Citation
Indispensable Role of Reactive Oxygen Species in the Pathogenesis of Type 1 Diabetes

Material Information

Title:
Indispensable Role of Reactive Oxygen Species in the Pathogenesis of Type 1 Diabetes
Creator:
Liu, Chao
Place of Publication:
[Gainesville, Fla.]
Florida
Publisher:
University of Florida
Publication Date:
Language:
english
Physical Description:
1 online resource (138 p.)

Thesis/Dissertation Information

Degree:
Doctorate ( Ph.D.)
Degree Grantor:
University of Florida
Degree Disciplines:
Medical Sciences
Immunology and Microbiology (IDP)
Committee Chair:
MATHEWS,CLAYTON ELWOOD
Committee Co-Chair:
DRIVER,JOHN P
Committee Members:
CHANG,LUNG-JI
NICK,HARRY S
Graduation Date:
8/9/2014

Subjects

Subjects / Keywords:
Antigens ( jstor )
Cytokines ( jstor )
Dendritic cells ( jstor )
Diabetes ( jstor )
Molecules ( jstor )
Oxidases ( jstor )
Peroxides ( jstor )
Reactive oxygen species ( jstor )
Superoxides ( jstor )
Type 1 diabetes mellitus ( jstor )
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
ctl -- dc -- diabetes -- nod-ncf1m1j -- ros
Genre:
bibliography ( marcgt )
theses ( marcgt )
government publication (state, provincial, terriorial, dependent) ( marcgt )
born-digital ( sobekcm )
Electronic Thesis or Dissertation
Medical Sciences thesis, Ph.D.

Notes

Abstract:
Reactive oxygen species (ROS) play an essential role in the pathogenesis of type 1 diabetes (T1D). By eliminating ROS generated by the phagocytic NADPH oxidase [NOX2], T1D onset is significantly reduced in the diabetes-prone NOD mouse model. However, the molecular mechanisms that are impacted by ROS in T1D pathogenesis remain unknown. Here, we provide evidence that inhibition of NOX2 activity in diabetogenic CD8+ T cells prevents beta cell destruction. Genetic ablation or chemical inhibition of NOX2 significantly suppressed the expression of the transcription factor T-bet, resulting in the ablation of pro-inflammatory cytokine and effector molecule production after anti-CD3 and anti-CD28 stimulation of CD8+ T cells. Further, we confirmed that the activation of mTOR complex 1 (mTORc1) was a critical event in ROS-regulated CD8+ T cell differentiation. mTORc1 was required for CD8+ T cell effector function in a T-bet dependent manner and the activation of mTORc1 was boosted by NOX2 generated ROS. These results indicate that by promoting mTORC1 activity, NOX2 plays anon-redundant role in TCR mediated CD8+ T cell effector function. In addition, our data showed that dendritic cells with deficient NOX2 are incapable of activating autoreactive CD8+ T cell activation. Instead of enhancing dendritic cell surface stimulatory molecule or promoting pro-inflammatory cytokine production upon various stimulations, ROS promote CD8+ T cell activation by facilitating autoantigen cross-presentation. NOX2 deficient NOD DCs showed a strong phagosomal acidification and antigen acidification, which lead to a quenched antigen tranlocation into the cytoplasm and deficient peptide uploading on MHC-1 molecules. This study confirmed that NOX2 is required for autoantigen cross-presentation in T1D, and diabtetogenic CD8+ T cell activation could be prevented by intervening the dendritic cell ROS production. ( en )
General Note:
In the series University of Florida Digital Collections.
General Note:
Includes vita.
Bibliography:
Includes bibliographical references.
Source of Description:
Description based on online resource; title from PDF title page.
Source of Description:
This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Thesis:
Thesis (Ph.D.)--University of Florida, 2014.
Local:
Adviser: MATHEWS,CLAYTON ELWOOD.
Local:
Co-adviser: DRIVER,JOHN P.
Electronic Access:
RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2015-08-31
Statement of Responsibility:
by Chao Liu.

Record Information

Source Institution:
UFRGP
Rights Management:
Applicable rights reserved.
Embargo Date:
8/31/2015
Classification:
LD1780 2014 ( lcc )

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Full Text

PAGE 7

Ncf1 m1J Ncf1 m1J In vitro Ncf1 m1J Ncf m1J NOD-Ncf1 m1J Ncf1 m1J

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In v itro In v ivo Ncf1 m1J in Vitro Ncf1 m1J

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Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J

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Ncf1 m1J Ncf1 m1J in vitro Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J in vivo

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Type 1 Diabetes

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Genetics of T ype 1 D iabetes human leukocyte antigen HLA

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INS

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VNTR VNTR VNTR VNTR PTPN22, Autoimmune P athogenesis

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Reactiv e Oxygen Species Metabolism of ROS

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Cellular Sources of Reactive Oxygen Species

