Article

Complement receptors C3aR and C5aR are required in the pathogenesis of experimental autoimmune uveitis † ‡ ‡ ‡ { Lingjun Zhang,*, Brent A. Bell, Minzhong Yu, Chi-Chao Chan,§ Neal S. Peachey, John Fung, † Xiaoming Zhang,*,1 Rachel R. Caspi,§ and Feng Lin ,1 † *Eye Research Institute, Tianjin Medical University Eye Center, Tianjin, China; Department of Immunology, Lerner Research ‡ { Institute, Department of Ophthalmic Research, Cole Eye Institute, and Digestive Disease Institute, Cleveland Clinic, Cleveland, Ohio, USA; and §Laboratory of Immunology, National Eye Institute, National Institutes of Health, Bethesda, Maryland, USA RECEIVED APRIL 16, 2015; REVISED AUGUST 27, 2015; ACCEPTED SEPTEMBER 3, 2015. DOI: 10.1189/jlb.3A0415-157R

ABSTRACT used to study the pathogenesis of autoimmune uveitis and to develop Recent studies have suggested that reagents inhibiting new therapies for this disease [2]. Adoptive transfer of activated T cells complement activation could be effective in treating from animals with EAU is sufficient to induce disease in na¨ıve mediated autoimmune diseases such as autoimmune recipients [3, 4], suggesting that this disease is mainly T cell mediated. uveitis. However, the precise role of the complement It has been established that autoreactive Th1 and Th17 cells are the ( and C5a receptors) in the primary mechanisms underlying the pathogenesis of EAU [5]. pathogenesis of autoimmune uveitis remains elusive and Although complement has been thought to function as an effector controversial. We induced experimental autoimmune system for [6], recent accumulating evidence indicates that uveitis in mice deficient or sufficient in both C3a and C5a in addition to its conventional roles in the innate , receptors and rigorously compared their retinal pheno- type using various imaging techniques, including indirect complement is also important in directly regulating T cell responses ophthalmoscopy, confocal scanning laser ophthalmos- in the adaptive immune response through different mechanisms copy, spectral domain optical coherence tomography, [7]. Among them, the complement receptors C3aR and C5aR have topical endoscopic fundus imaging, and histopathological been found to synergically augment T cell responses in several analysis. We also assessed retinal function using elec- studies in which C3aR and C5aR—whetheronAPCsoron troretinography. Moreover, we performed Ag-specific Tcells—have been shown to be required for a robust T cell TcellrecallassaysandTcelladoptivetransferexperi- response [8–12]. However, contradictory reports have shown that ments to compare pathogenic T cell activity between adeficiency of C5aR on dendritic cells actually promotes pathologic wild-type and knockout mice with experimental autoim- Th17 responses and that blockade of C5aR enhances Th17 mune uveitis. These experiments showed that C3a cellnumbersinanexperimentalmodelofasthma[13]. /-deficient mice developed much Several lines of previous studies have suggested that activated less severe uveitis than did control mice using all retinal fi examination methods and that these mice had reduced complement contributes signi cantly to the pathogenesis of EAU – pathogenic T cell responses. Our data demonstrate that [14 16]. Also, at least some of these studies [15, 16] have suggested both complement anaphylatoxin receptors are important that complement does so by regulating T cell responses, implying for the development of experimental autoimmune uveitis, an important role for C3aR and C5aR in the development of EAU. suggesting that targeting these receptors could be a valid However, surprisingly, a recent brief report [17] suggested that approach for treating patients with autoimmune uveitis. C3aR and C5aR are not required, because no retinal histopatho- J. Leukoc. Biol. 99: 447–454; 2016. logical difference was noted between WT and C3aR/C5aR KO mice

