Dual Function of the IRF8 in Autoimmune Uveitis: Loss of IRF8 in T Cells Exacerbates Uveitis, Whereas Irf8 Deletion in the Retina Confers Protection This information is current as of October 4, 2021. Sung-Hye Kim, Jenna Burton, Cheng-Rong Yu, Lin Sun, Chang He, Hongsheng Wang, Herbert C. Morse III and Charles E. Egwuagu J Immunol published online 10 July 2015

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 10, 2015, doi:10.4049/jimmunol.1500653 The Journal of Immunology

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

Sung-Hye Kim,* Jenna Burton,* Cheng-Rong Yu,* Lin Sun,* Chang He,* Hongsheng Wang,† Herbert C. Morse, III,† and Charles E. Egwuagu*

IFN regulatory factor 8 (IRF8) is constitutively expressed in and B cells and plays a critical role in the functional mat- uration 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 –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, Downloaded from 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 http://www.jimmunol.org/ 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. The Journal of Immunology, 2015, 195: 000–000.

he nine-member IFN regulatory factor (IRF) family of In fact, IRF8 inhibits the Th17 cell differentiation program transcription factors is characterized by an N-terminal through its interaction with the Th17 master transcription factor, DNA-binding domain that mediates binding to the core g T ROR- t (6), and integrates TCR and signaling pathways by guest on October 4, 2021 DNA sequence (GAAA) in the IFN-stimulated that drive differentiation of effector CD8+ T cells (7). and a C-terminal IRF-association domain, which facilitates het- IRF8 is also expressed by APCs, including microglia in the CNS, erodimerization with other members of the IRF family as well as macrophages, and dendritic cells, and plays crucial roles in myeloid ETS family members (1, 2). IRF8, also known as IFN consensus cell differentiation, macrophage activation, and functional matu- sequence-binding , is expressed almost exclusively in he- ration of CNS microglia (8–10). Furthermore, IRF8 regulates in- matopoietic cells of both the myeloid and lymphoid lineages (3). nate immune responses and influences the differentiation of Th It functions to a large extent by forming complexes with IRF1, cells by modulating transcription of cytokine in APCs, in- IRF2, IRF4, or PU.1, and, depending on its interaction part- cluding Il12a, which codes the cytokine IL-12p35, a common ners, IRF8 can act as a transcriptional repressor or activator. In subunit shared by IL-12 and IL-35. In collaboration with IRF1, the lymphocyte lineage, it is constitutively expressed by B cells IRF8 also activates transcription of Il27p28 and contributes to and regulates B cell lineage specification, commitment, and dif- mechanisms of ocular immune privilege by inducing retinal ferentiation (4). In contrast, IRF8 is not expressed in naive T cells, microglial cells and neurons to express IL-27 and complement but is rapidly induced in response to antigenic stimulation or TCR factor H (11–13). It is of note that increased expression of the activation (5), suggesting that it may be required for regulating immunosuppressive , IL-27 and IL-35, in the retina or genes involved in T cell differentiation and/or effector functions. brain mitigates experimental autoimmune uveitis (EAU) and ex- perimental autoimmune encephalomyelitis (EAE), animal models of uveitis, and multiple sclerosis, respectively (13–16). *Molecular Immunology Section, National Eye Institute, National Institutes of Health, Bethesda, MD 20892; and †Laboratory of Immunogenetics, National Institute Two recent studies have examined the contributions of IRF8 to of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD colitis and encephalitis. Mice with a global Irf8 knockout or 20852 T cell–specific deletion of the Irf8 (LCK-IRF8KO) devel- Received for publication March 18, 2015. Accepted for publication June 16, 2015. oped a more severe inflammation of the colon resulting from Address correspondence and reprint requests to Dr. Charles E. Egwuagu, Molecular enhanced expansion of Th17 cells (6). In the other report, EAE Immunology Section, National Eye Institute, National Institutes of Health, Building 10, Room 10N109A, 10 Center Drive, Bethesda, MD 20892-1857. E-mail address: clinical scores were found to be similar between WT and LCK- [email protected] IRF8KO mice, suggesting that the expression of IRF8 by T cells Abbreviations used in this article: EAE, experimental autoimmune encephalomyeli- does not have a consequential role in EAE (17). In this study, we tis; EAU, experimental autoimmune uveitis; ERG, electroretinogram; IRBP, inter- photoreceptor retinoid-binding protein; IRF, IFN regulatory factor; LN, lymph node; used CD4-Cre mice to generate mice with targeted deletion of p.i., postimmunization; Treg, T regulatory; WT, wild-type. Irf8 in T cells to rule out the possibility that different outcomes

