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Kinetic changes in microglia-related retinal transcripts in experimental autoimmune uveoretinitis (EAU) of B10.RIII mice

Journal of Neuroinflammation volume 22, Article number: 37 (2025) Cite this article

Abstract

In this study the retinal transcriptome was investigated during the development of experimental autoimmune uveoretinitis (EAU) in mice. EAU was induced by immunizing B10.RIII mice with human interphotoreceptor retinoid binding protein (hIRBP) 161–180 peptide. Genome-wide transcriptional profiles of EAU (day 7, 14 or 21 after immunization) and of control retinas were generated using DNA-microarrays and bioinformatic data mining. Microglia-associated transcripts were identified. Quantitative real-time polymerase chain reaction was performed to validate the expression of differentially expressed genes. Retinal transcript validation revealed that complement and interferon-related pathways, as well as gene clusters specific for antigen-processing and -presentation, and immunosuppression are involved during the course of the disease. Immunofluorescence analysis confirm that upregulated transcripts in EAU are also expressed by retinal microglia. Furthermore, the heterogenous expression patterns observed in retinal microglia, suggests the presence of different subpopulations of retinal microglia in EAU. This study expands our knowledge of the local immune processes involved in EAU pathology.

Introduction

Uveitis is an inflammatory disease affecting the uveal tissues of the eye, and is classified into several anatomical subgroups [1]. The prevalence of noninfectious uveitis in adults is estimated to be 121 per 100,000 [2]. Noninfectious posterior uveitis describes inflammation that primarily affects the retina or/and choroid [1, 3,4,5,6]. Chronic or severe disease courses often lead to uveitis-related ocular complications, such as cystoid macular edema, predisposing patients to a high risk of losing their vision and an impaired quality of life [5, 7, 8]. Currently, primary treatment is with corticosteroids, using conventional synthetic disease-modifying anti-rheumatic drugs (csDMARDs) if the response to treatment is insufficient [9,10,11]. Methotrexate (MTX) is the most commonly prescribed csDMARD for the long-term treatment of patients with insidious onset uveitis. For refractory cases, biologic (b) DMARDs targeting particular pro-inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) (adalimumab, ADA), interleukin (IL)-1 (anakinra), or IL-6 (tocilizumab) are given [12]. The anti-TNF-α monoclonal antibody ADA is approved for the treatment of non-infectious intermediate, posterior and panuveitis in adults [12,13,14]. The pathogenesis of this disease is poorly understood, however, which hinders treatment and prognosis. It is believed to involve both innate and adaptive immune responses. Biomarkers that indicate disease onset, severity, and progression could therefore aid in the appropriate management of uveitis.

Animal models such as experimental autoimmune uveoretinitis (EAU) in mice mimicking noninfectious posterior uveitis in humans are promising for understanding immunological mechanisms and identifying disease-specific targets. This model exhibits similar tissue damage, including retinal vasculitis, granulomas, folding, and detachment [15,16,17]. EAU can be studied in mice through a spontaneous transgenic model [18, 19] or through immunization using interphotoreceptor retinoid-binding protein (IRBP) peptide as a retinal antigen and complete Freund’s adjuvant (CFA) and/or pertussis toxin (PTX) as adjuvants. The peripheral and local immune response leading to EAU in mice was identified to be predominantly a Th1/Th17-driven immune response [20]. In the IRBPp161-180-induced EAU mouse model, cellular infiltration typically begins on day 9–10 after the immunization and peaks around day 14. The ocular cellular infiltrate consists mainly of activated macrophages and granulocytes, which further mediates local inflammatory processes and tissue destruction [20].

Retinal microglia are resident innate immune cells with pluripotent functions, including phagocytosis, antigen presentation, and release of immunomodulatory soluble factors. These factors might constitute a promising target for individualized, target-specific therapy [21, 22]. Retinal microglia significantly influence the development and course of retinal diseases, such as age-related macular degeneration (AMD), diabetic retinopathy, retinoschisis, retinitis pigmentosa, light damage model, and posterior uveitis [23,24,25,26,27,28,29,30,31,32,33]. There is controversy about whether retinal microglia can act as antigen presenting cells and thus play an immunoregulatory role in the disease induction [34,35,36]. However, studies in rats suggest that retinal microglia play a role during early stages of EAU pathogenesis through antigen presentation and secretion of proinflammatory cytokines such as IL-1β and TNF-α [28, 37,38,39]. Okuniki et al. demonstrated that retinal microglia play a crucial role in initiating the recruitment of circulating inflammatory immune cells which lead to EAU [28, 40].

To comprehend the mechanisms underlying autoimmune uveitis, previous studies have characterized the gene expression profile in the retina of EAU in mice, using gene microarray or RNA-sequencing (RNA-seq). The retinal transcriptome of B6 mice with established EAU that was induced via the adoptive transfer model [35, 41] or developed spontaneously in Aire−/− B6 mice [42] was analyzed in these studies. Additionally, retinal microRNAs were examined in B10.RIII mice during disease progression [43].

In contrast to previous studies, we have now collected retinal tissues from B10.RIII mice on days 7, 14, and 21 after EAU induction and from healthy controls for transcriptome analysis using gene microarrays.

As the EAU induction in B10.RIII mice leads to severe intracoular inflammation (mid EAU score 3), compared to the B6 model (induced by hIRBP-1-20; mid EAU-score 2) we could expect significant changes to measure in retinal transcriptome of B10.RIII mice [44]. This allowed us to obtain a kinetic overview of gene expression profiles associated with intraocular processes during the disease course. In this context we focused in particular on microglia-related transcripts.

Material and methods

EAU was induced in B10.RIII mice, which are highly susceptible to developing EAU and in which the disease course is well characterized [20]. Animals were housed in an air-conditioned environment with a 12-h light–dark schedule and had access to water and food ad libitum. All experimental procedures complied with the German animal welfare law, which is in line with the European Community law, and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The animal protocols used in this study were reviewed and approved by the governmental body responsible for animal welfare in the state of North Rhine-Westphalia Germany (application no. 81-02.04.2017.A430). EAU was induced in 8-week-old female B10.RIII mice (The Jackson Laboratory, Bar Harbor, USA) as previously described [45,46,47]. On day 0 mice were immunized with 100 μg of hIRBPp161-180 in an emulsion of 0.2 mL at a 1:1 vol/vol with CFA which contained 2.5 mg/mL H37RA. The emulsion was administered subcutaneously. A concomitant injection of 0.4 µg PTX was administered intraperitoneally to the mice. Mice were sacrificed on days 7, 14, or 21 post immunization (p.i.) and their eyes and retinas collected. EAU severity was exemplified using ocular histology on a scale of 0–4 [48]. Healthy mice were used as controls. Total RNA was extracted from the retinas of both healthy control- and EAU-mice, and subjected to microarray analysis (n = 2 each group) as previously described [49, 50]. The quality of isolated retinal RNA was assessed using the Agilent Bioanalyzer Platform (RNA 6000 nano kit).