after they were immunized with IRBP1–20 peptide to induce EAU. Introduction To understand further the mechanism by which complement regulates the pathogenesis of EAU and to clarify the importance of Autoimmune uveitis, a major cause of blindness, has an unclear the complement anaphylatoxin receptors in the development of etiology, and many patients fail to respond to treatment [1]. EAU, EAU, we compared the disease severity in C3aR/C5aR KO mice induced by IRBP peptide immunization in mice, has been widely and WT mice with EAU. In our experiments, IRBP1–20 immuniza- tion resulted in a mild and inconsistent disease with incomplete Abbreviations: AF = autofluorescence, C3aR = , C5aR = C5a penetrance, making the data difficult to analyze. In the present receptor, CC = choroidal complex, cSLO = confocal scanning laser ophthalmoscopy, EAU = experimental autoimmune uveitis, ERG = electro- retinography, IR = infrared, IRBP = interphotoreceptor retinoid-binding 1. Correspondence to: F.L., Department of Immunology/NE60, Lerner protein, KO = knockout, RFDF = red free dark field, RPE = retinal pigment Research Institute, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH epithelium, SD-OCT = spectral domain optical coherence tomography, 44195, USA. E-mail: [email protected]; X.Z., Eye Research Institute, Tianjin TEFI = topical endoscopic fundus imaging, VRI = vitreoretinal, WT = wild-type Medical University, Tianjin, China. E-mail: [email protected]