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500653 2 CONTEXTUAL ROLE OF IRF8 IN NEUROINFLAMMATION observed in the colitis and EAE models did not derive in part from a ground electrode (s.c. stainless steel needle) was positioned at the base use of the relatively “leaky” Lck-Cre mice for generating mice of the tail. Signals were differentially amplified and digitized at a rate of with Irf8 deletion in the T cell compartment. We also generated 1 kHz. Amplitudes of the major ERG components (a- and b-wave) were measured (Espion software; Diagnosys) using automated and manual two mouse strains with targeted deletion of Irf8 in retinal neurons methods. Immediately after ERG recording, imaging of the fundus was and microglia. We have used these strains to clarify the involve- performed, as described above. For each study, ERG data are based on ment of IRF8 in autoimmune disease and to investigate whether analyses of six mice (12 eyes) per group. IRF8 is a potential therapeutic target in uveitis and other CNS Analysis of CD4+ Th cells autoimmune diseases. Freshly isolated T cells or IRBP-stimulated T cells from the spleen, LN (cervical, axillary, inguinal), or retina were analyzed for expression of Materials and Methods inflammatory by FACS or an intracellular cytokine expression Mice assay, as described (17). In some experiments, naive T cells were activated by plate-bound anti-CD3 (10 mg/ml) and soluble anti-CD28 Abs (3 mg/ml) We derived mice with conditional deletion of Irf8 in CD4+ T cells (CD4- fl/fl in plates without exogenous cytokines. For intracellular cytokine detection, IRF8KO) or neurons (aCre-IRF8KO or RX-IRF8KO) by breeding Irf8 cells were restimulated for 5 h with PMA (20 ng/ml)/ionomycin (1 mM). mice with CD4-Cre (Taconic, Hudson, NY) mice or mice expressing the Golgi-stop was added in the last hour, and intracellular cytokine staining Cre-recombinase under the direction of a retina-specific promoter. For fl/fl was performed using BD Biosciences Cytofix/Cytoperm kit (BD Phar- targeted deletion of Irf8 in the neuroretina, we bred the Irf8 mouse strain mingen, San Diego, CA). FACS analysis was performed on a BD Bio- with either a-Cre transgenic mice (provided by Dr. P. Gruss, Max-Planck- sciences FACSCalibur using labeled mAbs and corresponding isotype Institute of Biophysical Chemistry, Gottingen, Germany), which expresses control Abs (BD Pharmingen). For lymphocyte proliferation assays, cells Cre-recombinase only in the retina (aCre-IRF8KO), or RX-Cre transgenic were cultured for 4 d in quintuplet cultures containing IRBP and CFSE. mice (provided by A. Swaroop, National Eye Institute, National Institutes

