp46+ +CD3-NKp46+ cells. Secondary antibodies alone were used as negative controls (data not shown).
67 A B Figure 3 3 IL 22 levels in saliva and sera samples of pS j S p atients Saliva (A) and s era (B) samples from pS j S patients (n = 31) or from healthy control volunteer subjects (n = 17) were tested for IL22 levels using the Luminex system. Horizontal bars represent the mean value within a group. indicates statistical significance using the MannWhitney U test.
68 A B C Figure 3 4 Correlation analysis of serum IL22 levels and saliva flow, focus score and disease duration. Positive correlation between IL22 levels and saliva flow was identified (p<0.05) in pSjS patients (n=31) (A), while no statiscial significant correlation was found between IL22 levels and focus score (n=16) (B) or disease duration (yrs) (n=24) (C). Both r and p values were identified using the Spearman r test. indicates statistical significance.
69 A B C D E Figure 3 5 Correlation between serum IL22 levels and clinical disease parameters in pSjS patients pSjS patients were evaluated for at least 35 clinical examination parameters. For this analysis, patients were separated into negative and positive groups for each cl inical parameter. Statistical comparison was determined for negative and positive groups of patients with serum IL 22 levels. Anti SSA/Ro (n=24) (A), anti SSB/La (n=24) (B), Anti SSA/Ro and anti SSB/La (n=16) (C), rheumatoid factor (n=22) (D), and hypergam maglobulinemia (n=24) (E).* indicates statistical significance using the MannWhitney U test.
70 A B C Figure 3 6 Expression of IL22 and ILC57BL/6.NODAec1R1Aec2 and C57BL/6 mice. Explanted salivary gland tissues from C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were fixed and embedded in paraffin. Sections were cut to 5 m and stained using either anti IL 22 or anti IL ntibodies were performed using rabbit IgG (A). Staining was developed by using DAB substrate and counterstaining was done with hematoxylin. Representative stained sections are shown with original magnification at 200X, stained for IL22 (B) and IL). indicates experiment not performed
71 A B C F igure 3 7 Expression of IL22 and ILC57BL/6.NODAec1R1Aec2 and C57BL/6 mice. Explanted lacrimal gland tissues from C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were fixed and embedded in paraffin. Sections were cut to 5 m and stained using either anti IL 22 or anti IL eveloped by using DAB substrate and counterstaining was done with hematoxylin. Representative stained sections are shown with original magnification at 200X, stained for IL22 (B) and IL). indicates experiment not performed.
72 A B C Figure 3 8 Expression of IL22 and ILC57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Explanted spleen tissues from C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were fixed and embedded in paraffin. Sections were cut t o 5 m and stained using either anti IL 22 or anti IL Isotype controls for both antibodies were performed using rabbit IgG (A). Staining was developed by using DAB substrate and counterstaining was done with hematoxylin. Representative stained secti ons are shown with original magnification at 200X, stained for IL22 (B) and IL* indicates experiment not performed.
73 A B C Figure 3 9 Expression of IL22 and ILAec1R1Aec2 and C57BL/6 mice. Explanted thymus tissues from C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were fixed using either anti IL 22 or anti IL Isotype controls for both antibodie s were performed using rabbit IgG (A). Staining was developed by using DAB substrate and counterstaining was done with hematoxylin. Representative stained sections are shown with original magnification at 200X, stained for IL22 (B) and IL* ind icates experiment not performed.
74 A B C Figure 3 10 Expression of IL22 and ILC57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Explanted lymph nodes from C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were fixed and embedded in paraffin. Sections were cut to 5 m and stained using either anti IL 22 or anti IL using DAB substrate and counterstaining was done with hematoxylin. Representative stained sections are shown with original magnification at 200X, stained for IL22 (B) and IL* indicates experiment not performed.
75 Figure 311 Immunofluorescence staining reveals subpopulations of NKp46+ cells within the salivary glands of C57BL/6.NOD Aec1R1Aec2 mice.Analysis was performed on salivary glands extracted from male C57BL/6.NOD Aec1R1Aec2 (n=3) and C57BL/6 (n=3) mice. Sections were stained with DAPI (blue), anti CD3 (purple), anti NKp46 (red), and anti RORt (green) and images were taken at 200X magnification. R ed arrow: +CD3+NKp46+ +CD3-NKp46+ cells. Secondar y antibodies alone were used as negative control with example shown of 20 wk C57BL/6.NOD Aec1R1Aec2 male.
