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Complement Anaphylatoxin Receptors C3ar and C5ar Are Required in the Pathogenesis of Experimental Autoimmune Uveitis † ‡ ‡ ‡ { Lingjun Zhang,*, Brent A

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Complement Anaphylatoxin Receptors C3ar and C5ar Are Required in the Pathogenesis of Experimental Autoimmune Uveitis † ‡ ‡ ‡ { Lingjun Zhang,*, Brent A Article Complement anaphylatoxin 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 T cell 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 anaphylatoxin receptors (C3a 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 antibodies [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 immune system, 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 antibody blockade of C5aR enhances Th17 mune uveitis. These experiments showed that C3a cellnumbersinanexperimentalmodelofasthma[13]. receptor/C5a receptor-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 = C3a receptor, 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,
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