CFSE dilution assays were performed using a commercially available Downloaded from of Health, Bethesda, MD), which expresses Cre-recombinase in the retina CFSE Cell Proliferation Kit (Molecular Probes, Eugene, OR). Briefly, as well as the retinal pigmented epithelium (RX-IRF8KO). Littermate fl/fl purified CD4 T cells were washed with labeling buffer (PBS/0.1% BSA) Irf8 mice on the C57BL/6J background were used as wild-type (WT) and stained with CFSE (5 mM) for 10 min at 37˚C. The cells were then controls. Mice were maintained and used in accordance with National Eye incubated in RPMI 1640 medium for 10 min and washed with CFSE la- Institute/National Institutes of Health Animal Care and Use Committee beling buffer (23) to remove excess CFSE, and the labeled cells were guidelines (ASP Protocol EY000262-19 and EY000372-14). counted and used for the CFSE dilution assay, as recommended by the Induction of experimental autoimmune uveoretinitis manufacturer. http://www.jimmunol.org/ Mice were immunized with 150 mg bovine interphotoreceptor retinoid- Quantitative and semiquantitative RT-PCR analyses binding protein (IRBP) and 300 mg human IRBP peptide (1–20) in Total RNA was extracted using the TRIzol reagent, according to the pro- 0.2 ml emulsion 1:1 v/v with CFA containing Mycobacterium tuberculosis cedures recommended by the manufacturer (Life Technologies, Gaithers- strain H37Ra (2.5 mg/ml), as previously described (14). The human IRBP burg, MD). All RNA samples were digested with RNase-free DNase I (Life peptide (1–20) is a highly purified peptide purchased from Sigma-Aldrich Technologies) and purified by phenol/chloroform extractions. RNA in- (St. Louis, MO), and R. Caspi (National Institutes of Health) provided the tegrity was verified by analysis of 18S and 28S rRNA expression on RNA bovine IRBP. Mice also received Bordetella pertussis toxin (0.2 mg/mouse) gels. RNA (10 mg), SuperScript III Reverse Transcriptase (Life Technol- concurrent with immunization, and clinical disease was established by ogies), and oligo(deoxythymidine)12–16 were used for first-strand synthe- fundoscopy or histological analysis, as described previously (18). For each sis, as previously described (20). First-strand synthesis containing each EAU experiment, 10 mice were used for the WT or IRF8KO strain. For mRNA sample, but no reverse transcriptase, was performed to control for by guest on October 4, 2021 FACS or intracellular cytokine staining, lymph node (LN) and spleen cells ∼ possible DNA contamination; failure to obtain RT-PCR products with any from 3 mice were pooled and analyzed. Fundoscopy and/or histology of the PCR amplimers confirmed the absence of DNA templates. All were used to monitor progression of pathology in the eyes at 14 or 21 d cDNA preparations used were suitable substrates for PCR amplification on postimmunization. Briefly, following i.p. injection of ketamine (1.4 mg/ the basis of efficient amplification of a b-actin sequence. Real-time PCR mouse) and xylazine (0.12 mg/mouse), pupils were dilated by topical was performed on an ABI 7500 (Applied Biosystems), and PCR param- administration of 1% tropicamide ophthalmic solution (Alcon, Fort Worth, eters were as recommended for the TaqMan Universal PCR kit (Applied TX). To avoid a subjective bias, evaluation of the fundus photographs was Biosystems). Primers and probes were purchased from Applied Bio- conducted without knowledge of the mouse identity by a masked observer. systems. At least six images (two posterior central retinal view; four peripheral retinal views) were taken from each eye by positioning the endoscope and Statistical analysis viewing from superior, inferior, lateral, and medial fields. Each individual lesion was identified, mapped, and recorded. The clinical grading system Statistical analyses were performed by independent two-tailed Student t for retinal inflammation by fundoscopy was based on changes at the retina, test. Data are presented as mean + SD. Asterisks denote p value (*p , optic nerve disc, and choroid, as previously established (19). For histology, 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001). eyes were harvested and fixed in 10% buffered formalin, and specimens were dehydrated through graded alcohols and embedded in paraffin. Serial vertical sections through the papillary-optic nerve plane were cut and Results stained with H&E, as described (18). Clinical scores and assessments of Generation and characterization of CD4-IRF8KO mice disease severity were based on pathological changes at the optic nerve disc We generated mice with targeted deletion of Irf8 in the CD4+ T cell and retinal tissues. compartment (CD4-IRF8KO) to investigate the function of IRF8 Electroretinogram recordings in T cells during the organ-specific autoimmune disease, uveitis. PCR analysis of tail DNA of mice from the cross between CD4- Before the electroretinogram (ERG) recordings, mice were dark-adapted fl/fl overnight and experiments were performed under dim red illumination. Cre and Irf8 mouse strains on C57BL/6J background con- Mice were anesthetized with a single i.p. injection of ketamine (1.4 mg/ firmed the generation of CD4-Cre/IRF8fl/fl double-positive mice mouse) and xylazine (0.12 mg/mouse), and pupils were dilated with (Fig. 1A). Use of the mouse CD4-Cre strain expressing the Cre Midrin P containing 0.5% tropicamide and 0.5% phenylephrine hydro- recombinase under the CD4 promoter element (CD4-Cre strain) chloride (Santen Pharmaceutical, Osaka, Japan). ERGs were recorded using + an electroretinography console (Espion E2; Diagnosys, Lowell, MA) that resulted in the targeted deletion of Irf8 in CD4 T cells, as pre- + generated and controlled the light stimulus. Dark-adapted ERG was viously reported, but also in CD8 T cells due to intrathymic recorded with a single flash delivered in a Ganzfeld dome with intensity of deletion at the double-positive stage (21, 22). Naive LN and 24–1 log cd s/m2 delivered in six steps. Light-adapted ERG was obtained splenic CD4+ T cells, purified by sorting from CD4-IRF8KO or with a 20 cd/m2 background, and light stimuli started at 0.3–30 cd s/m2 in five steps. Gonioscopic prism solution (Alcon Laboratories, Fort Worth, WT mice, were stimulated with anti-CD3/CD28 Abs for 4 d. TX) was used to provide good electrical contact and to maintain corneal Western blot analysis of the total cellular protein extract confirmed moisture. A reference electrode (gold wire) was placed in the mouth, and that the IRF8 protein was indeed absent, specifically in the CD4 The Journal of Immunology 3