76 A B Figure 3 1 2 IL 22 levels in saliva and sera samples of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Saliva (A) and sera (B) samples from C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice at 4, 8, 12, 16, and 20 wks (n=6) were tested for IL 22 levels by ELISA. Horizontal bars represent the mean value within a group. At all time points, IL22 was absent from both saliva and sera of SjS mouse models and normal controls.
77 A B C Figure 313. Flow cytometry gating examples. Gating for NKp46+CD3cells and their subsets (CD11b+/ -CD27+/ -) in lymphocyte populations harvested from spleen (A), thymus (B), lymph nodes (C), and peritoneal exudate (D) of 4, 8, 12, 16, 20, and 24 wk C57BL/6.NOD Aec1R1Aec2 mice and 4, 8, and 20 wk C57BL/6 mice in males (n=3) and females (n=3). Gating is also shown for CD4+CD3-NKp46-CD11bcells, representing LTi like cells, in spleen (E), thymus (F), lymph nodes (G), and peritoneal exudates (H) Example provided was conducted on a 4 wk male C57BL/6.NOD Aec1R1Aec2 mouse.
78 D E F Figure 313. (continued)
79 G H Figure 313 (continued)
80 A B Figure 3 14 NK cell populations present within the spleen of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Splenocytes from C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were treated with FITC conjugated rat anti mouse NKp46, PE conjugated hamster anti Cy7 conjugated rat anti mouse CD11b, and APC conj ugated hamster anti mouse CD27. The total NKp46+CD3NK cells are shown (A). The total NKp46+CD3NK cell population was then broken down into NKp46+CD3subpopulations (B), from most mature to least mature: CD11bhighCD27low, CD11bhighCD27high, CD11blowCD2 7high, CD11blowCD27low. *p<0.05, **p<0.01, ***p<0.005
81 A B Figure 3 15 NK cell populations present within the thymus of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Thymocytes from C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were treated with FITC conjugated rat anti mouse NKp46, PE conjugated hamster anti Cy7 conjugated rat anti mouse CD11b, and APC conjugated hamster anti mouse CD27. The total NKp46+CD3NK cells are shown (A). The total NKp46+CD3NK cell population was then broken down into NKp46+CD3subpopulations (B), from most mature to least mature: CD11bhighCD27low, CD11bhighCD27high, CD11blowCD27high, CD11blowCD27low. *p<0.05, **p<0.01, ***p<0.005
82 A B Figure 3 16 NK cell populations present within the lymph nodes of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Lymphocytes obtained from the lymph nodes of C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were treated with FITC conjugated rat anti mouse NKp46, PE conjugated hamster anti Cy7 conjugated rat anti mouse CD11b, and APC conjugated hamster anti mouse CD27. The total NKp46+CD3NK cells are shown (A). The total NKp46+CD3NK cell population was then broken down into NKp46+CD3subpopulations (B), from most mature to least mature: CD11bhighCD27low, CD11bhighCD27high, CD11blowCD27high, CD11blowCD27low. *p<0.05, **p<0.01, ***p<0.005
83 A B Figure 3 17 NK cell populations present within peritoneal exudates cells in C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Lymphocytes harvested from the peritoneal cavity of C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were treated with FITC conjugated rat anti mouse NKp46, PE conjugated hamster anti Cy7 conjugated rat anti mouse CD11b, and APC conjugated hamster anti mouse CD27. The total NKp46+CD3NK cells are shown (A). The total NKp46+CD3NK cell population was then broken down into NKp46+CD3subpopulations (B), from most mature to least mature: CD11bhighCD27low, CD11bhighCD27high, CD11blowCD27high, CD11blowCD27low. *p<0.05, **p<0.01, ***p<0.005
84 Figure 3 1 8 LTi like cell populations present within the spleen, thymus, lymph nodes, and peritoneal exudates cells of C57BL/6.NOD Aec1R1Aec2 and C57BL/6 mice. Lymphocytes harvested from the spleen, thymus, lymph node, and peritoneal cavity of C57BL/6.NOD Aec1R1Aec2 (4, 8, 12, 16, 20 wk) and C57BL/6 (4, 8, 20) mice were treated with FITC conjugated rat anti mouse NKp46, PE conjugated hamster anti Cy7 conjugated rat anti mouse CD11b, and APC conjugated rat anti mouse CD4. The total CD4+CD3-NKp46-CD11bLTi like cells are shown. *p<0.05, **p<0.01, ***p<0.005