Subsequently, the RNA was processed for analysis on Affymetrix GeneChip® Arrays (Mouse Gene 1.0/2.0 ST Array) following the Affymetrix standard protocol, as previously described [50]. The differentially expressed genes (DEG) between EAU and healthy control retinas were defined as those with a Log2-fold change (FC) in gene expression and a −Log10 > 1.3 (corresponds to a p value cutoff of <0.05) and visualized as volcano plots using the VolcaNoseR web tool [51]. Differential expression of selected genes was verified by qRT-PCR using 50 ng of cDNA template per reaction, as previously described [50]. Primer sequences are displayed in Supplemental Table 5. Gene Ontology (GO) enrichment analyses were performed using the ShinyGO 7.7 online tool [52]. Statistical analyses of EAU scores and qRT-PCR results were performed using MedCalc Version 12.4 (Ostend, Belgium).

To demonstrate the expression of selected proteins, immunofluorescence microscopy was conducted in accordance with the methodology as previously described [53]. In brief, mediosagittal sections of fixed and paraffin-embedded eyes were deparaffinized and antigen retrieval was conducted. Following a blocking step with 5% normal goat serum the sections were incubated with primary antibodies targeting S100A6 (monoclonal; rabbit anti-mouse, AbClonal, Germany), arginase-1 (Arg-1; monoclonal; rabbit anti-mouse, AbClonal, Germany), H2-Ab1 (polyclonal; rabbit anti-mouse, AbClonal, Germany) or TMEM119 (guinea pig anti-mouse SYSY, Göttingen, Germany).

After rinsing, the slides were incubated with the secondary antibody (1:500 in 1% BSA; goat anti-rat AF 564 antibody, Invitrogen, Germany; goat anti-rabbit AF596, or goat anti-guinea pig AF488, Invitrogen, Germany) for 30 min at room temperature, (RT). Cell nuclei were stained with DAPI (Sigma-Aldrich, Taufkrichen, Germany). Subsequently, the slides were coverslipped with Mowiol (Carl Roth, Karlsruhe, Germany). A minimum of two representative sections per eye (n = 2 per group) were utilized for the localization of positive cells. Sections lacking the primary antibody served as negative controls. The sections were examined under a fluorescence microscope (Axiophot, Carl Zeiss, Oberkochen, Germany) and three randomly selected regions per retina were photographed.

Results

EAU score

Histopathological evaluation revealed severe EAU at day 14 (2.4 ± 0.6) and 21 (2.2 ± 0.6) after inducing EAU (p < 0.05; Fig. 1). By contrast, no signs of ocular inflammation were observed in control mice (0 ± 0) or in EAU mice at day 7 p.i. (0 ± 0).

Fig. 1
figure 1

EAU score. A Histology of hematoxylin–eosin (HE)-stained cross sections of a healthy control-, and during disease course at day 7, 14 and 21 after EAU induction. B Histopathological EAU score of mice were assessed in control mice (n = 4) and on day 7 (n = 4), day 14 (n = 5) and day 21 (n = 7) after EAU induction. The mean EAU score of each group is indicated by a bar (Kruskal–Wallis test * p = 0.0144)

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Transcriptome analysis

The volcano graph displays the DEGs in the retina of mice with EAU on days 7, 14, and 21 after model induction. Only values with Log2-FC ≥ 2 and −Log10 p value >1.3 are shown (see Fig. 2; Supplemental Table 1). At day 7 p.i. glb1l3 was significantly upregulated (Fig. 2A). On day 14, 227 genes were upregulated and 75 downregulated (Fig. 2B). On day 21 after EAU induction, 686 genes were upregulated and 151 downregulated (Fig. 2C). The 14 genes with the highest Log2-FC at days 14 and 21 were labeled (Fig. 2B, C). Among them, Timp1, Serpina3n, Lcn2, and BC023105 were upregulated at both time points. On day 14, the following genes were upregulated: Snord118, Arg1, Gbp2, Irgm1, Irgm2, S100a6, Tgm1, Nlrc5, Edn2, and Cdc23. On day 21, the following genes were upregulated: Lyz2, H2-Ea-ps, H2-Aa, Cd74, Ms4a6c, C3, Cybb, Steap4, Gm11428, Mpeg1, and Ngp.

Fig. 2
figure 2

Transcriptome analysis. Volcano plot for retinal transcript dataset at A day 7, B day 14, and C day 21 after EAU induction. Volcano plot for the 28,000 genes from the genechip array. The volcano plots were generated with VolcaNoseR showing the Log2-FC value (x-axis) and −Log10 p value (y-axis). In each graph, every point represents an individual transcript. The vertical lines represent Log2-FC ≥ 2, both upregulated (right side) and downregulated (left side), and the horizontal lines represent a −Log10 p value ≥1.3 as the threshold cutoff. The top 14 candidates are labeled (Supplemental Table 1). Pathway enrichment analyses were performed with gene names of proteins with significantly higher abundance in EAU at D day 14 and E day 21. GO analyses were performed with Shiny GO 7.0, showing the 20 most significantly enriched functional categories from biological processes, cellular complex, or molecular function in EAU on D day 14 and E day 21. Size of the dots corresponds to more significant p values. Gene names which are clustered to respective pathways are given in Supplemental Table 3. All genes in the genechip array were used as the enrichment background

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Functional analysis of DEGs in the active and resolution phases of EAU

The functions of DEGs (p value −Log10 > 1.3) were analyzed by gene ontology (GO) enrichment analysis, which includes biological process (BP), molecular function (MF), and cellular component (CC). Figure 2D and E (Supplemental Table 2) shows the top 20 enriched GO terms in each category (p.adjust < 0.05). On day 14 after EAU induction, when intraocular inflammation peaks, transcripts involved in cytokine/chemokine response, particularly interferon-associated response, and complement-related pathways were upregulated. During the resolution phase on day 21, interferon-, cytokine-, and chemokine-related genes were upregulated, as were genes associated with myeloid cell activation, particularly macrophages and microglia. Additionally, genes involved in antigen-processing and antigen-presentation were upregulated, along with genes involved in positive or negative immune regulation. The DEGs that were downregulated at days 14 and 21 after EAU induction mainly contributed to the phototransduction and homeostasis of the retinal tissue, as shown in Supplemental Table 3.