0741-5400/16/0099-447 © Society for Leukocyte Biology Volume 99, March 2016 Journal of Leukocyte Biology 447 fi study, we used a recently de ned IRBP651–670 peptide, which is fundus. Algorithms within the software package were used for automatic real- time tracking and mean averaging of sequentially collected images to further superior to the IRBP1–20 peptide in terms of EAU penetrance and scoring [18]. We monitored EAU development by indirect enhance the signal-to-noise ratio. A manually controlled z-axis focus- adjustment knob on the system was used for linear axial translation of the ophthalmoscopy and examined the fundus in detail using cSLO, SD- cSLO imaging plane [25], which enabled focal plane advancement through OCT, TEFI, and retinal histopathological analysis. We also evaluated the retina, albeit with relatively low axial spatial resolution [25, 26]. The retinal function using ERG and assessed Ag-specific T cell responses RPE–CC and VRI interfaces were identified and served as reference points for using recall assays and T cell adoptive transfer experiments. In notation of the anterior and posterior retinal margins [25]. The adjustable contrast to the data obtained with the IRBP1–20 model of EAU, our focus was used to identify and document any retinal pathology observed using data have demonstrated that these 2 complement anaphylatoxin IR-cSLO and AF-cSLO imaging modes [21]. receptors are important in the pathogenesis of EAU, because they An SD-OCT system (840 HR SDOIS; Bioptigen, Inc., Morrisville, NC, USA) with a center operating wavelength of ;840 nm and an in-depth, axial are required for the development of pathogenic T cells. resolution of ;6 mm was used in the studies. Images were collected with a 50° field-of-view objective, which provides a lateral resolution of ;2.5 mmand fundus field of view of ;1.5 mm [27]. MATERIALS AND METHODS Conventional visible light fundus images were collected using a custom- fabricated TEFI apparatus that has been previously described [28]. This Animals system provides a fundus field of view of ;70°. C57BL/6J WT and C3aR/C5aR double-KO mice (backcrossed with C57BL/6J mice for .12 generations, 8–12 wk old) were developed and have been described Electroretinography in previous reports [10, 11, 19]. These mice were maintained under pathogen-free fl conditions in the animal facility of Lerner Research Institute, Cleveland Clinic Conventional strobe- ash ERG was used to evaluate the responses of the outer (Cleveland, OH, USA). The Institutional Animal Care and Use Committee of retina, as described previously [29, 30], using a UTAS-E3000 signal averaging Cleveland Clinic approved all the procedures involving mice, all of which were system (LKC Technologies, Gaithersburg, MD, USA). In brief, after keeping performed in accordance with the U.S. Department of Health and Human Services the mice in the dark overnight, darkness-adapted ERGs were recorded from 2 GuidefortheCareandUseofLaboratoryAnimals and institutional guidelines. the corneal surfaces of each mouse. Stimulus luminances, ranging from 3.6 to 2.1 log cd-s/m2, are presented in increasing order. The interstimulus interval was progressively increased from 4 to 90 s. For each stimulus Induction of EAU condition, $2 successive responses were averaged. After $7 min of light For induction of EAU, the WT and KO mice were immunized by adaptation, a series of flash luminances from 20.8 to 1.9 log cd-s/m2 was subcutaneous injection of 200 ml of an emulsion containing 200 mg of human presented at 2 Hz. Twenty-five successive responses were averaged for a single IRBP651–670 peptide LAQGAYRTAVDLESLASQLT [20] (custom synthesized stimulus condition. The a-wave amplitude was measured 5 ms after flash onset by GenScript USA Inc., Piscataway, NJ, USA) and 250 mgofMycobacterium from the prestimulus baseline. The b-wave amplitude was measured from the tuberculosis H37Ra in complete Freund’s adjuvant (Difco Laboratories, Inc., trough of the a-wave to the peak of the positive b-wave or, if no a-wave was Detroit, MI, USA). The emulsion was injected into each thigh (50 ml each) present, from the prestimulus baseline. and at the base of the tail (100 ml). A single dose of 500 ng of pertussis toxin (List Biologic Laboratories, Inc., Campbell, CA, USA) was given by T cell recall assays intraperitoneal injection after immunization on the same day. T cell recall assays were performed on d 14 (experiment 1) or d 20 5 Clinical and histopathological scoring of EAU (experiments 2 and 3). Splenocytes (4 3 10 ) from each of the immunized WT and KO mice were seeded in 96-well round-bottomed microtiter plates The mice were examined daily for clinical signs of EAU using a binocular without or with the presence of 20 mg/ml IRBP651–670 peptide or the same indirect ophthalmoscope (Keeler Instruments, Inc., Broomall, PA, USA). The concentration of a nonrelevant peptide (ovalbumin323–339), in a total volume pupils were dilated using a mixed ophthalmic solution of 0.5% tropicamide of 100 ml. Culture supernatants were collected after 72 h of incubation for and 1.25% phenylephrine hydrochloride. The disease severity was scored on IFN-g and IL-17 concentration measurement using conventional ELISA – a scale of 0 4, according to published criteria [2]. (BioLegend, San Diego, CA, USA). For retinal histopathological evaluation, the mice were euthanized, and the whole eyes were collected on d 14 or 20 after immunization and immersed in 10% formaldehyde in PBS buffer for fixation. The fixed tissues Adoptive transfer of uveitis were embedded in paraffin and processed. Sections of 5 mmwerecut At 14 d after immunization, the mice were euthanized. Splenocytes (; 1 3 through the pupil and optic nerve axis and stained with H&E. Retinal 108) from each of the WT and KO mice with EAU were cultured in the histopathological changes were graded in a blinded fashion at the National presence of 10 mg/ml IRBP peptide and 10 ng/ml IL-23 for 72 h in RPMI Eye Institute using 4 sections for each eye, according to previously published 1640 supplemented with 10% FBS, 2 mM glutamine, 100 U/ml penicillin, and scoring criteria [2]. 100 mg/ml streptomycin (Life Technologies, Carlsbad, CA). After incubation, blasting T cells (activated Ag-specific T cells) were enriched by Ficoll Retinal imaging centrifugation and counted. The same number (5 3 106 cells per mouse) of the in vitro–activated WT or KO T cells were infused by intraperitoneal Retinal imaging was performed using cSLO, SD-OCT, and TEFI in accordance injection into na¨ıve WT recipient mice. The clinical scores of these mice with published protocols after general anesthesia [21, 22]. cSLO (Heidelberg were recorded under indirect ophthalmoscopy examination, and eye Retina Angiograph II; Heidelberg Engineering, Carlsbad, CA, USA) collects sections were prepared for histopathological examinations at the end of the both reflectance and fluorescence information from the posterior segment of experiment. the mouse [23]. Equipped with a 55° wide-field objective lens, the system provides an estimated field of view of ;1.6 mm in mice [24]. Infrared cSLO (l = 815 nm illumination/reflection), infrared dark field cSLO (l =815nm Statistical Analysis illumination/crossed polarized filtered reflection), RFDF-cSLO (l =488nm The clinical scores and ERG results were assessed by repeated-measures illumination/crossed polarizer filtered reflection), visible AF-cSLO (l =488 ANOVA. Scores from the 2 eyes of 1 mouse were averaged and considered nm excitation/500–680 nm emission), and infrared autofluorescence cSLO a single event, as an independent variable. Student’s t test was used for 2 sets of (l = 795 nm excitation/.800 nm emission) modes were used to image the data. Data are expressed as the mean 6 SD, except for adoptive transfer