T cell compartment of the CD4-IRF8KO mice (Fig. 1B). This resulting in substantial destruction of retinal cells, development of result is consistent with previous studies showing that, whereas more retinal folding, serous retinal detachment, vasculitis, retini- IRF8 is not constitutively expressed in naive T cells, expression is tis, choroiditis, and vitritis. These pathological features are hall- rapidly induced in WT CD4+ T cells following TCR activation (5). marks of severe acute uveitis (18, 23, 24), further reflected by the To further characterize the phenotype of the CD4-IRF8KO T cells, clinical scores shown in Fig. 2B. we examined whether the loss of IRF8 affected their proliferative IRF8 deficiency in CD4+ T cells promotes the expansion of capacity. Naive CD4+ T cells from WT or CD4-IRF8KO mice Th17 cells during EAU were stimulated with anti-CD3/CD28 Abs for 4 d under non- polarizing condition. Analysis of thymidine incorporation indi- Previous studies of the LCK-IRF8KO mouse strain suggested that cated a significant increase in the proliferative response of T cells T cell–specific Irf8 disruption promotes colitis by increasing Th17 from CD4-IRF8KO mice as compared with cells from WT mice expansion. By comparison, no significant differences in the de- (Fig. 1C). We further found that a higher proportion of IRF8- velopment or severity of EAE were observed between WT and deficient CD4+ T cells produced IL-2 than their WT counter- LCK-IRF8KO mice (6, 17). To investigate the mechanistic basis parts (Fig. 1D), providing suggestive evidence that IRF8 may for the development of a more severe uveitis in CD4-IRF8KO function in vivo to restrain T lymphocyte proliferation in response compared with WT mice, we analyzed the immune phenotype + to Ag stimulation. Consistent with the observed effects of IRF8 and cytokine expression profile of CD4 T cells in the LN of both on the proliferation of CD4+ T cells, we also found that CD4- mouse strains. We isolated LN cells of unimmunized mice or IRF8KO T cells exhibited an enhanced activation phenotype, as IRBP-immunized WT or CD4-IRF8KO mice, stimulated the cells indicated by increased proportion of cells expressing CD44 and with IRBP for 4 d in medium containing CFSE, and performed CD25, the high-affinity IL-2R (Fig. 1E). The increased prolifer- intracellular cytokine-staining analysis. Consistent with previous Downloaded from ation and activation phenotype exhibited by the CD4-IRF8KO studies, development of EAU in the WT mice was associated with T cells compared with the WT T cells were accompanied by an increase in Th1 and Th17 cells (13). In keeping with results of the increase in the percentage of the activated CD4+ T cells under- colitis study (6), our data revealed a significant increase in Th17 f/f going (Fig. 1F) and a corresponding elevation in the cells in the LN of CD4-IRF8KO mice as compared with IRF8 expression of proapoptotic genes (Fig. 1G). Consistent with find- mice during EAU (Fig. 3). This result suggests that enhanced + ings of a recent study showing increased production of IL-17 in severity of EAU in mice with IRF8-deficient CD4 T cells derived, http://www.jimmunol.org/ activated CD4+ T cells derived from LCK-IRF8KO mice (6), we in part, from the expansion of Th17 cells. T regulatory (Treg) cells observed a marked increase in cells expressing high intracellular have recently been shown to bring about resolution of autoim- levels of IL-17 in cultures of CD4-IRF8KO cells (Fig. 1H). We mune uveitis (25). We therefore examined whether severe EAU in also stimulated CD4+ T cells from WT or CD4-IRF8KO mice with the CD4-IRF8KO mice could have resulted from perturbation anti-CD3/CD28 Abs for 4 d under the Th17-polarizing conditions. of the levels of Treg cells. Compared with EAU in the WT mice, + The cells were then analyzed by an intracellular cytokine-staining the percentage of Foxp3 Treg cells was significantly reduced in assay. Interestingly, the increased proportion of IL-17+ cells was the LN of CD4-IRF8KO compared with WT mice. However, the + accompanied by a reduction in the frequency of Th17 cells difference in the levels of the Foxp3 T cells between the WT and by guest on October 4, 2021 expressing ROR-gt at high levels and an increased proportion of CD4-IRF8KO mice is relatively small, and thus, the extent to the IL-17–expressing CD4+ T cells that were ROR-gtlow (Fig. 1I). which the modest reduction in Treg cells contributes to disease + Taken together, these findings suggest that functions of IRF8 in exacerbation in mice with IRF8-deficient CD4 T cells is not clear T cells may include the regulation of proliferation and protection (Fig. 3). of T cells from activation-induced cell death. Loss of Irf8 in retinal microglial cells and neurons confers CD4-IRF8KO mice develop more severe EAU protection from uveitis To investigate the impact of deleting IRF8 in CD4+ T cells on In previous reports, we showed that retinal cells express IRF8 development of uveitis, we induced EAU in WT (Irf8fl/fl) and during EAU and that IRF8 expression is localized to the photo- CD4-IRF8KO mice by active immunization with IRBP, a 140-kDa receptors and microglial cells in the ganglion cell layer of the ret- retinal glycoprotein produced by photoreceptor cells (18). Initial ina (12, 13, 26). In view of recent reports that microglial cells of signs of uveitis in the C57BL/6J EAU model are generally ob- the brain express IRF8 and that activation of microglia by IRF8 served by day 12 postimmunization (p.i.) with full-blown uveitis exacerbates neuroinflammation (9, 10, 17), we sought to deter- occurring between days 16 and 22 p.i. with a disease incidence of mine whether the loss of IRF8 in the retina would confer pro- 100% (18). Disease progression was monitored by fundoscopy tection from EAU similar to EAE (17). In the EAE model, mice and confirmed histologically by assessing the extent of lym- with global deletion of Irf8 or loss of IRF8 in and phocyte infiltration of the neuroretina and ocular pathology. macrophage are resistant to EAE (6). In this study, it was most Fundoscopic images obtained on days 14 and 21 p.i. showed the expedient to address the role of IRF8 in mechanisms that regulate development of ocular inflammation in the WT mouse eyes ocular inflammation in the retina by use of a Cre-lox strategy to characterized by blurred optic disc margins and enlarged juxta- generate mice that lack IRF8 in the retina. We generated mice with papillary areas, retinal vasculitis with moderate cuffing, vitreitis, a targeted deletion of Irf8 in the retina using two mouse strains choroiditis, and yellow-whitish retinal and choroidal infiltrates with overlapping patterns of Cre recombinase expression in the (Fig. 2A). In contrast, the fundus images reveal more severe dis- retina (27, 28). The Pax6/aCre mouse strain specifically expresses ease in the eyes of CD4-IRF8KO mice with significantly higher the Cre-recombinase in photoreceptors, retinal neurons, microglia clinical scores compared with the eyes of WT mice (Fig. 2A). cells, and all retinal cell types with the exception of amacrine cells Histological analysis of retinas from eyes harvested 21 d p.i. (27), whereas the Rx-Cre mouse that expresses Cre recombinase underscored the severity of EAU in the eyes of CD4-IRF8KO under direction of the medaka fish promoter element mediates mice. Compared with EAU in the immunized WT mice, the dis- inactivation of in the developing retina (28). ease in the CD4-IRF8KO mice was characterized by the infiltra- PCR analysis of tail DNA of mice from the cross between Pax6/ tion of massive numbers of inflammatory cells into the retina, aCre-Cre or Pax6-Cre and Irf8fl/fl mouse strains on the C57BL/6 4 CONTEXTUAL ROLE OF IRF8 IN NEUROINFLAMMATION Downloaded from http://www.jimmunol.org/