85 A B Figure 3 19 IL 22 treatment of HSG cells results in halt in proliferation but not apoptosis. HSG cells were treated with IL22 and apoptosis was observed using the mitochondrial membrane potential assay. Apoptotic cells appear green and live cells appear as either red or red and green. (A) HSG cells were treated with IL22 at varying concentrations (0, 1, 3, 10, 30, or 100 ng/mL) and apoptosis was observed at the given time points (2, 6, 24, or 48 hr) via fluorescence microscopy. (B) HSG cells were treated with IL22 at varying concentrations (0, 10, 50, 100, or 500 ng/mL) and apoptosis was observed at the given time points (24 or 48 hr) using flow cytometry, where the green apoptotic cells are represented by the FL1H channel and the red live cells are represented by t he FL2H channel.
86 Figure 3 20 IL 22 treatment of HSG cells results in decreased proliferation. A total of 5x104 HSG cells were incubated overnight in a 24 well plate in DMEM/10% FBS/1% P/S. Cells were then treated with 0, 10, 50, 100 or 500 ng/mL of IL 22 for 6, 24, or 48 hr. At 4 hr before time point, MTT solution was added and at time point, MTT solvent was added to dissolve purple MTT crystals. Contents from each well were transferred to an ELISA plate and absorbance values were read using an ELISA plate reader at wavelength 570 nm, with background absorbance at 655 nm subtracted. *p<0.05, **p<0.01
87 A B Figure 3 21 IL 22 induces cell cycle arrest. A total of 5x104 HSG cells were incubated overnight in a 24 well plate in DMEM/10% FBS/1% P/S. Cells were then either treated with 1 00 ng/mL of IL 22 for 72 hours or left untreated. Analysis was performed upon treatment with Vybrant DyeCycle Ruby stain according to manufacturers instructions. (A) Cell cycle analysis showed an increased amount of cells in the G2M phase of the cell cycle, as compared with the untreated control. Unstained sample measurement s are also included. (B) Quantitative comparison of the percentage of cells within the G2 M phase for both untreated and treated cells.
88 CHAPTER 4 DI SCUSSION In the present study, it was investigated whether IL22 was present in the LSG of pSjS patients and whether the detected levels of IL22 in saliva and sera of pSjS patients might correlate with clincial manifestations used to classify pS j S. The C5 7BL/6.NOD Aec1R1Aec2 SjS mouse model was then examined to determine if it also demonstrated increased expression of IL22 in the salivary glands as well as in other relevant organs. Saliva and serum levels of IL22 were investigated and two lesser known IL22producing cell populations (NKp46+CD3-CD11bhigh/lowCD27high/low NK cells and CD4+CD3-NKp46-CD11bLTi cells) were examined for their presence in multiple tissues. Functional assays were then performed on HSG cells treated with IL22 to determine its ef fects on apoptosis and proliferation. Results indicate that in human SjS patients significant numbers of cells within the lymphocytic infiltrates, glandular ducts, and/or myoepithelial layers stained positive for both IL22 and its receptor, IL22R 1/2. At the same time, levels of IL22 were clearly elevated in the sera of pSjS patients, while only a minority of patients (4 of 31) had detectable levels in their salivas. Although several sera from participants used as controls in this study contained detectable levels of IL22, they remained lower than pSjS patients. Analyses to determine associations between serum IL22 levels and various clinical manifestations of pSjS revealed positive and statistically significant correlations between IL22 levels and sal iva flow, anti SSB/La, both anti SSA/Ro and anti SSB/La, RF, hypergammaglobulinemia, and autoimmune thyroiditis. Correlations with anti SSA/Ro alone and joint inflammation showed weak, but not statistically significant, associations.