Microglia-related transcripts

To identify microglia-related genes differentially expressed during EAU pathogenesis, the top 50 regulated transcripts (Log2 FC gene expression ≥2 and p value −Log10 > 1.3) on days 14 and 21 p.i. were compared with a microarray dataset of primary activated microglial cells isolated from degenerating retinoschisin-deficient (Rs1h-/Y) retinas [54]. This dataset identified 1110 differentially expressed microglia-specific transcripts during retinal degeneration that are associated with microglial reactivity. Of these, 20 transcripts were identified in EAU mice on day 14 and 21, and 23 transcripts in EAU on day 21, which matched the transcriptomic profiles of activated microglia in P21 Rs1h-/Y mice.

Of 899 DEGs, 76 EAU related transcripts were differentially expressed at day 14 p.i., only, and another 114 transcripts were found to be differentially expressed at both time points, day 14 and 21. Finally, 666 differentially expressed transcripts associated with pathological changes in EAU were exclusively differentially expressed on day 21 p.i. (Fig. 3A; Supplemental Table 4). With regard to the Rs1h-/Y related genes, 20 genes were significantly differentially expressed at day 14 and day 21 p.i. and 23 genes at day 21 only (Fig. 3A; Supplemental Table 4).

Fig. 3
figure 3

Microglia and EAU-related DE genes in retinal transcriptome of EAU mice. A Venn diagram comparing the expression of the top DE transcripts (Log2-FC gene expression >2 and p value <0.05) from EAU d14 (blue circle) and d21 (red circle) with a microarray dataset from primary activated microglial cells isolated from degenerating Rs1h-/Y retinas (green circle) to identify microglia-specific genes in EAU (Supplemental Table 4). Microglia-associated genes identified in the subset of DE genes are written in italic. B Genes selected for qRT-PCR analysis are grouped into the subclasses: complement, antigen presentation, interferon, inflammation, and immunoregulation. n-fold gene expression is displayed as box-plot (Log10 n-fold gene expression; target gene). Statistical differences were analyzed via * ANOVA (parametric), or.# Kruskal–Wallis test (nonparametric; p < 0.05)

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Out of the 899 DEGs, 27 transcripts related to EAU and/or microglia were selected for validation by qRT-PCR (Supplemental Table 5). The relative mRNA levels were estimated and depicted as log fold change in box plots (Fig. 3B).

Although no histopathological inflammatory signs were visible, several pro-inflammatory genes, including C3, H2-Ab1, H2-Aa, S100A6, Tgm2, and Steap, and particularly those related to the interferon response (Nlrc5, If1204, Cybb, and Ifitm), as well as anti-inflammatory genes (Arg1, Gm11428, and Serpina3n) were significantly upregulated at day 7 p.i. compared to naïve mice. No altered expression on the transcript Mpeg1 was found in qRT-PCR analysis during EAU.

All other selected transcripts verified by qRT-PCR were significantly upregulated on day 14 and remained significantly elevated until day 21 after inducing EAU. Compared to day 14, the transcripts H2-Ab1, H2-Aa, S100a6, and Arg1 were down-regulated in the resolution phase of EAU on day 21.

Immunofluorescence

To prove the retinal expression of selected transcripts in EAU, immunofluorescence analysis of S100A6, Arg-1 and H2-Ab1 together with the microglia-specific marker TMEM119 [55, 56] were conducted on sections of naïve mouse eyes and mouse eyes with EAU at d21 p.i. (Fig. 4). In naïve eyes, no expression of S100A6, Arg-1 and H2-Ab1 were detected in TMEM119 positive retinal microglia. A weak positive Arg-1 signal was detected in the outer plexiform layer of naïve eyes. In EAU eyes (day 21 p.i.) elevated expression of S100A6 was detected in retinal tissue. The S100A6 signal was not co-localized with TMEM119-positive retinal microglia (Fig. 4A). In contrast, immunofluorescence staining showed that Arg-1 and H2-Ab1 were expressed by cells of the cellular infiltrate. Furthermore, the staining was co-localized with TMEM119-positive retinal microglia (Fig. 4B, C). However, a subset of TMEM119-positive retinal microglia was negative for Arg-1 and H2-Ab1.

Fig. 4
figure 4

Localization of EAU-related proteins in microglia of the naïve retina compared to the retina with EAU. The retinal expression of A S100A6, B Arg-1, C H2-Ab1 (AF594; red) and the microglia marker TMEM119 (AF488; green) in naïve mice (n = 2) and mice with EAU (day 21; n = 2) was determined by immunofluorescence analysis. Cell nuclei were stained with DAPI (blue). Co-localized expression of analysed proteins with TMEM119-positive retinal microglia was marked with white asterisks. Representative pictures are displayed

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Discussion

The present study analyzed the kinetic changes in the retinal transcriptome in B10.RIII mice during EAU development. This model exhibits intraocular inflammation characterized by a self-limiting monophasic disease course [15, 57, 58] which includes a prodromal phase between days 5–11, a peak at day 14 p.i., and a remission phase at day 21 p.i. During the prodromal phase, although elevated numbers of CD4+ and CD11b+ cells can be detected in the retina, no histopathological inflammatory signs are visible [58]. Despite the absence of histological signs of ocular inflammation on day 7, we detected a significant change in the retinal transcriptome in the present study. Specifically, upregulation of b-galactosidase (glb1l3) was observed. The expression of β-galactosidase was also elevated in inflammatory, activated microglia in the context of neuronal degeneration [59]. In the present study, qRT-PCR validation of gene expression indicated significant upregulation of additional transcripts involving innate immune processes, such as the complement system, antigen presentation, interferon response, and immune regulation, in the subclinical induction phase of EAU at day 7 p.i. Other studies have described oxidative stress and translocation of the alarmin HMGB1 at early EAU timepoints [60, 61]. The early upregulation (d7 p.i.) of S100A6 as an alarmin in the present study, and elevated expression in resident retinal cells of EAU mice at day 21 p.i. in immunofluorescence analysis are in line with the previous sc-RNA-seq study of Heng et al. showing an increased expression of S100a6 in müller cells, astrocytes and perivascular cells of Aire−/− mice with EAU compared to healthy WT mice (https://jacobheng.shinyapps.io/uveoretinitis/) [42]. This underscores the early activation of the localimmune response in EAU although overt signs of ocular inflammation are not present [62]. Upregulation of Serpina3n was found in endothelial cells of the inner blood retinal barrier in murine EAU [41], and in uveal epithelial cells in the early phase of endotoxin induced uveitis (EIU) in rats [62]. The upregulation of Serpina3n at day 7 p.i in the current study suggests it is involved in early disease development.