FIGURE 1. Generation and characterization of IRF8 conditional KO mice. Irf8fl/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). Naive CD4+ T cells from LN and spleens of WT or F8 generation CD4-IRF8KO mice (after eight 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 6 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 by guest on October 4, 2021 percentages of IL-2–expressing CD4+ T cells (D) or percentages of activated CD4+ T cells (E). Histogram presented in (D) and (E) represents percentages of IL-2–expressing cells or CD44highCD25+ 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 and I) Naive CD4+ T cells from WT or CD4-IRF8KO mice were stimulated with anti-CD3/CD28 Abs for 4 d 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-gt (mean and SEM, n = 3). Results are rep- resentative of at least three independent experiments. *p , 0.05, **p , 0.01 (Student two-tailed t test). background confirmed the generation of Rx-IRF8KO (Fig. 4A) confirmatory evidence that expression of IRF8 by retinal cells, and aCre-IRF8KO (Fig. 4B) mice with targeted deletion of IRF8 including retinal microglial cells, may promote neuroinflammation in retinal cells. To directly examine the effects of loss of IRF8 in and contribute to the immunopathogenic mechanisms that underlie retinal cells, we induced EAU in WT (Irf8fl/fl), aCre-IRF8KO, the development of uveitis. and RX-IRF8KO mice by active immunization with IRBP in CFA and monitored the disease by fundoscopy or histology. We first Upregulation of anti-inflammatory cytokines in the retina established that IRF8 transcripts were indeed absent in retinal correlates with amelioration of EAU in mice with retina- cells by isolating RNA from retinal cells of WT, RX-Irf8KO, or specific disruption of Irf8 aCre-Irf8KO mice, and then analyzing for expression of IRF8 Production of IL-12 family cytokines such as IL-12 and IL-23 by mRNA by RT-PCR. We show that, although IRF8 transcripts were APCs has been implicated in pathogenic mechanisms that mediate detected in the retina of WT mice, they were not detectable in EAU and EAE (29, 30), whereas IL-27 and IL-35 are thought retinal cells of RX-Cre-IRF8KO or aCre-IRF8KO mice (Fig. 4C). to contribute to the suppression of both diseases (13–16). We Fundus images and histological analyses of the day 21 retina show therefore examined whether mitigation of EAU in aCre-IRF8KO a significant reduction in EAU pathology in the aCre-IRF8KO mice and RX-IRF8KO mice derived in part from perturbations in the (Fig. 4D). Compared with EAU in the immunized WT mice, we levels of pro- and/or anti-inflammatory cytkines in the retina detected fewer numbers of inflammatory cells in the vitreous, less during EAU. We obtained cells from the retina of unimmunized retinal infolding, as well as reduction in other key features of severe C57BL/6J mice or inflamed eyes of IRBP-immunized WT or uveitis—vasculitis, retinitis, choroiditis, and vitritis. Similar reduc- aCre-IRF8KO mice. RNA isolated from the cells was analyzed by tions in pathological features of EAU are also observed in day 21 quantitative PCR to assess the transcript levels of cytokines that retina of Rx-IRF8KO mice immunized with IRBP (Fig. 4E). The regulate inflammation. In line with published data, we detected reduced disease observed in both Irf8-deficient mouse strains, as elevated levels of IFN-g and IL-17 transcripts in the retina of WT reflected by significantly lower clinical scores, provided strong mice with EAU (Fig. 5A). In contrast, the levels of these proin- The Journal of Immunology 5

