Dual Function of the IRF8 Transcription Factor in Autoimmune Uveitis: Loss of IRF8 in T Cells Exacerbates Uveitis, Whereas Irf8 Deletion in the Retina Confers Protection

Abstract

IFN regulatory factor 8 (IRF8) is constitutively expressed in monocytes and B cells and plays a critical role in the functional maturation of microglia cells. It is induced in T cells following Ag stimulation, but its functions are less well understood. However, recent studies in mice with T cell-specific Irf8 disruption under direction of the Lck promoter (LCK-IRF8KO) suggest that IRF8 directs a silencing program for Th17 differentiation, and IL-17 production is markedly increased in IRF8-deficient T cells. Paradoxically, loss of IRF8 in T cells has no effect on the development or severity of experimental autoimmune encephalomyelitis (EAE), although exacerbating colitis in a mouse colitis model. In contrast, mice with a macrophage/microglia-specific Irf8 disruption are resistant to EAE, further confounding our understanding of the roles of IRF8 in host immunity and autoimmunity. To clarify the role of IRF8 in autoimmune diseases, we have generated two mouse strains with targeted deletion of Irf8 in retinal cells, including microglial cells and a third mouse strain with targeted Irf8 deletion in T cells under direction of the nonpromiscuous, CD4 promoter (CD4-IRF8KO). In contrast to the report that IRF8 deletion in T cells has no effect on EAE, experimental autoimmune uveitis is exacerbated in CD4-IRF8KO mice and disease enhancement correlates with significant expansion of Th17 cells and a reduction in T regulatory cells. In contrast to CD4-IRF8KO mice, Irf8 deletion in retinal cells confers protection from uveitis, underscoring divergent and tissue-specific roles of IRF8 in host immunity. These results raise a cautionary note in the context of therapeutic targeting of IRF8.

Figure 1

Generation and characterization of IRF8 conditional KO mice. Irf8^fl/fl mice were crossed with CD4-Cre mice to generate mice with deletion of IRF8 in CD4⁺ T cells (CD4-IRF8KO). The CD4-IRF8KO mice were identified by PCR analysis of mouse tail genomic DNA (A). Naïve CD4⁺ T cells from lymph nodes and spleens of WT or F8 generation CD4-IRF8KO mice (after 8 cycles of brother-sister mating) were stimulated with anti-CD3/CD28 Abs and analyzed by western blotting (B) and thymidine incorporation assays (C). Data shown in (C) are mean ± SEM from five replicate cultures. *P < 0.05. The immunophenotype of the stimulated T cells was characterized by FACS and intracellular cytokine staining assays. Numbers in quadrants indicate percentages of IL-2-expressing CD4⁺ T cells (D) or percentages of activated CD4⁺ T cells (E). Histogram presented in (D) and (E) represent percentages of IL-2-expressing cells or CD44^HiCD25⁺ cells, respectively (Mean and SEM, N=3). CD4⁺ T cells from the WT or CD4-IRF8KO mice were stimulated with anti-CD3/CD28 Abs for 48 h and analyzed for apoptosis using the annexin V assay (F) and RT-PCR (G). Numbers in quadrants and histogram in F, indicate percentages of CD4⁺ T cells undergoing apoptosis or necrosis (Mean and SEM, N=3). (H, I) Naïve CD4⁺ T cells from WT or CD4-IRF8KO mice were stimulated with anti-CD3/CD28 Abs for 4 days under Th17-polarizing condition. The cells were then analyzed by FACS for intracellular cytokine staining. Numbers in quadrants and histograms indicate percentages of CD4⁺ T cells expressing IL-17 and/or ROR-γt Mean and SEM, N=3). Results are representative of at least 3 independent experiments. *P < 0.05, **P < 0.01, (Student’s two tailed t-test).

Figure 2

IRF8KO mice develop more severe EAU. EAU was induced in WT or CD4-IRF8KO mice by immunization with IRBP in CFA and disease progression was analyzed by fundoscopy or histology. (A) Fundus images of eyes harvested 14 days or 21 days post-immunization were taken using an otoendoscopic imaging system. Assessment of the severity of the inflammatory disease (EAU scores) was based on changes at the optic nerve disc and retinal vessels or tissues. Black arrow indicates inflammation with blurred optic disc margins and enlarged juxtapapillary areas; blue arrows indicate retinal vasculitis with moderate cuffing; white arrow shows yellow-whitish retinal and choroidal infiltrates. (B) For histological analysis, eyes were harvested 21 days post-immunization. Histologic sections through the retina were stained with hemotoxylin and eosin and EAU scores were determined as described (18). White arrow heads indicate the presence of inflammatory cells in vitreous (V); blue astericks indicate retinal folds. Results represent at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test (two tailed)).