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Cybb Cyba Neutrophil cytosolic factor 1 Ncf1

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Redox Signals and Oxidative Stress

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Ka

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ROS Promote P ro -inflammatory Responses

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Reactive Oxygen Species in T cells

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PTPN22 PTPN22

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MAPK

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Reactive Oxygen Species in Type 1 Diabetes

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Ncf1 m1J ALR mice and T1D Insulin Dependent Diabetes Idd IL Suppressor of Superoxide Production Susp Susp Susp

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NADPH oxidase and T1D phox Ncf1 m1J Ncf1 m1J Ncf1 m1J Scid phox Ncf1 m1J Ncf1 m1J

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Ncf1 m1J Ncf1 m1J

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Summary Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J in vitro Ncf1 m1J

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Background

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Ncf1 m1J Scid Materials and Methods Animals Prkdc scid Scid Rag1 tm1Mom Rag1 -/Rag1 tm1Mom Rag1 -/Rag1 -/-

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Rag1 -/Ncf1 m1J Materials Adoptive Transfer Ncf1 m1J

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Scid 1 2 Ncf1 m1J Ncf1 m1J 3 Ncf1 m1J 4 Ncf1 m1J Purification and Cell Culture of CD8 + T cells Ncf1 m1J CD8 + T cell proliferation Detection of cytokine production and secretion by CD8 + T cells

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Quantitative Real-Time Quantitative PCR Cell-Mediated Lymphocytoxicity Assay Rag1

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Immunoblot , Measurement of mTOR Complex Activity

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Determination of Rheb-GTP levels after T Cell Activation Statistics t P Results Delayed Transfer of Diabetes with NODNcf1 m1J Splenocytes and Purified CD4 + and CD8 + T cells. Ncf1 m1J Scid Scid Ncf1 m1J Ncf1 m1J Ncf1 m1J

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Ncf1 m1J P Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J CD8 + T cells in NODNcf1 m1J Mice Exhibit a Reduced Production of Pro-inflammatory Cytokines and Effector Molecules Ncf1 m1J Ncf1 Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J

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Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J The NOX2 Inhibitor, Apocynin, Suppresses the Production of Effector Molecules in CD8 + T Cells Ncf1 p22 phox

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Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J H 2 O 2 Instead of Superoxide Promotes NOD CD8 + T cell Function In vivo

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Ncf1 m1J

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NADPH Oxidase is Required for Diabetogenic T Cell K Rag1 -/Ncf1 in vitro Rag1 -/

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NADPH Oxidase has no Impact on Effector Molecule Production aft er PMA/Ionomycin Stimulation Ncf1 m1J Ncf1 m1J

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The mTORc1 is Required for CD8 + T Cell Effector Molecule Production Through T-bet NADPH Oxidase Enhances mTORc1 Activity during CD8 + T Cell Activation

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Ncf1 m1J Ncf1 m1J Redox Regulation of mTORc1 activity in CD8 + T cell Ncf1 m1J

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Ncf1 m1J ROS promotes mTORc1 activity via suppressing TSC1/2 Ncf1

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in vivo Ncf1 m1J Discussion

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Scid Ncf1 m1J -Ncf1 m1J

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Ncf1 m1J ClC3,

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Ncf1 m1J Ncf1 m1J Ka

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Ncf1 Ncf1 m1J

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Ncf1 m1J Scid Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J P P P P P P P P

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Ncf1 m1J p* p **p ***p

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*p **p ***p

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* p **p ***p

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*** p

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*p ** p ***p

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Ncf1 m1J Ncf1 m1J *p

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Ncf1 m1J

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Background

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Ncf1 m1J Scid Ncf1 m1J

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Scid Ncf1 m1J Ncf1 m1J Materials and Methods Animals Rag1 tm1Mom AI4 Rag1 -/Rag1 tm1Mom Rag1 -/Rag1 -/Rag1 -/Rag1 -/Ncf1 m1J Materials

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Cell Culture Quantitative Real-Time Quantitative PCR

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Flow Cytometry Measurement of Phagocytosis

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Measurement of phagosomal pH Phagosomal Antigen Degradation Assay

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Detection of Antigen Translocation Purification of T cells Measurement of T Cell Proliferation In vitro Cross Presentation Assays in vitro

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Ncf1 m1J Statistics t P Results Dendritic Cell Maturation is Normal in NODNcf1 m1J Ncf1 m1J

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Ncf1 m1J Ncf m1J Ncf m1J Ncf m1J

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Lack of NOX2 does not Impact Production of Type 1 Interferons by DC Ncf m1J Ncf m1J Ncf1 m1J

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Comparable Dendritic Cell Phagocytosis in NOD and NODNcf m1J Ncf m1J Ncf1 m1J Enhanced Phagosomal Acidification in NOD-Ncf1 m1J DCs Slc11a1 Idd5