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 A

progression was analyzed by fundoscopy or histology. ( ) Fundus images of eyes harvested 14 or 21 d postimmunization were taken using an otoen- Downloaded from doscopic 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 d postimmunization. Histologic sections through the retina were stained with H&E, and EAU scores were determined, as described (18). White arrowheads indicate the presence of inflammatory cells in vitreous (V); blue asterisks indicate retinal folds. Results represent at least three independent experiments. **p , 0.01, ***p , 0.001 (Student two-tailed t test). http://www.jimmunol.org/ flammatory cytokines were significantly lower in the retinas of and lymphocyte lineage commitment (31, 32). In the EAE model, Cre-IRF8KO compared with those of WT mice during EAU IRF8 produced by activated microglia exacerbated neuroin- (Fig. 5A). We also detected a significant increase in IL-10 tran- flammation (17) by upregulating the production of IL-12 and IL- scripts in the retina of aCre-IRF8KO mice (Fig. 5A). This sug- 23 while downregulating IL-27, resulting in the induction of gested that the marked reduction of EAU in Irf8 null mouse strains a cytokine milieu that favored the expansion of pathogenic Th17 derived in part from enhanced production of the immunosup- cells. We therefore examined whether loss of IRF8 expression in pressive cytokine, IL-10, and reductions in expression of the in- the retina modulates expression of IL-12 family cytokines in the flammatory Th1 and Th17 cytokines in the target tissue. IL-12 retina during EAU. We found that expression of transcripts for by guest on October 4, 2021 family cytokines have profound influences on Ag presentation Ebi3, a cytokine subunit shared by IL-27 (IL-27p28/Ebi3) and IL-

FIGURE 3. Th17 cells are expanded in CD4-IRF8KO mice during EAU. (A) The immunophenotype of freshly isolated LN cells of unimmunized mice or IRBP-immunized WT or CD4-IRF8KO mice was characterized by FACS and intracellular cytokine-staining assays. Numbers in quadrants indicate per- centages of IL-17–, ROR-gt–, and/or IFN-g–expressing CD4+ or CD8+ T cells. (B) LN cells of unimmunized mice or IRBP-immunized WT or CD4- IRF8KO mice were labeled with CFSE and stimulated with IRBP for 4 d 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-g, or Foxp3. Statistical analysis of the percentage of cytokine-expressing T cells was based on analysis of six mice per group. Results represent at least three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 (Student two-tailed t test). 6 CONTEXTUAL ROLE OF IRF8 IN NEUROINFLAMMATION

FIGURE 4. Loss of Irf8 in neuroretinal cells confers protection from uveitis. (A and B) PCR genotype analysis of tail DNA isolated from WT Irf8f/f or heterozygous Irf8f/2 mice (A and B), RX-Cre-Irf8KO (A)oraCre-Irf8KO (B) mice. (C) RNA was isolated from retinal cells of WT, RX-Cre-Irf8KO, aCre- Irf8KO mice and analyzed for IRF8 expression by RT-PCR. The white lines in (A) and (C) indicate where parts of the image were joined. (D and E)EAU was induced in WT, aCre-Irf8KO (D), or RX-Cre-Irf8KO mice (E) by active immunization with IRBP in CFA. Progression and severity of EAU were Downloaded from 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) is indicated. Clinical scoring was based on changes at the optic nerve disc, retinal vessels, and surrounding tissues, as described in Materials and Methods. Bottom panels, Eyes were enucleated on day 21 post- immunization, fixed in formalin, and 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. Results are representative of at least three independent experiments. ****p , 0.0001 (Student two-tailed t test). Ch, choroid; GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; OpN, optic nerve; RPE, retinal pigment epithelial layer. http://www.jimmunol.org/