448 Journal of Leukocyte Biology Volume 99, March 2016 www.jleukbio.org Zhang et al. Complement receptors C3aR/C5aR required for EAU results, which are presented as the mean 6 SEM. Statistical significance was set SD-OCT (Fig. 2K). These lesions were often interconnected at P , 0.05. when closer to the optic nerve and occasionally formed a semicontiguous ring around it (Fig. 2A and F). Beyond the initial ring, most lesions were isolated as individual spots and/ RESULTS or retinal infoldings. SD-OCT imaging also showed prominent fi fi changes to the outer retinal architecture in 75% of the WT De ciency of C3aR/C5aR results in signi cantly mice examined, including complete loss of external limiting reduced EAU clinical scores membrane and inner segment/outer segment bands. Hyper- fi We rst immunized 8 WT and 8 KO mice with IRBP651–670 reflective retinal infoldings originate from the RPE apical peptide to induce EAU. For the next 14 d, we examined the surface, extending into the outer nuclear layer, and displacing retina by indirect ophthalmoscopy and recorded the clinical inner retina lamina including outer plexiform layer, inner scores daily, in accordance with previously published scoring nuclear layer, and inner plexiform layer. KO mice rarely criteria [2]. These experiments showed that although WT mice exhibited any of these features, and if they did, the features fl developed retinal in ammation, KO mice were protected, which were substantially less in magnitude than in the WT mice (Fig. fi was indicated by the signi cantly lower clinical scores for the KO 2B, G–J, and L). mice than for the WT mice (Fig. 1A). We repeated the experiment 2 more times with 5 mice per group and monitored Anaphylatoxin receptors C3aR/C5aR reduce ERG the mice until d 19. These experimental results showed that the sensitivity in the uveitic mouse retina KO mice consistently developed less severe EAU than the WT ERG is another sensitive assay to evaluate EAU severity from mice (Fig. 1B and C). These data indicate that the anaphylatoxin a different perspective [33, 34]. We used strobe flash ERGs to receptors C3aR/C5aR are indeed required for the pathogenesis compare the outer retinal function of WT and KO mice for of EAU. retinal injury assessment 14 d after immunization as a comple- mentary approach for the imaging assays used. These experi- Retinal imaging assessments of C3aR/C5aR-sufficient ments showed that under every intensity of flash stimulus, all vs. C3aR/C5aR-deficient mice with uveitis ERG amplitudes of both a- and b-waves under the dark-adapted In addition to using indirect ophthalmoscopy examinations to condition and the b-wave under the light-adapted condition were assess retinal inflammation, we evaluated EAU severity in one all significantly lower in the WT mice with EAU than in the KO half of the WT and KO mice on d 14 by cSLO and SD-OCT, 2 mice with EAU (Fig. 3). This finding suggests that the function of imaging techniques widely used in the clinic to evaluate uveitis photoreceptors and bipolar cells in both rod and cone pathways in patients [31, 32]. We also used TEFI to obtain visible light of WT mice was damaged more severely than in KO mice, a result fundus images for comparison. In agreement with previous consistent with the augmented EAU severity in WT mice, which results obtained from indirect ophthalmoscopy examinations, was assessed by indirect ophthalmoscopy, cSLO, SD-OCT, and these imaging assessments revealed the consistent presence of TEFI (Figs. 1 and 2). multiple retinal pathologic features in the WT but not in the KO mice (Fig. 2), including 1) numerous chorioretinal lesions Anaphylatoxin receptors C3aR/C5aR increase observed by TEFI (Fig. 2A vs. B), 2) increased AF features histopathological scores in EAU mice observed using AF-cSLO in the inner retina (Fig. 2D vs. H) in We next evaluated EAU severity by retinal histopathological close proximity to the VRI, adjacent to the retinal vasculature analysis. At the end of each experiment, the eyes were harvested, and nerve fibers, and in the ganglion cell layer, 3) asymmetric, and paraffin-embedded ocular sections were prepared for H&E hyperreflective features (Fig. 2E vs. I) observed using IR-cSLO staining. The slides were examined and scored in a blinded that circumvent and/or extend from the optic nerve, 4) fashion at the National Eye Institute in accordance with the elongated AF lesions observed in the outer (Fig. 2F vs. J) retina established mouse EAU histopathological scoring criteria [2]. by AF-cSLO, and 5) reflective foci within the vitreous chamber These examinations found that in all 3 experiments, the KO mice and prominent retinal infoldings observed by SD-OCT (Fig. 2K displayed significantly reduced histopathological scores com- vs. L). The hyperreflective (Fig. 2A) and AF (Fig. 2F) lesions pared with the WT mice after EAU induction (Fig. 4), in seen using TEFI, RFDF-cSLO (image not shown), and AF-cSLO accordance with all previous retinal imaging and function assays correlated directly with the retinal infoldings observed using (Figs. 2 and 3).