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Slc11a1 Slc11a1 Ncf1 m1J Ncf1 m1J phox Accelerated Antigen Degradation in NODNcf1 m1J DCs Ncf1 m1J

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Ncf1 m1J Ncf1 m1J Ncf1 m1J Impaired Antigen Translocation without NOX2 Derived ROS Ncf1 m1J Ncf1 m1J Ncf1 m1J

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Deficient Cross Presentation in ph47 phox Deficient DCs in vitro Ncf1 m1J Ncf1 m1J Ncf1 Ncf1 m1J Ncf1 Ncf1 m1J Discussion Ncf1 m1J

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Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J Ncf1 m1J

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Ncf1 m1J Slc11a1 Cybb Ncf1 m1J

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Ncf1 m1J

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Ncf1 m1J

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Ncf1 m1J Ncf1 m1J

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Ncf1 m1J Ncf1 m1J

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Ncf1 m1J Ncf1 m1J Ncf1 m1J

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Ncf1 m1J *p **p ***p

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Ncf1 m1J *p ** p ***p

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Ncf1 m1J Ncf1 m1J * p **p ***p

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Background

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in vitroin vivo Materials and Methods Animals Ncf1 m1J/m1J Ncf1 m1J Ncf1 m1J Prkdc scid Scid Ncf1 m1J Ncf1

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Materials Adoptive co-transfer of splenocytes Ncf1 m1J Ncf1 m1J Scid Flow cytometry

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In vitro suppression assay In vivo suppression assay Ncf1 m1J

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Ncf1 m1J Scid Statistical analysis. Results Splenocytes from p47 phox Deficient NOD Mice Do Not Suppress T1D Ncf1 m1J Ncf1 m1J Scid

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Ncf1 m1J Ncf1 m1J Suppression Capacity of Regulatory T cells from NODNcf1 m1J Showed No Deficient in Vitro in vitro in vitro Ncf1 Ncf1 m1J Ncf1 m1J Ncf1 in vitro Ncf1 m1J Non-Diabetogenic Regulatory T cells Are Not Deficient with NOX2 Mutation

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Ncf1 m1J Ncf1 m1J NODNcf1 m1J Regulatory T Cells Showed a Trend of Decreased Suppression in BDC2.5 T Cell Induced Diabetes in vivo Ncf1 m1J Ncf1 m1J Rag1 -/

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Ncf1 m1J Ncf1 m1J Ncf1 m1J in vivo Discussion Ncf1 m1J Ncf1 in vivo

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Ncf1 m1J Ncf1 Ncf1 m1J in vitro in vivo

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in vivo Ncf1 m1J in vitro Ncf1 m1J in vitro in vivo Ncf1 m1J

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in vitro

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Ncf1 m1J . Ncf1 m1J . Scid

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Ncf1 m1J

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Ncf1 m1J

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Ncf1 m1J in vivo Ncf1 m1J Rag1 /

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Ncf1 Ncf1

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Ncf1 Ncf1 N cf 1 Ncf1 m1J Ncf1 m1J Scid Ncf1 m1J

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Scid Ncf1 m1J

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Clin Dev Immunol 2012 Trends Immunol 30 Clin Mol Allergy9 Nat Chem Biol 4 Antioxid Redox Sign 15 Journal of Biological Chemistry 244 J Phys Chem A 106 Archives of Biochemistry and Biophysics 423 Nat Rev Drug Discov 6 Antioxid Redox Sign 8 Postepy Hig Med Dosw (Online) 60 Science300 Mol Cell Endocrinol 250 Sci STKE 2006 Antioxid Redox Sign 14 Nat Chem Biol 7

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Journal of Biological Chemistry 282 Journal of Biological Chemistry 283 Biochem J 417 Biochemistry-Moscow+ 70 Semin Cell Dev Biol 12 Journal of Biological Chemistry 278 Nat Genet 38 Acta Biochim Pol 46 Biochim Biophys Acta 1366 Exp Gerontol 45 Immunity38 Ann N Y Acad Sci 991 Free Radic Biol Med 47

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Free Radic Biol Med 49 Nat Rev Immunol4 Seminars in Immunopathology 30 Cell Immunol 88 Science 242 J Immunol 163 Journal of Biological Chemistry 269 Embo Journal 21 Nat Struct Biol 8 J Clin Invest 96 J Leukoc Biol 79 Mol Cell 15

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Journal of Biological Chemistry 273 Free Radic Biol Med 44 J Clin Invest 73 Biochem Pharmacol 64 Arch Biochem Biophys 422 Trends in Plant Science 7 Am J Physiol Regul Integr Comp Physiol 287 Arterioscler Thromb Vasc Biol 24 Molecular and Cellular Biology 26 Biochem Biophys Res Commun 351 J Immunol 146 Nature Immunology 5

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Proc Natl Acad Sci U S A 91 Transpl Immunol 10 Diabetes 58