35 (IL-12p35/Ebi3), was .3-fold higher in the retinas of aCre- patients and animals. Changes in the ERG occurring during EAU IRF8KO compared with WT mice (Fig. 5B). In contrast, we did are indicative of alterations in visual function (33). We analyzed not observe any differences between WT and CD4-IRF8KO mice scotopic (dark adaptation) and photopic (light adaptation) ERG for expression of IL-12p40, a cytokine subunit shared by IL-12 responses in EAU mice at day 21 postimmunization. We observed (IL-12p35/IL-12p40) and IL-23 (IL-23p19/IL-12p40). Taken to- similar ERG responses after dark or light adaptation in unimmu- gether, these results suggest that mitigation of ocular pathology in nized WT and a-CRE-IRF8KO mice (Fig. 6A). EAU develop-

aCre-IRF8KO mice during EAU may derive in part from in- ment was associated with a marked reduction in photopic b-wave by guest on October 4, 2021 creased expression of IL-27 and IL-35. amplitudes (Fig. 6B). Importantly, the lowest amplitudes were ob- served in the retinas of WT mice with EAU. The light-adapted Loss of Irf8 in the retina prevents the decline of retinal function b-wave amplitudes of the retinas of Irf8-deficient mice with EAU during uveitis were higher than that of the retinas of WT mice with disease, but Electroretinography measures electrical potential change of the lower than the amplitudes for retinas of unimmunized WT mice retina in response to light stimulation arising predominantly from (Fig. 6B). These functional changes reflect corresponding patho- changes in the neural activity of photoreceptors and second-order logical lesions documented for these mouse strains by histological neurons in the retina and is a well-established tool for uncovering findings and fundus changes and confirm results of previous studies gross physiologic changes in the intact retina. Thus, it is routinely showing that rod and cone functions, as measured by ERG, are used to analyze layer-by-layer changes of retinal function in perturbed during the ocular inflammation associated with EAU (33).

FIGURE 5. Anti-inflammatory cytokines are upregulated in the retina of IRF8-defi- cient mice during EAU. EAU was induced in WT or aCre-Irf8KO mice by active im- munization with IRBP in CFA. Retina iso- lated from the eyes of the mice on day 21 postimmunization was digested with colla- genase, and retina cells were analyzed for the expression of IFN-g, IL-17, and IL-10 (A) and IL-35 (EBI3, IL-12p35) or IL-12 (IL-12p40) (B) by quantitative RT-PCR. Re- sults represent at least three independent experiments. *p , 0.05, **p , 0.01, ****p , 0.0001 (Student two-tailed t test). The Journal of Immunology 7

FIGURE 6. Visual function is altered in IRF8KO mice during EAU. (A and B) Visual function of WT or aCre-Irf8KO mice was analyzed by ERG. (A) Downloaded from ERG response after dark or light adaptation was analyzed in unimmunized mice (three mice or six eyes; n = 6). (B) ERG response after light adaptation was analyzed in IRBP/CFA-immunized mice with EAU. Data are presented as the mean 6 SEM of five 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 a-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 a-CRE-IRF8KO mice (10 eyes at each time point, n = 10). *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 (Student two-tailed t test). http://www.jimmunol.org/

Discussion significant inhibition of Th1 cells. It is therefore noteworthy that IRF8 is known primarily as a transcription factor that plays crit- the marked decrease of Th1 cells in CD4-IRF8KO mice with ical roles in regulating myeloid and B cell lineage commit- exacerbated uveitis is consistent with the notion that Th1 cells ment and maturation (4, 8–10). However, its functional role in may have a protective role in uveitis (13, 39). In contrast to the T lymphocytes other than CD8+ T cells is not well understood. In pathogenic roles played by Th17 cells in human and mouse uveitis this study, we generated mice with a targeted deletion of Irf8 in the (13, 39), Treg cells are immunosuppressive and have recently been CD4+ T cell compartment (CD4-IRF8KO) and used them to in- shown to be effective in ameliorating EAU (25). We show in this vestigate the contribution of IRF8 to the regulation of CD4+ T cell work that the enhanced EAU pathology seen in CD4-IRF8KO also by guest on October 4, 2021 effector functions with particular emphasis on their role in regu- correlated with a small, but significant reduction in the levels of lating immunopathogenic mechanisms that mediate the CNS au- Foxp3+ Treg cells. However, it is doubtful that the modest re- toimmune diserase, uveitis (34, 35). CD4-IRF8KO CD4+ T cells duction in Treg cells played a major role in the disease exacer- proliferated more than T cells from WT mice. The increased bation observed in the IRF8-deficient mice. It is also not clear how proliferation was accompanied by elevated levels of CD4+ T cells loss of IRF8 results in disparate effects on Th17 and Th1 cells as undergoing apopotosis and upregulated expression of proapoptotic well as Treg cells, three important CD4+ T subsets that play genes. This suggests that a major function of IRF8 in CD4+ T cells critical roles in the development or regulation of autoimmune may be to restrain proliferation and protect CD4+ T cells from diseases. Clues to the possible involvement of IRF8 in the regu- activation-induced cell death. It is, however, of note that IRF8 is lation of Th cell effector functions or lineage specification can be also absent from CD8+ T cells because of Cre recombinase- informed by recent reports showing that TCR signaling activates mediated deletion of Irf8 at the double-positive (CD4+CD8+) pioneering transcription factors such as BATF and IRF4 to recruit stage of T cell development in the thymus. We and others have inflammation-associated transcription factor to target gene struc- shown that CD4-Cre mice do indeed mediate targeted gene de- tures during Th cell differentiation (40, 41). In the context of the letion in both CD4+ and CD8+ T cells (36–38). However, in this Th17 differentiation program, proinflammatory environmental study, we have focused on effects of Irf8 deletion in CD4+ T cells cues, such as hypoxia, have recently been shown to stabilize the because EAU is predominantly mediated by CD4+ T cells and, in Th17 lineage while simultaneously destabilizing the Treg lineage particular, Th17 and Th1 cells. by promoting the interactions of hypoxia-inducible factor 1a with We further show that the effect of IRF8 on host immunity is ROR-gt and Foxp3, respectively (41, 42). In contrast, interactions contextual, depending on cell type and the target organ system of IRF8 with ROR-gt have the specific effect of silencing Th17 responding to IRF8 signaling. CD4-IRF8KO mice developed more developmental pathways (6). Data presented in this work indi- severe disease characterized by the development of hallmark cate that IRF8 may antagonize T cell activation and prolifer- features of severe uveitis and enhanced expansion of uveitogenic ation, raising the possibility that competition between IRF8 and Th17 cells. These results are consistent with a previous report of hypoxia-inducible factor 1a for binding to lineage-specific master exacerbated colitis in LCK-IRF8KO mice lacking IRF8 in T cells transcription factors may result in diametrically opposed effects during the later stages of T cell development (6). However, in on Th cell development and effector functions. However, because contrast to the mouse colitis model in which severe colitis in the IRF8 activities are dependent on its interactions with other tran- LCK-IRF8KO mice resulted from increased effects of Th17 cells scription factors (1), its exact role in host immunity is difficult to without perturbations of Th1 cells, exacerbation of ocular in- discern, as it is highly dependent on these partners and their ability flammation in the CD4-IRF8KO mice was also associated with to recruit IRF8 to active chromatin loci. 8 CONTEXTUAL ROLE OF IRF8 IN NEUROINFLAMMATION