Figure 1. C3aR/C5aR KO mice have decreased clinical scores in EAU. WT and KO mice were immunized to induce EAU, and the degree of manifestation of clinical uveitis was examined using a binocular indirect ophthalmoscope (n = 8 in each group for the first experiment, n = 5 in each group for the second experiment, and n = 5 in each group for the third experiment). Clinical scores from the 2 eyes of each mouse were averaged and consid- ered as a single event. *P , 0.05. DKO, double knockout; EXP., experiment.

www.jleukbio.org Volume 99, March 2016 Journal of Leukocyte Biology 449 Figure 2. Representative ocular pathology exam- ples documented by in vivo imaging. Hyperre- flective lesions were observed in WT (A) but not KO (B) mice using TEFI. cSLO images further isolated these features as hyperreflective and/or AF changes to both the inner (C–H) and outer (E–J) retina. Numerous foci can be seen adjacent to the retinal vessels and in the avascular space of WT (D, arrows) vs. KO mice (H, downward- pointing arrows). Hyperreflective streaks (arrows) can be observed emanating from the optic disk at ;30°, 150°, and 270°. Other hyperreflective areas (circled) can be seen (E), which were rarely observed (circled) in KO mice (I). AF-cSLO (F) revealed AF lesions (arrows) previously shown by TEFI (A). KO mice (J) were void of AF lesions resembling those observed in WT mice (F). SD- OCT B-scans ranging from the posterior lens to beyond the choroid revealed more reflective foci in the WT mice (K, down arrows) compared with those in the KO mice (L). The foci likely represented clusters of active inflammatory cells or cellular debris in the vitreous cavity (VC) in close proximity to the VRI (K). SD-OCT image field of view = 1 mm 3 1.5 mm.

