Immune-mediated inflammation, a major cause of vision loss, is not fully understood. This project is aimed at collecting new information concerning the mechanisms, cells and molecules involved in the pathogenic process of ocular inflammation. The collected information is to be used for development of new approaches for treatment of this group of eye diseases. Research in FY2012 focused mainly on three issues: (i) analysis of dissimilarities between T-helper (Th) cell lines generated by either the commonly used activation with anti-CD3/CD28 antibodies, or with the physiological activation mode by the specific antigen presented by antigen-presenting cells (APC);(ii) examining the features of natural- and induced-T-regulatory cells (nTreg and iTreg, respectively) with specificity toward an ocular antigen;(iii) further dissection of the surprising dual effect of DAP12 on development of EAU and related immune responses. Summary Targeted at learning about pathogenic processes of inflammatory eye diseases, this project focused in FY 2012 mainly on populations of T-cells involved in these processes. Specifically, we extended our study in FY 2012 by focusing on the following issues: (1) Dissimilarities between lines of Th cells generated by activation with anti-CD3/CD28 antibodies, or by the specific antigen presented by APC;(2) collecting information on different subpopulations of Treg cells;(3) further dissecting the surprising dual effect of DAP12 on immunopathogenic responses. 1) Dissimilarities between Th lineages generated by different mechanisms. Lineages of Th are generated by activation of nave CD4 cells concurrently with polarization by the lineage-specific cytokine(s). It is assumed that in vivo, the activation is provided by interaction of the nave CD4 cells with their specific Ag, presented by APCs. Since only miniscule proportions of CD4 cells with specificity toward tested Ags exist in preparations of CD4 cells from WT animals, the activation by Ag and APC has been commonly replaced by exposure of the CD4 cells to antibodies against two cellular molecules that participate in the physiological process of activation, namely, CD3 and CD28. The availability of TCR transgenic (Tg) mice, in which the majority of T-cells recognize the same antigen, made it possible, however, to generate lines of polarized Th cells by activation of nave CD4 cells by either the specific antigen presented by APC, or by the anti-CD3/CD28 antibodies. These transgenic mice made it possible to compare between lines of polarized cells generated by either one of the two modes of activation. We compared between lineages of Th1, Th9 and Th17, generated by either one of the activation modes, and found remarkable differences between the two lineages of each of the three phenotypes in the following biological activities: capacities to migrate to and proliferate in the recipient spleen and, importantly, to induce inflammation in the recipient mouse eyes expressing the target antigen;the expression profiles of certain chemokines, chemokine receptors and major surface antigens;and, notably, the pattern of gene expression, as monitored by microarray analysis. Our findings thus underscore the limitations of biological observations made with Th lineages generated by only activation of CD4 cells with the anti-CD3/CD28 Abs. 2) Differences among T-regulatory (Treg) populations generated by different modes. Specific Tregs, that inhibit immune response against self antigens, have the potential of being used to specifically inhibit pathogenic immune responses and are the focus of intensive research effort. The induction of Treg requires the cells to be activated during the process of polarization toward acquisition of the immune suppressive capacity. The activation process used in the majority of studies have been by antibodies against CD3/CD28, but other studies have employed specific activation by the antigen presented by APC. To our knowledge, no publication has compared Treg cells generated by these two procedures. Using our system of TCR transgenic mice (see above) made it possible to make such a comparison. In general, Treg cells induced by antigen and APC were more suppressive than those induced by activation with the antibodies. The inhibitory effects of the two lineages of Tregs were tested in cultures of nave CD4 during acquisition of polarity or in cultures of polarized Th1, Th9 or Th17 cells responding to the specific antigen (HEL). In all systems, Th17 exhibited the highest level of susceptibility, whereas Th1 cells were the most resistant and Th9 cells were intermediate in their susceptibility. 3) Resolution to the enigmatic dual effects of DAP-12 on immunopathogenic responses. DAP-12 (DNAX-activating protein of 12kDa) plays a major role in the immune response by transducing a variety of activation signals. The role of DAP-12 in the etiology of pathogenic autoimmunity has been controversial by the molecules capacity to either enhance or suppress experimental autoimmune diseases in different studies. In the present study we compared DAP-12 null mice and wild type (WT) controls for their susceptibility to EAU induction and immune responsiveness. Mice were reared in two animal facilities, with low (A) or high (B) standards. Although disease development in WT control mice from both facilities was comparable, DAP-12 null mice from facility A were superior to their WT controls in both the severity of their ocular inflammation and the levels of proliferation and cytokines produced by their lymphocytes. In contrast, DAP-12 null mice from facility B were inferior to their controls by both parameters. Our data thus provide a possible resolution to the controversy concerning the role of DAP-12: deficiency in this molecule can either promote or suppress the pathogenic response, depending on the environment. Further, we suggest that differences between the environments of the two animal facilities affected the immune responsiveness by modifying the microbial flora of the mice and consequently, their immune capacity.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIAEY000069-35
Application #
8556801
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
35
Fiscal Year
2012
Total Cost
$1,188,321
Indirect Cost
Name
U.S. National Eye Institute
Department
Type
DUNS #
City
State
Country
Zip Code
Hinshaw, Samuel J H; Ogbeifun, Osato; Wandu, Wambui S et al. (2016) Digoxin Inhibits Induction of Experimental Autoimmune Uveitis in Mice, but Causes Severe Retinal Degeneration. Invest Ophthalmol Vis Sci 57:1441-7
Wandu, Wambui S; Tan, Cuiyan; Ogbeifun, Osato et al. (2015) Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response. PLoS One 10:e0128906
Richard, Arianne C; Tan, Cuiyan; Hawley, Eric T et al. (2015) The TNF-family ligand TL1A and its receptor DR3 promote T cell-mediated allergic immunopathology by enhancing differentiation and pathogenicity of IL-9-producing T cells. J Immunol 194:3567-82
Richard, Arianne C; Tan, Cuiyan; Hawley, Eric T et al. (2015) Correction: The TNF-Family Ligand TL1A and Its Receptor DR3 Promote T Cell-Mediated Allergic Immunopathology by Enhancing Differentiation and Pathogenicity of IL-9-Producing T Cells. J Immunol 195:5839-40
Zhao, Chan; Shi, Guangpu; Vistica, Barbara P et al. (2014) Induced regulatory T-cells (iTregs) generated by activation with anti-CD3/CD28 antibodies differ from those generated by the physiological-like activation with antigen/APC. Cell Immunol 290:179-84
Shi, Guangpu; Vistica, Barbara P; Nugent, Lindsey F et al. (2013) Differential involvement of Th1 and Th17 in pathogenic autoimmune processes triggered by different TLR ligands. J Immunol 191:415-23
Nugent, Lindsey F; Shi, Guangpu; Vistica, Barbara P et al. (2013) ITE, a novel endogenous nontoxic aryl hydrocarbon receptor ligand, efficiently suppresses EAU and T-cell-mediated immunity. Invest Ophthalmol Vis Sci 54:7463-9
Montalvo, Vanessa; Quigley, Laura; Vistica, Barbara P et al. (2013) Environmental Factors Determine DAP12 Deficiency To Either Enhance or Suppress Immunopathogenic Processes. Immunology :
Vistica, Barbara P; Shi, Guangpu; Nugent, Lindsey et al. (2012) SLAT/Def6 plays a critical role in the pathogenic process of experimental autoimmune uveitis (EAU). Mol Vis 18:1858-64
Tan, Cuiyan; Gery, Igal (2012) The unique features of Th9 cells and their products. Crit Rev Immunol 32:1-10

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