89 An important aspect of S j S is the role B cells play in the development of disease (120, 125, 126) Although S j S is characterized by lym phocytic infiltrations in the salivary glands, a major component of interest is the formation of germinal center (GC) like structures that tend to correlate with disease progression and severity (119) These GC like structures contain immunoglobulins (i.e., anti SSA/Ro anti SSB/La, RF and anti musc arinic type III receptor antibodies) that, no doubt, contribute to the immunological assault against the acinar and ductal tissues, and at the same time act as biomarkers of disease status. A recently published study by Hsu et al. (127) has shown in an ani mal model of autoimmunity that IL17 is indispensable for the maintenance and formation of GC like structures. Similarly, Doreau et al. (128) have demonstrated that IL17 alone or in combinat ion with BAFF (B cells activating factor) can influence the survival, proliferation and differentiation of B lymphocytes, at the same time maintain the existence of self reactive B cells. It is interesting to note, then, that while both TH17 cells and NK cells can secrete both IL17 and IL22, this is also true of LTi cells whose functions include the formation of lymphoid organs and subsequent development of GCs (129131) Furthermore, Cella et al. (121) have recently shown that NK 22 cells can also secret BAFF. These fact s point to the possibility that the intercommunication and interaction of IL22 and IL17 might mediate B cell immunity through proliferation and survival of B lymphocytes, thereby contributing to the pathology and clinical outcome of S j S in both humans and animal models of S j S. This concept is supported indirectly by the current results wherein the IL22 levels correlate with those antibodies currently used as diagnostic markers of disease.
90 Whole saliva produced by salivary glands is a critical factor in oral health. S j S patients with dry mouth can manifest rampant dental caries, tooth decay and incr eased oral microbial infections Whole saliva contains a number of antimicrobial products, defensin, lysozyme, lactoferrin, lipocalins and mucins primarily MUC5b and MUC7 (120, 132) Interestingy, IL22 in conjunction with IL17, acts on targeted tissues defensins, lipocalins and S100 calcium binding proteins (55, 75, 88, 96) Furthermore, IL 22producing NKp46+ cells can contribute to epithelial cell resistance to injury from bacterial/viral infections or autoimmune attack mediated by pathogenic immune cells, as well as promote tissue remodeling (51) Therefore, the paradox of IL17 and IL22 needs to be resolved as well as chacterizing the immunological and tissue homeostatis i n order to fully understand the etiology of S j S Several seminal studies clearly demonstrated the presence of TH17 in labial salivary gland biopsies of S j S patients and signficantly elevated levels of IL17 in saliva and sera samples of S j S patients (30, 31, 133) We and others have proposed that IL17 directly contributes to the gross pathology and clinical outome of S j S, specifically the formation of LF in the salivary glands and the decrease in saliva flow in animal of models and human patients of S j S. Since then we have accumulated sufficient evidence to support th e hypothesis that IL17 directly involves in the pathogenesis of S j S (accepted manuscript). However, until now, there have been little or no data presented on whether IL22 also contributes to the pathology observed in S j S. One important aspect is the possibility that IL 22 acts as a potential counter balance to IL17, providing a possible protective role, as reported for some baterial/viral infections and
91 inflammatory bowel disease and hepatitis experimental animal models (109, 134, 135) Proliferation and apoptosis assays of HSG cells treated with IL22 in vitro demonstrated that the cytokine effectively halted the cell cycle at the G2M phase, stopping proliferati on when IL22 was presented at concentrations higher than 100 ng/mL for at least 24 hrs. It is likely that this occurred by IL22 downregulating the proproliferative signaling mediator ERK1/2 and AKT phosphorylation. Apoptosis was not induced by IL22 treatment, however. These results taken together indicate the therapeutic potential of IL 22 to be an inhibitor of tumor grow th, however, it is unlikely that IL22 would be able to inhibit lymphocytic infiltration growth as i mmune cells lack IL22R 1, and so c annot be targets of IL22, indicating that IL22 should not have an effect on a population