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. 2012:18:1858-64.
Epub 2012 Jul 7.

SLAT/Def6 plays a critical role in the pathogenic process of experimental autoimmune uveitis (EAU)

Barbara P Vistica  1 , Guangpu Shi, Lindsey Nugent, Cuiyan Tan, Amnon Altman, Igal Gery
Affiliations

SLAT/Def6 plays a critical role in the pathogenic process of experimental autoimmune uveitis (EAU)

Barbara P Vistica et al. Mol Vis. 2012.

Abstract

Purpose: SWAP 70-like adaptor of T cells (SLAT; aka Def6) is a recently discovered guanine nucleotide exchange factor for Rho guanosine triphosphate (GTP)ases that has been previously shown to play a role in cluster of differentiation(CD)4+ T cell activation, T-helper (Th)1/Th2/Th17 differentiation and development of experimental autoimmune encephalomyelitis. Here, we investigated the role of SLAT/Def6 in the development of experimental autoimmune uveitis (EAU), an animal model for several uveitic conditions in humans.

Methods: SLAT/Def6 deficient ("KO") mice and C57BL/6 controls were immunized with interphotoreceptor retinoid-binding protein (IRBP), along with pertussis toxin. The development of ocular inflammation was determined by both fundoscopy and histological examination. Lymphoid cells from draining lymph nodes were cultured with IRBP to measure lymphocyte proliferation and release of cytokines. Purified dendritic cells were tested for their capacity to present antigen to responding lymphocytes. In addition, the lymphoid cells were tested for the expression of forkhead box P3 (FoxP3), using conventional methods, and the activity of T-regulatory cells was determined by their capacity to inhibit in vitro proliferative responses. Serum anti -IRBP antibody levels were measured by enzyme-linked immunosorbant assay (ELISA). quantitative polymerase chain reaction (qPCR) was used to determine the transcript levels of cytokines in inflamed eyes.

Results: SLAT/Def6 KO mice had significantly reduced EAU compared to controls. Cells isolated from draining lymph nodes of SLAT/Def6 KO mice exhibited impaired proliferation and production of Th1 and Th17 signature cytokines (interferon [IFN]-γ and interleukin [IL]-17, respectively) when compared with cells isolated from control mice. qPCR of inflamed eyes detected similar levels of IFN-γ transcript in control and SLAT/Def6 KO mice, whereas the IL-17 transcript levels in eyes of the SLAT/Def6 KO mice were lower than in eyes of the controls. The SLAT/Def6 KO mice resembled their wild type (WT) controls, however, in the levels of their serum antibody against IRBP, the antigen presenting capacity of their dendritic cells, the proportion of cells expressing Foxp3 and the immunosuppressive activity of their T-regulatory cells.

Conclusions: SLAT/Def6 KO mice exhibit reduced capacity to develop ocular inflammation and cellular activity when immunized with IRBP. Our study provides new data showing that SLAT/Def6 plays a major role in the T cell-mediated autoimmune processes that bring about the inflammatory eye disease, EAU.

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Figures

Figure 1
Figure 1
SLAT/Def6 deficient mice are inferior to their WT controls in developing EAU. A: Disease scores of individual mice of the two mouse lines. Horizontal lines are mean ±SEM. A summary of repeated experiments comparing the response in mice of the two lines. ** p<0.005. B: Sections of representative eyes of the two groups. The changes in the eye of the control mouse are markedly more severe and mainly include infiltration of inflammatory cells in various ocular tissues, as well as retinal folding and loss of photoreceptor cells (score: 2+). Only minor infiltration is seen in the SLAT/Def6 deficient mouse eye (score: 0.5).
Figure 2
Figure 2
SLAT/Def6 KO mice are inferior to their WT controls in their cellular response to the immunizing antigens. A: Proliferative assay of a representative experiment; similar data were obtained in 2 other repeated experiments. IRBP was added at the indicated concentrations (μg/ml). PPD was added at 5 μg/ml. * p<0.05, **p<0.005. B: Levels of IL-17 and IFN-γ secreted by lymph node cells of SLAT/Def6 KO mice cultured with IRBP at 10 μg/ml. Data of two experiments, with supernatants collected on day 2 or 4 of culture. ns=not significant; *p<0.05.
Figure 3
Figure 3
Unlike their defective lymphocyte responses, the SLAT/Def6 KO mice resemble their WT controls in their dendritic cell functional activity. DC preparations [14] from the KO mice or their WT controls were tested for their capacity to present IRBP to purified CD4 from syngeneic mice. The response was measured by thymidine incorporation assay [13].
Figure 4
Figure 4
SLAT/Def6 deficiency selectively affects the involvement of Th17 cells in the EAU pathogenic process. Expression of IL-17 and IFN-γ was determined by qPCR in eyes of SLAT/Def6 KO mice and their WT controls. The data are expressed as the ratio between IL-17 and IFN-γ transcript levels in eyes of WT and KO mice, in each of the two experiments.
Figure 5
Figure 5
SLAT/Def6 KO mice resemble their WT controls in their Treg proportions and activities. A: Immunofluorescence analyses of lymph node cells in which the proportion of cells expressing FoxP3 was determined by gating on whole lymph node cells, or on the CD4 population. B: The Treg functional activity of the KO mice is similar to that of the WT controls.
Figure 6
Figure 6
SLAT/Def6 KO mice resemble their WT controls in their production of anti-IRBP antibody. Sera were collected on days 8 or 14 post-immunization and the antibody levels were determined by ELISA.
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