It has been frequently shown that complement factors, and their receptors are important modulators during EAU development [63,64,65,66]. Thus, elevated retinal C3AR1, C4b expression has been shown in scRNA-seq analysis in Aire−/− mice developing autoimmune uveitis spontaneously [42]. Early upregulation of retinal transcripts associated with innate immunity such as C3 and Serping1 (Serine peptidase inhibitor, clade G, member 1) has also been demonstrated in experimental models of optic nerve crush [67], and in the EAU adoptive transfer model in C57BL/6 mice [41]. Whereas the previous studies focused on the phase of fully developed uveoretinitis, the present study showed an early induction of C3 expression and thereby confirms that the complement cascade is involved in the early phase of disease development. The elevated expression of their regulators C3AR1, A2M, and Serping 1 at the peak and resolution phases may reflect their importance in counter-regulating the local immune response. Indeed, these results underscore the relevant role that innate immunity plays in the induction and disease course of EAU.

The EAU model exhibits an inflammatory peak at day 14 p.i., which is characterized by a massive cellular infiltration of leukocytes in particular, neutrophils, monocytes and T cells, as well as pathological changes in the retinal tissue such as folding, detachment, and Dahlen Fuchs nodules. The immune response in the classical EAU model is driven by Th1/Th17 cells [15, 58, 68]. At day 14 p.i., a predominance of intraocular Th17 cells over Th1 cells has been reported, which changes to a Th1 phenotype during the subsequent resolution phase. The resolution phase at day 21 p.i. is characterized by a declining cellular infiltration, with a shift from Th17 to Th1 cells, while pathological destruction in the retinal tissue persists [15, 20, 57, 58]. An elevated expression of T cell-related transcripts of Th17 cells in particular could be shown in the study of Lipski et al. [35]. In that study, dissociated, and sorted retinal cells of C57BL/6 mice, with EAU induced via adoptive transfer of antigen-specific T cells, were analyzed via RNA-sequencing [41]. In a scRNA-seq approach, total number of immune cells, including monocytes, T cells, B cells, and NK cells in the retinal tissue of Aire−/− mice with spontaneous uveoretinitis were low compared to the cells of retinal origin [42]. In the current study, no significantly elevated T-cell-specific retinal transcripts were detected. Notably, Th17-related transcripts were less prominent in the current study. This might be explained by the fact that whole retinal tissue comprising cells of retinal origin and infiltrating immune cells were analyzed, whereby the majority of cells were of retinal origin as previously shown [42]. Another possible explanation is the plasticity of Th17 cells, which can convert into Th1 cells, as described for d21 after EAU induction [15]. In particular, interferon-related transcripts, indicating a Th1-driven immune response, were significantly upregulated during ongoing disease, as shown in the spontaneous uveitis models [42].

With regard to the previous studies [41, 42], similar transcripts could be shown in mice with EAU related to both innate and adaptive immunity, antigen presentation, cytokines/chemokines, and complement system. At least the broad upregulation of interferon-related transcripts and transcripts involved in antigen presentation confirms that T cells are involved in the pathogenesis of EAU. Consistently with those results, we found a significant upregulation of transcripts participating in antigen processing and presentation at day 7 p.i. The absence of obvious inflammatory infiltration at this time indicates that resident cells in the retina may be activated, and participate in creating a proinflammatory environment and antigen presentation to infiltrating T cells. Previous studies have shown that the induction of MHC class II in macrophages and resident microglia is strongly associated with disease severity in EAU which supports this assumption [35].

The early upregulation (d7 p.i.) of H2-Ab1, H2Aa as MHC-II molecules in the present study, and elevated expression of H2-Ab1 in retinal microglia and infiltrating cells of EAU mice at day 21 p.i. in immunofluorescence analysis, are in line with the previous sc-RNA-seq study of Heng et al. showing an increased expression of H2-Ab1 and H2Aa in monocytes/macrophages and retinal microglia of Aire−/− mice with EAU compared to healthy WT mice (https://jacobheng.shinyapps.io/uveoretinitis/) [42] Comparison of the transcripts identified in the present study with those of activated retinal microglia in Rs1h-/Y mice [54] suggests that there is a potential relationship between the expression of complement and antigen-presenting associated retinal transcripts in EAU and retinal microglia.

Furthermore, the immunofluorescence analysis of the current study demonstrated a heterogenous expression of H2-Ab1 in retinal microglia of eyes with EAU, pointing to the presence of different M1/M2 phenotypes.

Microglia are notably activated by the interferon pathway, involving the transcription factor Irf8 [69,70,71,72]. In the current study, Irf8 is already upregulated at day 7 p.i., with subsequently enhanced expression at days 14 and 21. Irf8 has a divergent and cell-specific role in EAU. Depletion of Irf8 in CD4 T cells is associated with an activated Th17 phenotype and a reduced regulatory T cell population, resulting in enhanced EAU scores. Conversely, depletion of Irf8 in macrophages and microglia protects against disease in the EAU model [73]. This is consistent with the role of antigen presenting cells (APC) expressing Irf8 in the experimental autoimmune encephalomyelitis (EAE) model, wherein it is necessary to prime pathogenic Th17 cells to induce autoimmune EAE [74]. The early upregulation of Irf8 and other interferon-related transcripts in the current study is comparable to the transcriptome of activated retinal microglia detected in Rs1h-/Y mice [54], indicating an early activation of retinal myeloid cells in EAU. In a study analyzing retinal scRNA-seq in Aire−/− mice with spontaneous autoimmune uveitis, transcripts related to the complement system, interferon response, antigen presentation, and immune regulation were found to be elevated in resident retinal cells such as Müller cells and microglial cells. This suggests that resident cells play an active role in stimulating the local autoimmune processes [42]. In consistency with those findings, the GO enrichment analysis in the current study indicated that the complement system, antigen presentation-associated processes, interferon-related pathways, and microglia/macrophages in particular in response to interferon-associated pathways are activated. This suggests that resident retinal cells are involved in local inflammatory processes, interacting with the antigen-specific local immune responses of infiltrating Th1 cells. As the EAU model in B10.RIII mice is a self-limiting disease model, transcripts reflecting downregulation of inflammation were significantly expressed at d21 p.i. As shown in the GO enrichment analysis, genes associated with a cellular response to IFN-β were predominantly enriched, and genes pointing to a negative regulation of innate immunity were also upregulated. Indeed, the immunoregulatory role of IFN-β in EAU has been shown previously [75]. Regarding the alignment of our analysis with the transcriptome of activated retinal microglia in Rs1h-/Y mice [54], we observed an increase in the expression of immunoregulatory transcripts with immunosuppressive properties such as Arg1 and Gm11428 (also known as Wfdc17) [21, 76], which are also expressed in monocytes/macrophages and retinal microglia of Aire−/− mice with EAU (https://jacobheng.shinyapps.io/uveoretinitis/) [42].