Adoptive transfer of in vitro–activated C3aR/C5aR- C3aR/C5aR-deficient T cells are less potent than WT T cells for deficient T cells induces ameliorated EAU inducing EAU. C3aR and C5aR are present on T cells, especially activated fi T cells [11], and on APCs, such as dendritic cells and Immunologic assays for IRBP-speci c Th1 and [35]. To assess the function of the T cells from Th17 responses WT and C3aR/C5aR KO mice with EAU, we compared the In addition to the clinical and histopathological evaluation of efficacy of the same number of WT and KO T cells in inducing disease, we performed Ag-specific recall assays to assess the retinal inflammation in na¨ıve recipient mice. Using an pathologic IRBP-specific Th1 and Th17 responses in both WT established protocol [36, 37], we activated the IRBP-specific and KO mice with EAU [15]. After incubating splenocytes from T cells in vitro by culturing splenocytes from WT and KO mice WT and KO mice with EAU in the presence of the immunizing with EAU using the same concentrations of IRBP peptide, IRBP peptide or a nonrelevant peptide, we measured the levels together with IL-23, for 3 d. We then enriched the activated of IFN-g and IL-17 (signature for Th1 and Th17 (blasted) T cells by Ficoll centrifugation and adoptively trans- response) in the culture supernatants using conventional ferred a fixed number of the in vitro–activated (blasted) WT or ELISA. In keeping with the markedly reduced clinical, KO T cells into na¨ıve recipient mice. We evaluated EAU functional, and histopathological manifestations of the retinas, development by indirect ophthalmoscopy. Adoptive transfer of theKOmiceconsistentlyshowedsignificantly reduced IBRP- activated WT T cells induced more severe retinal inflammation specificIFN-g and IL-17 responses compared with the re- than did the same number of KO T cells (Fig. 5), showing that sponses of the WT mice (Fig. 6), suggesting that C3aR and

450 Journal of Leukocyte Biology Volume 99, March 2016 www.jleukbio.org Zhang et al. Complement receptors C3aR/C5aR required for EAU

Figure 3. ERG examination of WT and KO mice in EAU. Strobe flash ERGs were used to evaluate the function of photoreceptors and bipolar cells. Representative ERG waveforms were selected from 4 WT mice and 5 KO mice under either dark- or light-adapted conditions (A). B) The intensity- response curves of the a-wave and b-wave in the dark-adapted condition and the b-wave in the light-adapted condition from WT and KO mice with EAU. According to repeated-measures ANOVA, which summarized the responses under a series of stimulus intensities, significant differences were present between the examined WT and KO mice in ERG a- and b-wave amplitudes in the dark-adapted condition and b-wave amplitudes in the light-adapted condition (a-wave: degrees of freedom = 1, 7; F = 6.249; P = 0.041; b-wave in dark-adapted condition: degrees of freedom = 1, 7; F = 7.566; P = 0.028; and b-wave in light-adapted condition: degrees of freedom = 1, 7; F = 6.930; P = 0.034).

C5aR are required for development of a robust autoreactive retina function and less-intense IRBP-specific Th1/Th17 re- T cell response necessary to induce EAU. sponses in KO mice than in WT mice, suggesting that both C3aR and C5aR are important in the pathogenesis of EAU. DISCUSSION Complement is an important part of the innate immune system, which primarily serves as the body’s first defense against In the present project, using WT and C3aR/C5aR KO mice, we pathogen invasion [38]. After activation, the complement- studied the significance of the complement anaphylatoxin activation product and its derivatives bind to the target cells, receptors in the pathogenesis of immunization-induced EAU. facilitating phagocytosis, a process known as opsonization. Using multiple imaging techniques, including indirect ophthal- Complement activation also leads to the formation of membrane moscopy, cSLO, SD-OCT, TEFI, and retinal histopathological attack complexes, which directly assault the target cells. In analysis, we consistently found that KO mice had less severe addition to these cell-bound complement-activation products, retinal inflammation than WT mice after immunization. These (including C3a and C5a) are produced and imaging results are consistent with the results from ERG and released into the fluid phase during complement activation. immunologic assays, which showed better preservation of outer After binding to their respective receptors C3aR and C5aR, both