Figure 3

Th17 cells are expanded in CD4-IRF8KO mice during EAU (A) The immunophenotype of freshly isolated lymph node (LN) cells of un-immunized mice or IRBP-immunized WT or CD4-IRF8KO mice was characterized by FACS and intracellular cytokine staining assays. Numbers in quadrants indicate percentages of IL-17-, ROR-γt-and/or IFN-γ-expressing CD4⁺ or CD8⁺ T cells. (B) LN cells of un-immunized mice or IRBP-immunized WT or CD4-IRF8KO mice were labeled with CFSE and stimulated with IRBP for 4 days before analysis by FACS for intracellular cytokine staining. The cells were gated on CD3/CD4 cells and numbers in quadrants indicate percentages of CD4⁺ T cells expressing IL-17, IFN-γ or Foxp3. Statistical analysis of the percentage of cytokine-expressing T cells was based on analysis of 6 mice per group. Results represent at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test (two tailed)).

Figure 4

Loss of Irf8 in neuroretinal cells confers protection from uveitis. (A, B) PCR genotype analysis of tail DNA isolated from WT Irf8^f/f or heterozygous Irf8^f/− mice (A, B), RX-Cre-Irf8KO (A) or αCre-Irf8KO (B) mice. (C) RNA was isolated from retinal cells of WT, RX-Cre-Irf8KO, αCre-Irf8KO mice and analyzed for IRF8 expression by RT-PCR. (D, E) EAU was induced in WT, αCre-Irf8KO (D) or RX-Cre-Irf8KO mice (E) by active immunization with IRBP in CFA. Progression and severity of EAU was monitored by funduscopy or histology. (Top panels). Fundus images were taken using an otoendoscopic imaging system, and the development of papillitis (black arrows), retinal vasculitis (blue arrows) and inflammatory infiltrates (white arrows) are indicated. Clinical scoring was based on changes at the optic nerve disc, retinal vessels, and surrounding tissues as described in the methods section. (Bottom panels). Eyes were enucleated on day 21 post-immunization, fixed in formallin, embedded in paraffin and sections were stained with H&E. Histologic sections through the retina were stained with H&E and EAU scores were determined as described (18). Infiltrated inflammatory cells in the vitreous (V) are denoted by black arrows; blue asterisks indicate retinal folds. OpN (optic nerve), GCL (ganglion cell layer), INL (inner nuclear layer), ONL (outer nuclear layer), RPE (retinal pigment epithelial layer), CH (choroid). Results are representative of at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test (two tailed)).

Figure 5

Anti-inflammatory cytokines are upregulated in the retina of IRF8-deficient mice during EAU. EAU was induced in WT or αCre-Irf8KO mice by active immunization with IRBP in CFA. Retina isolated from the eyes of the mice on day 21 post-immunization were digested with collagenase and retina cells were analyzed for the expression of IFN-γ, IL-17, IL-10, IL-35 (EBI3, IL-12p35) or IL-12 (IL-12p40) by qRT-PCR. Results represent at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test (two tailed)).

Figure 6

Visual function is altered in IRF8KO mice during EAU. (A, B) Visual function of WT or αCre-Irf8KO mice was analyzed by electroretinography (ERG). (A) ERG response after dark or light adaptation was analyzed in unimmunized mice (3 mice or 6 eyes; N=6). (B) ERG response after light adaptation was analyzed in IRBP/CFA immunized mice with EAU. Data are presented as the mean ± SEM of 5 mice from two individual experiments. Gold asterisks; ERG comparison between unimmunized and immunized wild type mice (10 eyes at each time-point, N=10). Blue asterisks; ERG comparison between unimmunized wild type mice and immunized α-CRE-IRF8KO mice (10 eyes at each time-point, N=10). Black asterisks; ERG comparison between IRBP/CFA immunized wild type mice and IRBP/CFA immunized α-CRE-IRF8KO mice (10 eyes at each time-point, N=10). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s two-tailed t-test.