We verified the elevated gene-expression of Arg-1 in the present study, and showed an elevated expression of Arg-1 in retinal microglia and infiltrating cells of EAU mice at day 21 p.i. in immunofluorescence analysis. Similar to the H2-Ab1 expression, subsets of Arg-1 positive and negative retinal microglia were shown. This indicates the presence of M2 phenotype of local microglia/myeloid cells at the remission phase of EAU, and may reflect their modulating role in the self-limiting EAU disease course.

To gain a deeper understanding of the intraocular pathophysiology of EAU in mice, further studies with transgenic animals with B6 background are warranted. Also the remission phase of EAU should be investigated on day 28 p.i. or later until the disease is in full remission. Further, the influence of CFA, an adjuvant used for EAU induction on retinal transcriptome and in particular retinal microglia should be addressed as previous studies showed a CFA induced activation of microglia in the CNS [77].

A significant limitation of the study is that the gene chip array analysis of the retina did not permit the determination of the specific cells in which gene regulation occurs. The assignment of microglia-associated genes is insufficient to draw conclusions regarding cell-specific changes in gene expression, as claimed in this study regarding retinal microglia. Such an analysis could have been conducted through previously cell sorting or, much more effectively, through single cell RNA sequencing.

Although retinal transcriptomic changes were not analyzed on a single-cell level, our results confirm the findings of a previous study [42], and showed the presence of activated retinal microglia, with different phenotypes in EAU. Furthermore, we could show that proinflammatory mechanisms involving the activation of resident innate immune cells, in particular modulation of retinal microglia-associated genes point to an activated status already before clinical signs of disease can be detected Our results supporting the assumption that retinal microglia have the ability to polarize in pro- and anti-inflammatory M1 or M2 phenotypes, allowing them to shape the local immune response in retinal tissue [78].

To gain more knowledge about the local mechanisms before diseases onset, at the peak, and during remission phase of the disease, a study at single-cell level including the retinal tissue architecture via spatial transcriptomics [79] would be highly desirable.

Availability of data and materials

No datasets were generated or analysed during the current study. The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

EAU:

Experimental autoimmune uveoretinitis

hIRBP:

Human interphotoreceptor-retinoid-binding protein

p.i.:

Post immunization

References

  1. Jabs DA, Nussenblatt RB, Rosenbaum JT, Standardization of Uveitis Nomenclature (SUN) Working Group. Standardization of uveitis nomenclature for reporting clinical data. Results of the First International Workshop. Am J Ophthalmol. 2005;140:509–16.

    Article  PubMed  Google Scholar 

  2. Thorne JE, Suhler E, Skup M, Tari S, Macaulay D, Chao J, et al. Prevalence of noninfectious uveitis in the United States: a claims-based analysis. JAMA Ophthalmol. 2016;134:1237–45.

    Article  PubMed  Google Scholar 

  3. Rothova A, Berendschot TTJM, Probst K, van Kooij B, Baarsma GS. Birdshot chorioretinopathy: long-term manifestations and visual prognosis. Ophthalmology. 2004;111:954–9.

    Article  PubMed  Google Scholar 

  4. Rothova A, Suttorp-van Schulten MS, Frits Treffers W, Kijlstra A. Causes and frequency of blindness in patients with intraocular inflammatory disease. Br J Ophthalmol. 1996;80:332–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Vidovic-Valentincic N, Kraut A, Hawlina M, Stunf S, Rothova A. Intermediate uveitis: long-term course and visual outcome. Br J Ophthalmol. 2009;93:477–80.

    Article  CAS  PubMed  Google Scholar 

  6. Wakefield D, Chang JH. Epidemiology of uveitis. Int Ophthalmol Clin. 2005;45:1–13.

    Article  PubMed  Google Scholar 

  7. Murphy CC, Greiner K, Plskova J, Frost NA, Forrester JV, Dick AD. Validity of using vision-related quality of life as a treatment end point in intermediate and posterior uveitis. Br J Ophthalmol. 2007;91:154–6.

    Article  PubMed  Google Scholar 

  8. Suttorp-Schulten MS, Rothova A. The possible impact of uveitis in blindness: a literature survey. Br J Ophthalmol. 1996;80:844–8.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Becker MD, Smith JR, Max R, Fiehn C. Management of sight-threatening uveitis: new therapeutic options. Drugs. 2005;65:497–519.

    Article  CAS  PubMed  Google Scholar 

  10. Jabs DA, Akpek EK. Immunosuppression for posterior uveitis. Retina. 2005;25:1–18.

    Article  PubMed  Google Scholar 

  11. Jabs DA, Rosenbaum JT, Foster CS, Holland GN, Jaffe GJ, Louie JS, et al. Guidelines for the use of immunosuppressive drugs in patients with ocular inflammatory disorders: recommendations of an expert panel. Am J Ophthalmol. 2000;130:492–513.

    Article  CAS  PubMed  Google Scholar 

  12. Dick AD, Rosenbaum JT, Al-Dhibi HA, Belfort R, Brézin AP, Chee SP, et al. Guidance on noncorticosteroid systemic immunomodulatory therapy in noninfectious uveitis: fundamentals of care for uveitis (FOCUS) initiative. Ophthalmology. 2018;125:757–73.

    Article  PubMed  Google Scholar 

  13. Jaffe GJ, Dick AD, Brézin AP, Nguyen QD, Thorne JE, Kestelyn P, et al. Adalimumab in patients with active noninfectious uveitis. N Engl J Med. 2016;375:932–43.

    Article  CAS  PubMed  Google Scholar 

  14. Ramanan AV, Dick AD, Beresford MW. Adalimumab for uveitis in juvenile idiopathic arthritis. N Engl J Med. 2017;377:789–90.