www.jleukbio.org Volume 99, March 2016 Journal of Leukocyte Biology 451 Figure 4. C3R/C5aR KO mice showed reduced histopathological scores in EAU. Paraffin-embedded ocular sections were prepared and stained by H&E. The slides were examined in a blinded fashion at the National Eye Institute, and histo- pathological scores were assigned. A) Histopath- ological scores of WT and KO mice in EAU from 3 independent experiments. Each dot represents the average score of 2 eyes from 1 mouse. *P , 0.05. B) Representative retinal histologic images of WT and KO mice in EAU. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer.

of which are G-protein–coupled receptors, C3a and C5a can leading to enhanced T cell responses [10, 42]. In addition, it has activate and recruit leukocytes to the site of complement been demonstrated that treating mice with an anti-C5 mAb, activation to facilitate inflammation and clear up the invading which inhibits C5 activation and C5a release, reduced the disease pathogens [39]. In addition to these conventional roles of severity of EAU [16]. All these data associating complement with complement in fighting infections as a part of the innate the pathogenesis of EAU, and other studies showing the immune system, accumulating evidence suggests that comple- importance of C3aR and C5aR in regulating T cell responses in ment is also important in regulating T cell responses from the other disease models [43], strongly suggest that C3aR/C5aR would . C3aR and C5aR have received much be required for the development of EAU. Moreover, the results of attention as key players for complement to directly regulate T cell studies using a similar ocular disease model, experimental responses, because both APCs and T cells express these autoimmune anterior uveitis, also strongly suggest that comple- anaphylatoxin receptors [40]. It has been demonstrated that ment and, potentially C3a/C5a, should be integrally involved in during APC and T cell interaction, both partners locally produce autoreactive T cell response development and ocular inflamma- complement components, and complement activation occurs tion [44, 45]. Our data support and expand this interpretation inside or near the immune synapse to produce C3a and C5a [11]. through imaging, functional, histopathological, and immunologic These locally produced C3a and C5a components, after binding evidence that C3aR/C5aR KO deficiency reduces disease severity to C3aR and C5aR on APCs, promote the surface expression of and the associated immunologic effector responses. MHC and costimulatory molecules, facilitating priming [10]. At Surprisingly, a recent brief report has suggested that C3aR/ the same time, they also bind to C3aR and C5aR on T cells; this C5aR KO mice with the same C57BL/6 background did not show binding inhibits activation-induced T cell apoptosis, facilitates any defect in developing EAU compared with control WT mice Th1 and Th17 effector T cell differentiation [11], and inhibits [17]. The exact reasons behind this apparent discrepancy are the development of the immunosuppressive Foxp3+ T-regulatory unclear; however, housing conditions could have played a role, cells [19, 41], leading to more robust T cell responses. because environmental factors, including the gut microbiome, EAU has been established as a primarily T cell mediated model has been found to affect the pathogenesis of many disease for autoimmune posterior uveitis [2]. Although the conventional role of complement (e.g., membrane attack complex formation and opsonization) is not likely to significantly contribute to the pathogenesis of the disease, it is possible that complement is still important through its newly discovered T cell regulatory activities. It has been reported that C3-deficient mice and mice overexpressing a soluble form of the complement inhibitor Crry show signs of less severe EAU after immunization as assessed by histopathologic examination [14]. However, the potential effect of these genetic changes on the immune response associated with EAU was not examined in this study. We have shown that mice deficient in the cell-surface complement inhibitor CD55, in which local complement activation is therefore insufficiently Figure 5. C3aR/C5aR KO T cells have defect in inducing EAU after controlled, had enhanced retinal inflammation in EAU and adoptive transfer. The same numbers of in vitro restimulated and activated (blasting) T cells from WT and KO EAU mice were adoptively augmented IRBP-specific Th1/Th17 responses [15]. These transferred into na¨ıve recipient mice. The respective clinical scores were results are consistent with previous reports showing that in the recorded (A), and the representative retina images of these adoptively absence of CD55, augmented levels of C3a and C5a are produced transferred mice are presented (B) (n = 10 in each group). Data are that interact with C3aR and C5aR on APCs and/or T cells, presented as mean 6 SEM.*P , 0.05.