of immune cells. However, u pon staining human LSG sections for ILstaining was observed which raised speculation as to the specificity of the anti body. Although blasting the antibody sequence against both IL22R 1 and IL22R 2 showed no overlap, further investigation of the sequence revealed high conservation of amino acids between binding sequences. Therefore, since it is known that IL22R 1 does not exist within immune cells, it can be assumed that the staining may be due to the presence of IL22R 2 which is known to be present in activated CD4+ T cells and in activated CD19+ B cells. Importantly, this leads one to question how IL22 may interact with IL 22R 2 within lymphocytic infiltrates. Studies have shown that IL 22R 2 does bind to IL22 and neutralizes IL22 activity in vitro It may be possible that instead of the typical IL22 receptor complex which consists of IL22R 1/IL 10R 2 a similar complex may be made from IL 22R 2 in combination with IL10R 2 or even with IL22R 2
92 triggers the migration of IL22R 2 for IL22 binding. In order to further investigate such a claim, costaining for CD4+ T cells and CD19+ B cells should be performed using an antibody specific for IL. It is also possible that IL22R 2 is instead acting as a decoy binding protein which the cells are secreting to neutralize the effects of I L 22, but this claim requires further examination. Notably, if IL 22 is able to bind to a receptor complex containing IL 22R 2 this would allow the cytokine to bind both activated CD4+ T cells and activated CD19+ B cells. This could allow IL22 to have an effect on the infiltrating lymphocytic population, exerting a protective effect on the salivary and lacrimal glands to prevent further gland destruction. Furthermore, although IL22R 1 is not expressed on immune cells, its expression is quite promiscuous in keratinocytes and epithelial cells of multiple organs including the skin, kidney, digestive and respiratory system (69) Paradoxically, IL17R is predominantly expressed by immune cells (45) Therefore, it is also possible that the influx of TH17 cells, which produce both IL17 and IL22, may have restricted function if only IL22 is capable of transducing its signal through the epithelium. Again, whether this signal transduction pathway elicits destruction or protection remains to be determined. Clearly, the interconnection between IL22 and IL17A/F in the context of micro enviornment or their interaction could dictate which role is exhibited and predicated. For instance, gut commensal microflora could promote the differentation of IL 22 producing cell populations which are characterized by NKp46 expression including +CD3-NKp46+ LTi like +CD3-NKp46LTi and an uncharacterized +CD3+NKp46+ cell populations (136138) +CD3+NKp46+ cells
93 have yet to be extensively studied, they may behave similar to NKT cells due to their CD3+NKp46+ phenotype; however, they may develop into a possible subset of LTi cells (136) In any event, it is imperative to examine the microenviornment of the salivary glands that leads to the generation of these unique cells populations and further elucidate the functions of each of these cell populations and their roles in the pathogenesis of S j S. Perhaps the most pertinent aspect of the current study is the fact that in human SjS patients the serum levels of IL22 show high correlations with each of the major criteria important in the diagnoses of S j S, that is, reduced saliva (and/or tear) flow, detection of anti SSA/Ro and/or anti SSB/La and leukocyte infiltrations of the salivary glands (as determined by histological examination of gland biopsies). Commonly, not all these criteria are meet by each patient. Thus, the current study appears to offer an additio nal parameter, one that has the potential to supplant those criteria dependent on analyses of serum markers. Analyses of a much larger cohort of patients and various sets of control cohorts will permit both confirmation and refinement of possible correlati on. With the possibility of IL22 being a key player in the human SjS disease phenotype, it became imperative to determine if the leading SjS mouse models also expressed increased IL22 levels. Using the C57BL/6.NOD Aec1R1Aec2 and C57BL/6.NOD Aec1Aec2 mous e models, IL 22 expression was investigated within the salivary and lacrimal glands. Remarkably, IL22 was not expressed in the salivary or lacrimal glands of the SjS mouse models, confirmed by both immunohistochemical staining (salivary and lacrimal glands) and Real Time PCR analysis (salivary glands).