    Article  PubMed  Google Scholar 

  15. Amadi-Obi A, Yu C-R, Liu X, Mahdi RM, Clarke GL, Nussenblatt RB, et al. TH17 cells contribute to uveitis and scleritis and are expanded by IL-2 and inhibited by IL-27/STAT1. Nat Med. 2007;13:711–8.

    Article  CAS  PubMed  Google Scholar 

  16. Forrester JV, Liversidge J, Dua HS, Dick A, Harper F, McMenamin PG. Experimental autoimmune uveoretinitis: a model system for immunointervention: a review. Curr Eye Res. 1992;11(Suppl):33–40.

    Article  PubMed  Google Scholar 

  17. Nussenblatt RB. The natural history of uveitis. Int Ophthalmol. 1990;14:303–8.

    Article  CAS  PubMed  Google Scholar 

  18. Horai R, Silver PB, Chen J, Agarwal RK, Chong WP, Jittayasothorn Y, et al. Breakdown of immune privilege and spontaneous autoimmunity in mice expressing a transgenic T cell receptor specific for a retinal autoantigen. J Autoimmun. 2013;44:21–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. DeVoss J, Hou Y, Johannes K, Lu W, Liou GI, Rinn J, et al. Spontaneous autoimmunity prevented by thymic expression of a single self-antigen. J Exp Med. 2006;203:2727–35.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Caspi RR. Understanding autoimmune uveitis through animal models. The Friedenwald Lecture. Invest Ophthalmol Vis Sci. 2011;52:1872–9.

    Article  PubMed  Google Scholar 

  21. Aslanidis A, Karlstetter M, Scholz R, Fauser S, Neumann H, Fried C, et al. Activated microglia/macrophage whey acidic protein (AMWAP) inhibits NFκB signaling and induces a neuroprotective phenotype in microglia. J Neuroinflammation. 2015;12:77.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Karlstetter M, Nothdurfter C, Aslanidis A, Moeller K, Horn F, Scholz R, et al. Translocator protein (18 kDa) (TSPO) is expressed in reactive retinal microglia and modulates microglial inflammation and phagocytosis. J Neuroinflammation. 2014;11:3.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Blank T, Goldmann T, Koch M, Amann L, Schön C, Bonin M, et al. Early microglia activation precedes photoreceptor degeneration in a mouse model of CNGB1-linked retinitis pigmentosa. Front Immunol. 2017;8:1930.

    Article  PubMed  Google Scholar 

  24. Gehrig A, Langmann T, Horling F, Janssen A, Bonin M, Walter M, et al. Genome-wide expression profiling of the retinoschisin-deficient retina in early postnatal mouse development. Invest Ophthalmol Vis Sci. 2007;48:891–900.

    Article  PubMed  Google Scholar 

  25. Gupta N, Brown KE, Milam AH. Activated microglia in human retinitis pigmentosa, late-onset retinal degeneration, and age-related macular degeneration. Exp Eye Res. 2003;76:463–71.

    Article  CAS  PubMed  Google Scholar 

  26. Penfold PL, Madigan MC, Gillies MC, Provis JM. Immunological and aetiological aspects of macular degeneration. Prog Retin Eye Res. 2001;20:385–414.

    Article  CAS  PubMed  Google Scholar 

  27. Prinz M, Schmidt H, Mildner A, Knobeloch K-P, Hanisch U-K, Raasch J, et al. Distinct and nonredundant in vivo functions of IFNAR on myeloid cells limit autoimmunity in the central nervous system. Immunity. 2008;28:675–86.

    Article  CAS  PubMed  Google Scholar 

  28. Rao NA, Kimoto T, Zamir E, Giri R, Wang R, Ito S, et al. Pathogenic role of retinal microglia in experimental uveoretinitis. Invest Ophthalmol Vis Sci. 2003;44:22–31.

    Article  PubMed  Google Scholar 

  29. Rungger-Brändle E, Dosso AA, Leuenberger PM. Glial reactivity, an early feature of diabetic retinopathy. Invest Ophthalmol Vis Sci. 2000;41:1971–80.

    PubMed  Google Scholar 

  30. Weber BHF, Schrewe H, Molday LL, Gehrig A, White KL, Seeliger MW, et al. Inactivation of the murine X-linked juvenile retinoschisis gene, Rs1h, suggests a role of retinoschisin in retinal cell layer organization and synaptic structure. Proc Natl Acad Sci USA. 2002;99:6222–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zeng H, Green WR, Tso MOM. Microglial activation in human diabetic retinopathy. Arch Ophthalmol. 2008;126:227–32.

    Article  PubMed  Google Scholar 

  32. Zhou T, Huang Z, Sun X, Zhu X, Zhou L, Li M, et al. Microglia polarization with M1/M2 phenotype changes in rd1 mouse model of retinal degeneration. Front Neuroanat. 2017;11:77.

    Article  PubMed  PubMed Central  Google Scholar 

  33. O’Koren EG, Yu C, Klingeborn M, Wong AYW, Prigge CL, Mathew R, et al. Microglial function is distinct in different anatomical locations during retinal homeostasis and degeneration. Immunity. 2019;50:723–37.e7.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Yamasaki R, Lu H, Butovsky O, Ohno N, Rietsch AM, Cialic R, et al. Differential roles of microglia and monocytes in the inflamed central nervous system. J Exp Med. 2014;211:1533–49.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Lipski DA, Dewispelaere R, Foucart V, Caspers LE, Defrance M, Bruyns C, et al. MHC class II expression and potential antigen-presenting cells in the retina during experimental autoimmune uveitis. J Neuroinflammation. 2017;14:136.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Wlodarczyk A, Løbner M, Cédile O, Owens T. Comparison of microglia and infiltrating CD11c+ cells as antigen presenting cells for T cell proliferation and cytokine response. J Neuroinflammation. 2014;11:57.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Gullapalli VK, Zhang J, Pararajasegaram G, Rao NA. Hematopoietically derived retinal perivascular microglia initiate uveoretinitis in experimental autoimmune uveitis. Graefes Arch Clin Exp Ophthalmol. 2000;238:319–25.

    Article  CAS  PubMed  Google Scholar 

  38. Ishimoto S, Zhang J, Gullapalli VK, Pararajasegaram G, Rao NA. Antigen-presenting cells in experimental autoimmune uveoretinitis. Exp Eye Res. 1998;67:539–48.