Another surprising observation in this study is that IRF8 ex- expansion of pathogenic T cells that mediate neuroinflammation pression by T cells may have different effects depending on the (17). We show in this work that the loss of IRF8 in retinal organ-specific autoimmune disease. For example, EAU and EAE microglia confers protection from severe uveitis. These observa- are two well-characterized models of the human CNS autoimmune tions thus highlight the multiple roles played by IRF8 in host diseases, uveitis and multiple sclerosis, respectively, and both immunity and raise a cautionary note in the context of therapeutic diseases share essential immunopathological mechanisms in terms targeting of IRF8. of critical roles of Th17 cells in their etiology (35, 43). It is therefore surprising that loss of IRF8 in CD4+ T cells exacerbates Acknowledgments EAU, but does not have a significant role in EAE (17). A possible We thank Dr. Haohua Qian and Yichao Li (Visual Function Core, National explanation for the different outcomes may relate to the different Eye Institute, National Institutes of Health) for ERG technical assistance strategies used to delete IRF8 in T cells. In this study, we deleted and Rafael Villasmil (National Eye Institute/National Institutes of Health Irf8 in T cells using the CD4-Cre mice with highly restricted FLOW Cytometry Core facility) for cell sorting and FACS analysis. We expression of the Cre recombinase in the CD4 compartment (21, also thank Rashid M. Mahdi (Molecular Immunology Section, National 22), whereas, in the EAE model, the Lck-Cre mice was used to Eye Institute) for technical assistance. delete Irf8 in T cells. It is of note that the Lck-Cre mouse strain does not faithfully recapitulate the expression pattern of the cor- Disclosures responding endogenous Lck gene, which can result in off-target The authors have no financial conflicts of interest. expression of the Cre recombinase (21, 22, 44). Nonetheless, if the difference derives from use of CD4-Cre versus the Lck-Cre mice, References Downloaded from it is difficult to reconcile the fact that colitis was exacerbated, 1. Lohoff, M., and T. W. Mak. 2005. Roles of -regulatory factors in whereas development or severity of EAE was not affected despite T-helper-cell differentiation. Nat. Rev. Immunol. 5: 125–135. the use of Lck-IRF8KO mice in both studies (6). 2. Tamura, T., H. Yanai, D. Savitsky, and T. Taniguchi. 2008. The IRF family transcription factors in immunity and oncogenesis. Annu. Rev. Immunol. 26: Data presented in this work showing that the loss of IRF8 in 535–584. retinal cells prevents the decline in retinal function during uveitis 3. Driggers, P. H., D. L. Ennist, S. L. Gleason, W. H. Mak, M. S. Marks, B. Z. Levi, extend our previous observation that the expression of IRF8 by J. R. Flanagan, E. Appella, and K. Ozato. 1990. An interferon gamma-regulated

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