94 Furthermore, IL22 was entirely absent from both the saliva and sera of C57BL/6.NOD Aec1R1Aec2 mice and normal C57BL/6 controls and NKp46++ subsets were present at much lower numbers than seen in humans within the salivary gland. If one considers the potential necessity of a viral infection to induce autoimmunity, the apparent lack of IL22 in mouse models is understandable. For instance, assuming that upon viral invasion of cytomegalovirus into the sali vary gland the release of IL22 from IL 22secreting cells such as NK and LTi cells occurs. As mouse models are maintained under a pathogenfree environment, they would therefore develop disease as a result of genetic predisposition and not from a viral t rigger, so would not exhibit increased IL22 secretion. In order to confirm this claim, however, further studies are needed. Immunohistochemical staining of the spleen, thymus, and lymph nodes did however indicate positive IL22 staining was present in both C57BL/6.NOD Aec1R1Aec2 mice and normal C57BL/6 controls Therefore, flow cytometric analysis was performed on the lymphocyte populations from these organs as well as from the perit oneal exudate. Several significant changes were observed between male C57BL/6.NOD Aec1R1Aec2 models and C57BL/6 controls, however, the overall differences were unremarkable. Female C57BL/6.NOD Aec1R1Aec2 models and C57BL/6 controls showed similar results. However, comparison between male and female C57BL/6.NOD Aec1R1Aec2 mice showed that males tended to have much higher levels of NKp46+CD3NK cells within the spleen and lymph nodes, and generally peaked in the spleen in males at 8 wks compared to 12 wks in females and in the thymus in males at 8 wks compared to 24 wks in females. Similarly, in C57BL/6 controls, in the spleen the males had higher total NK cell levels than females and and
95 both genders had a constant level over time (male and female data for 12, 16, 24 wks not performed). It is possible that in C57BL/6.NOD Ae c1R1Aec2 mice the disease process does not affect the NK and LTi like cell populations within the spleen, thymus, lymph node, and peritoneal exudate, but instead these cell populations are influenced more by gender, as demonstrated by comparison with the C57BL/6 controls. The question becomes what is the mechanism by which gender and hormones may manage the movement of NK and LTi like cells within the mouse, however, additional studies using larger sample sizes for both males and females is necessary. As d emonstrated by the vast range of available mouse models, S j S is a highly complex disease whose etiology is still not well understood. It is likely that S j S pathogenesis involves an intricate relationship between genetics and environmental factors which can provoke both innate and adaptive immunity, hormone secretion, and the autonomic nervous system into triggering the initiation and progression of the disease. Animal models demonstrate a variety of potential pathologies for the disease, ranging from overpr oduction of inflammatory cytokines to exposure by exocrine glandtargeting viruses. Therefore, these animal models provide a useful tool in observing the different stages in the glandular pathophysiological abnormality to the loss of immune tolerance and eventually to the onset of overt or clinical disease. In addition, they can serve as great tools in designing diagnoses, as well as in prevention and treatment therapies. Each mouse model possesses its own advantages, as well as pitfalls, and no ideal model for the study of S j S currently exists. Spontaneous models naturally develop S j S and appear most similar to the human S j S disease, but still have their drawbacks Knock out animal models can also be useful, allowing observation of the importance a
96 particular protein, regulatory mechanism, or cell type has in disease development, leading to improved treatment options. However, no S j S mouse model fulfills all of the necessary characteristics of the human disease, and such discrepancies may cause progr ess in the field to come to a standstill. A better model is needed. Furthermore, by identifying the presence of not only T cells, but also NK cells and LTi cells within the lymphocytic infiltrates of human SjS patients such data may further our understanding of how the over production of IL22 is stimulated within each IL22producing cell population. As such, treatments for SjS can be optimized to either halt IL22 secretion from each cell type if its effects are shown to be proinflammatory, or to stimul ate IL22 production if it demonstrates a protective effect in SjS. Future studies are critical in understanding the pathophysiological function of IL22, encompassing both its localized and systemic effects in SjS.
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111 BIOGRAPHICAL SKETCH Tegan Noel Lavoie received her Bachelor of Science in chemistry from Florida Atlantic University in May 2009 and her Master of Science in medical sciences from the University of Florida in May 2011. She is the oldest of three children to Lisa Graham and currently resides in Gainesville, FL. After graduation, she plans on returning to graduate school to earn her Ph.D. in chemistry, specializing in total synthesis of small organic compounds. In the future, Ms. Lavoie would like to become a pri ncipal investigator at a universit y and teach graduatelevel organic synthesis courses in addition to conducting research synthesizing small, naturally inaccessible products used for new drug discovery.