452 Journal of Leukocyte Biology Volume 99, March 2016 www.jleukbio.org Zhang et al. Complement receptors C3aR/C5aR required for EAU

Figure 6. C3aR/C5aR mice show reduced Ag- specific Th1 and Th17 responses in EAU. Spleno- cytes were incubated with or without 10 mg/ml IRBP peptide or the same concentration of ovalbumin (OVA)323–339 peptide for 3 d. The levels of IFN-g, a signature for Th1 cells, and IL- 17, a signature cytokine for Th17 cells, were measured by ELISA. Results shown are from 3 independent experiments. *P , 0.05. EXP., experiment.

models, including EAU [46–48]. Also, the histopathological AUTHORSHIP analysis was the only assay used to compare the severity of EAU L.Z., B.A.B., M.Y., and C.-C.C. performed the experiments, between WT and KO mice for the whole study [17]. Thus, any analyzed data, and edited the manuscript. N.S.P., J.F., X.Z., and effects on disease (onset, kinetics) up to that point would have R.R.C. analyzed data and reviewed and/or edited the manu- been missed. Finally, IRBP – is a very weak , resulting in 1 20 script. F.L. designed the experiments, analyzed the data, and borderline disease scores and incomplete penetrance, making prepared the manuscript. data in which disease inhibition is expected difficult to interpret [2]. In the present study, in addition to rigorously examining the ACKNOWLEDGMENTS retina using various imaging and histopathological techniques, we evaluated retinal function using ERG and autoreactive T cell This work was supported in part by U.S. National Institutes of activities by Th1/Th17 recall assays and T cell adoptive transfer Health National Institute of Arthritis and Musculoskeletal and Skin experiments. Moreover, we used the IBRP651–670 peptide, a more Diseases (Grant AR061564) and National Institute of Diabetes and recently discovered epitope that is uveitogenic in C57BL/6J Digestive and Kidney Diseases (Grant DK10358), and the mice, and induces more robust and reproducible EAU in WT Foundation Fighting Blindness, Research to Prevent Blindness, the

C57BL/6J mice than does the IRBP1–20 peptide [18]. This new Wolf Family Foundation, and the Llura and Gordon Gund immunization protocol should be valuable in overcoming the Foundation. L.Z. was supported in part by China National Natural shortcomings of the previous protocol using the IRBP1–20 peptide Science Foundation Grant 8142800018. We thank Catherine for EAU studies, especially for conditions in which reduced EAU Doller from Visual Sciences Research Center at Case Western severity is anticipated in the experimental subjects. Reserve University (EY11373) for histology and Christine Kassuba In conclusion, consistent with accumulating evidence suggesting from Cleveland Clinic for help with manuscript preparation. that C3aR and C5aR are positive regulators for pathogenic T cell development, using different imaging studies, function studies, and DISCLOSURES immunologic assays to study EAU in WT and C3aR/C5aR KO mice, The authors declare no conflicts of interest. we found that these complement receptors are required in the fi pathogenesis of EAU. These ndings identify C3aR and C5aR as REFERENCES important receptors necessary for the full development of EAU and 1. Selmi, C. (2014) Diagnosis and classification of autoimmune uveitis. the associated Th1 and Th17 effector responses, suggesting that Autoimmun. Rev. 13, 591–594. 2. Caspi, R. R. (2003) Experimental autoimmune uveoretinitis in the rat antagonists of these complement receptors should be examined for and mouse. Curr. Protoc. Immunol. Chapter 15, Unit 15.6. doi:10.1002/ their potential as new drugs for the treatment of autoimmune uveitis. 0471142735.im1506s53.

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