    Article  CAS  PubMed  Google Scholar 

  39. Zhang J, Wu GS, Ishimoto S, Pararajasegaram G, Rao NA. Expression of major histocompatibility complex molecules in rodent retina. Immunohistochemical study. Invest Ophthalmol Vis Sci. 1997;38:1848–57.

    CAS  PubMed  Google Scholar 

  40. Okunuki Y, Mukai R, Nakao T, Tabor SJ, Butovsky O, Dana R, et al. Retinal microglia initiate neuroinflammation in ocular autoimmunity. Proc Natl Acad Sci USA. 2019;116:9989–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lipski DA, Foucart V, Dewispelaere R, Caspers LE, Defrance M, Bruyns C, et al. Retinal endothelial cell phenotypic modifications during experimental autoimmune uveitis: a transcriptomic approach. BMC Ophthalmol. 2020;20:106.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Heng JS, Hackett SF, Stein-O’Brien GL, Winer BL, Williams J, Goff LA, et al. Comprehensive analysis of a mouse model of spontaneous uveoretinitis using single-cell RNA sequencing. Proc Natl Acad Sci USA. 2019;116:26734–44.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ishida W, Fukuda K, Higuchi T, Kajisako M, Sakamoto S, Fukushima A. Dynamic changes of microRNAs in the eye during the development of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 2011;52:611–7.

    Article  CAS  PubMed  Google Scholar 

  44. Fan N-W, Li J, Mittal SK, Foulsham W, Elbasiony E, Huckfeldt RM, et al. Characterization of clinical and immune responses in an experimental chronic autoimmune uveitis model. Am J Pathol. 2021;191:425–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Hennig M, Bauer D, Wasmuth S, Busch M, Walscheid K, Thanos S, et al. Everolimus improves experimental autoimmune uveoretinitis. Exp Eye Res. 2012;105:43–52.

    Article  CAS  PubMed  Google Scholar 

  46. Busch M, Bauer D, Hennig M, Wasmuth S, Thanos S, Heiligenhaus A. Effects of systemic and intravitreal TNF-α inhibition in experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci. 2013;54:39–46.

    Article  CAS  PubMed  Google Scholar 

  47. Bauer D, Busch M, Pacheco-López G, Kasper M, Wildschütz L, Walscheid K, et al. Behavioral conditioning of immune responses with cyclosporine A in a murine model of experimental autoimmune uveitis. NeuroImmunoModulation. 2017;24:87–99.

    Article  CAS  PubMed  Google Scholar 

  48. Agarwal RK, Caspi RR. Rodent models of experimental autoimmune uveitis. Methods Mol Med. 2004;102:395–419.

    CAS  PubMed  Google Scholar 

  49. Dirscherl K, Karlstetter M, Ebert S, Kraus D, Hlawatsch J, Walczak Y, et al. Luteolin triggers global changes in the microglial transcriptome leading to a unique anti-inflammatory and neuroprotective phenotype. J Neuroinflammation. 2010;7:3.

    Article  PubMed  PubMed Central  Google Scholar 

  50. Karlstetter M, Lippe E, Walczak Y, Moehle C, Aslanidis A, Mirza M, et al. Curcumin is a potent modulator of microglial gene expression and migration. J Neuroinflammation. 2011;8:125.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Goedhart J, Luijsterburg MS. VolcaNoseR is a web app for creating, exploring, labeling and sharing volcano plots. Sci Rep. 2020;10:20560.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Ge SX, Jung D, Yao R. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics. 2020;36:2628–9.

    Article  CAS  PubMed  Google Scholar 

  53. Bauer D, Böhm MRR, Wu X, Wang B, Jalilvand TV, Busch M, et al. Crystallin β-b2 promotes retinal ganglion cell protection in experimental autoimmune uveoretinitis. Front Cell Neurosci. 2024;18:1379540.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Weigelt K, Ernst W, Walczak Y, Ebert S, Loenhardt T, Klug M, et al. Dap12 expression in activated microglia from retinoschisin-deficient retina and its PU.1-dependent promoter regulation. J Leukoc Biol. 2007;82:1564–74.

    Article  CAS  PubMed  Google Scholar 

  55. Bennett ML, Bennett FC, Liddelow SA, Ajami B, Zamanian JL, Fernhoff NB, et al. New tools for studying microglia in the mouse and human CNS. Proc Natl Acad Sci USA. 2016;113:E1738–46.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Satoh J, Kino Y, Asahina N, Takitani M, Miyoshi J, Ishida T, et al. TMEM119 marks a subset of microglia in the human brain. Neuropathology. 2016;36:39–49.

    Article  CAS  PubMed  Google Scholar 

  57. Jiang HR, Lumsden L, Forrester JV. Macrophages and dendritic cells in IRBP-induced experimental autoimmune uveoretinitis in B10RIII mice. Invest Ophthalmol Vis Sci. 1999;40:3177–85.

    CAS  PubMed  Google Scholar 

  58. Kerr EC, Raveney BJE, Copland DA, Dick AD, Nicholson LB. Analysis of retinal cellular infiltrate in experimental autoimmune uveoretinitis reveals multiple regulatory cell populations. J Autoimmun. 2008;31:354–61.

    Article  CAS  PubMed  Google Scholar 

  59. Kitchener EJA, Dundee JM, Brown GC. Activated microglia release β-galactosidase that promotes inflammatory neurodegeneration. Front Aging Neurosci. 2023;15:1327756.

    Article  CAS  PubMed  Google Scholar 

  60. Jiang G, Sun D, Yang H, Lu Q, Kaplan HJ, Shao H. HMGB1 is an early and critical mediator in an animal model of uveitis induced by IRBP-specific T cells. J Leukoc Biol. 2014;95:599–607.

    Article  PubMed  PubMed Central  Google Scholar 

  61. Khurana RN, Parikh JG, Saraswathy S, Wu G-S, Rao NA. Mitochondrial oxidative DNA damage in experimental autoimmune uveitis. Invest Ophthalmol Vis Sci. 2008;49:3299–304.

    Article  PubMed  Google Scholar 

  62. Takamiya A, Takeda M, Yoshida A, Kiyama H. Expression of serine protease inhibitor 3 in ocular tissues in endotoxin-induced uveitis in rat. Invest Ophthalmol Vis Sci. 2001;42:2427–33.

    CAS  PubMed  Google Scholar 

  63. Copland DA, Hussain K, Baalasubramanian S, Hughes TR, Morgan BP, Xu H, et al. Systemic and local anti-C5 therapy reduces the disease severity in experimental autoimmune uveoretinitis. Clin Exp Immunol. 2010;159:303–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Read RW, Szalai AJ, Vogt SD, McGwin G, Barnum SR. Genetic deficiency of C3 as well as CNS-targeted expression of the complement inhibitor sCrry ameliorates experimental autoimmune uveoretinitis. Exp Eye Res. 2006;82:389–94.

    Article  CAS  PubMed  Google Scholar 

  65. Zhang L, Bell BA, Li Y, Caspi RR, Lin F. Complement component C4 regulates the development of experimental autoimmune uveitis through a T cell-intrinsic mechanism. Front Immunol. 2017;8:1116.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Zhang L, Bell BA, Yu M, Chan C-C, Peachey NS, Fung J, et al. Complement anaphylatoxin receptors C3aR and C5aR are required in the pathogenesis of experimental autoimmune uveitis. J Leukoc Biol. 2016;99:447–54.

    Article  CAS  PubMed  Google Scholar 

  67. Templeton JP, Freeman NE, Nickerson JM, Jablonski MM, Rex TS, Williams RW, et al. Innate immune network in the retina activated by optic nerve crush. Invest Ophthalmol Vis Sci. 2013;54:2599–606.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Luger D, Silver PB, Tang J, Cua D, Chen Z, Iwakura Y, et al. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J Exp Med. 2008;205:799–810.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Masuda T, Iwamoto S, Mikuriya S, Tozaki-Saitoh H, Tamura T, Tsuda M, et al. Transcription factor IRF1 is responsible for IRF8-mediated IL-1β expression in reactive microglia. J Pharmacol Sci. 2015;128:216–20.

    Article  CAS  PubMed  Google Scholar 

  70. Masuda T, Nishimoto N, Tomiyama D, Matsuda T, Tozaki-Saitoh H, Tamura T, et al. IRF8 is a transcriptional determinant for microglial motility. Purinergic Signal. 2014;10:515–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Masuda T, Tsuda M, Yoshinaga R, Tozaki-Saitoh H, Ozato K, Tamura T, et al. IRF8 is a critical transcription factor for transforming microglia into a reactive phenotype. Cell Rep. 2012;1:334–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Minten C, Terry R, Deffrasnes C, King NJC, Campbell IL. IFN regulatory factor 8 is a key constitutive determinant of the morphological and molecular properties of microglia in the CNS. PLoS ONE. 2012;7: e49851.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Kim S-H, Burton J, Yu C-R, Sun L, He C, Wang H, et al. 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. J Immunol. 2015;195:1480–8.

    Article  CAS  PubMed  Google Scholar 

  74. Yoshida Y, Yoshimi R, Yoshii H, Kim D, Dey A, Xiong H, et al. The transcription factor IRF8 activates integrin-mediated TGF-β signaling and promotes neuroinflammation. Immunity. 2014;40:187–98.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Sun M, Yang Y, Yang P, Lei B, Du L, Kijlstra A. Regulatory effects of IFN-β on the development of experimental autoimmune uveoretinitis in B10RIII mice. PLoS ONE. 2011;6: e19870.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Karlstetter M, Walczak Y, Weigelt K, Ebert S, Van den Brulle J, Schwer H, et al. The novel activated microglia/macrophage WAP domain protein, AMWAP, acts as a counter-regulator of proinflammatory response. J Immunol. 2010;185:3379–90.

    Article  CAS  PubMed  Google Scholar 

  77. Raghavendra V, Tanga FY, DeLeo JA. Complete freunds adjuvant-induced peripheral inflammation evokes glial activation and proinflammatory cytokine expression in the CNS. Eur J Neurosci. 2004;20:467–73.

    Article  PubMed  Google Scholar 

  78. Jin N, Sha W, Gao L. Shaping the microglia in retinal degenerative diseases using stem cell therapy: practice and prospects. Front Cell Dev Biol. 2021;9: 741368.

    Article  PubMed  PubMed Central  Google Scholar 

  79. Choi J, Li J, Ferdous S, Liang Q, Moffitt JR, Chen R. Spatial organization of the mouse retina at single cell resolution by MERFISH. Nat Commun. 2023;14:4929.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This project was funded in part by the uveitis section of the German Society of Ophthalmology (DOG), Munich, Germany and supported by grants from the Deutsche Forschungsgemeinschaft (DFG) (ME4050/12-1, ME4050/13-1) and by a grant from the Bundesministerium für Bildung und Forschung (BMBF) ‘Lipid Immune Neuropathy Consortium’. The DOG, DFG and BMBF had no role in the design of this study, its execution, analyses, interpretation of the data, or writing of the manuscript.

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Author notes

  1. Maren Kasper and Marcus Karlstetter shared first authorship and contributed equally in the experimental study and the wirting of the mansucript.

Authors and Affiliations

  1. Department of Ophthalmology at St. Franziskus Hospital, Ophtha-Lab, Hohenzollernring 74, 48145, Münster, Germany

    Maren Kasper, Lena Wildschütz, Martin Busch, Dirk Bauer & Arnd Heiligenhaus

  2. Chair of Experimental Immunology of the Eye, Department of Ophthalmology, University of Cologne, Cologne, Germany

    Marcus Karlstetter, Rebecca Scholz & Thomas Langmann

  3. Department of Neurology, University Hospital Münster, Münster, Germany

    Gerd Meyer zu Hörste

  4. Institute for Experimental Ophthalmology, Westfalian Wilhelms-University of Münster, Münster, Germany

    Solon Thanos

  5. University of Duisburg-Essen, Duisburg, Germany

    Arnd Heiligenhaus

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Contributions

MK, MK, LW, TL, AH designed the research project. MK, MK, LW, RS, MB, DB, performed the research, collected and analyzed the data. MK, MK, TL, and AH wrote the manuscript. ST, GMZH participated in critical reviewing of the manuscript. All authors read and approved the manuscript.

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Correspondence to Maren Kasper.

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Ethics approval and consent to participate Animals were housed at the animal facilities in accordance with European guidelines. Animal treatment conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Ethics approval was obtained from the Ethics Committee [North Rhine-Westphalia State Agency for Nature, Environment, and Consumer Protection (LANUV)].

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Kasper, M., Karlstetter, M., Wildschütz, L. et al. Kinetic changes in microglia-related retinal transcripts in experimental autoimmune uveoretinitis (EAU) of B10.RIII mice. J Neuroinflammation 22, 37 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12974-025-03358-x

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Journal of Neuroinflammation

ISSN: 1742-2094