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. 2020 Nov:114:102507.
doi: 10.1016/j.jaut.2020.102507. Epub 2020 Jun 24.

Autoimmunity to neuroretina in the concurrent absence of IFN-γ and IL-17A is mediated by a GM-CSF-driven eosinophilic inflammation

So Jin Bing  1 , Phyllis B Silver  1 , Yingyos Jittayasothorn  1 , Mary J Mattapallil  1 , Chi-Chao Chan  1 , Reiko Horai  2 , Rachel R Caspi  3
Affiliations

Autoimmunity to neuroretina in the concurrent absence of IFN-γ and IL-17A is mediated by a GM-CSF-driven eosinophilic inflammation

So Jin Bing et al. J Autoimmun. 2020 Nov.

Abstract

IFN-γ and IL-17A can each elicit ocular autoimmunity independently of the other. Since absence of IFN-γ or IL-17A individually failed to abolish pathology of experimental autoimmune uveitis (EAU), we examined EAU development in the absence of both these cytokines. Ifng-/-Il17a-/- mice were fully susceptible to EAU with a characteristic eosinophilic ocular infiltrate, as opposed to a mononuclear infiltrate in WT mice. Retinal pathology in double-deficient mice was ameliorated when eosinophils were genetically absent or their migration was blocked, supporting a pathogenic role for eosinophils in EAU in the concurrent absence of IFN-γ and IL-17A. In EAU-challenged Ifng-/-Il17a-/- mice, ocular infiltrates contained increased GM-CSF-producing CD4+ T cells, and supernatants of retinal antigen-stimulated splenocytes contained enhanced levels of GM-CSF that contributed to activation and migration of eosinophils in vitro. Systemic or local blockade of GM-CSF ameliorated EAU in Ifng-/-Il17a-/- mice, reduced eosinophil peroxidase levels in the eye and in the serum and decreased eosinophil infiltration to the eye. These results support the interpretation that, in the concurrent absence of IFN-γ and IL-17A, GM-CSF takes on a major role as an inflammatory effector cytokine and drives an eosinophil-dominant pathology. Our findings may impact therapeutic strategies aiming to target IFN-γ and IL-17A in autoimmune uveitis.

Keywords: Eosinophils; Experimental autoimmune uveitis; GM-CSF; IFN-γ; IL-17A; Neuroinflammation.

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Figures

Fig. 1.
Fig. 1.
Ifng−/−Il17a−/− mice are susceptible to EAU and show eosinophil dominant infiltration in the eyes. WT, Ifng−/−, Il17a−/−, and Ifng−/−Il17a−/− mice on C57BL/6 background were immunized with 150 μg IRBP and 300 μg p1–20. (A, B) EAU scores were evaluated by fundoscopy (A) and histology (B) 21 days after immunization. (C) Representative histopathology of uveitic retina in each group with H&E stain. Original mag, 20x (left) and 150x (right). Arrows show eosinophils. (D) CD45+ live cells in the EAU eyes on day 16–18 were analyzed by algorithm-based t-SNE visualization and FlowSOM clustering based on their surface marker expression. Shown are the scheme for the cell-type cluster (left most), and a representative plot and parts of whole graph from each group. (E) The number of SiglecF+ (Eosinophils), Ly6C−Ly6G+ (Neutrophils) or Ly6C+Ly6G− (Monocytes) in CD11b+ gates, and CD4+ or CD8+ T cells in the eyes. (A) Data combined from 4 experiments (WT; n = 41, Ifng−/−; n = 26, Il17a−/−; n = 27, Ifng−/−Il17a−/−; n = 34). (B–E) Representative data from 4 independent experiments, and each group contains at least 3 mice. Data shown as mean ± SEM. Significance was determined using Mann-Whitney U test (A, B), ANOVA (E). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, NS; not significant.
Fig. 2.
Fig. 2.
Eosinophils contribute to the pathogenesis of EAU in the absence of IFN-γ and IL-17A. (A–C) WT, ΔdblGATA, Ifng−/−Il17a−/−, Ifng−/−Il17a−/−ΔdblGATA mice on C57BL/6 background were immunized with 100 μg of p651–670. (A, B) EAU scores were evaluated by (A) fundoscopy, and (B) histology with H&E stain. Original mag, 20x. (C) The number of SiglecF+, Ly6C−Ly6G+, or Ly6C+Ly6G− cells in the CD45+CD11b+CD11c− gates from eyes on day 16. (D) The frequency of CCR3+ cells in eye-infiltrating eosinophils was determined by FACS. (E, F, G) WT and Ifng−/−Il17a−/− mice were s.c. treated with 200 μg of a CCR3 antagonist (SB328437) every day from one day before immunization. (E) The frequency of CCR3+ eosinophils and total eosinophils in eye-infiltrating cells were determined by FACS. (F) Fundoscopy score on day 15 days after immunization. (G) Levels of Eosinophil Peroxidase (EPO) in the serum were measured by LEGENDplex on day 16. (A) Data combined from 3 experiments (WT; n = 14, ΔdblGATA; n = 10, Ifng−/−Il17a−/−; n = 14, Ifng−/−Il17a−/− × ΔdblGATA; n = 5). (B–G) Representative data from 2 independent experiments, and each group contains at least 3 mice. Data shown as mean ± SEM. Significance was determined using Mann-Whitney U test (A, F), or unpaired t-test (C, D, E, G). *p < 0.05, **p < 0.01.
Fig. 3.
Fig. 3.
Production of GM-CSF is enhanced in cells that lack both IFN-γ and IL-17A. (A) On day 18 after EAU immunization of WT and Ifng−/−Il17a−/− mice on C57BL/6 background with IRBP and p1–20, eye-infiltrating cells were collected and intracellular cytokine staining was performed after a PMA/ionomycin pulse. Representative FACS plots (left), frequencies (middle) or numbers (right) of GM-CSF positive cells gated on CD4+ T cells. (B) GM-CSF secretion in supernatants of splenocytes was measured by ELISA after in vitro recall stimulation with IRBP. (C) WT and Ifng−/− mice were treated i.p. with 600 μg of anti-IL-17A Ab (MM17F3) or isotype control (mouse IgG1) every other day starting on day −1 relative to EAU immunization with IRBP and p1–20. (D, F) GM-CSF in splenocyte culture supernatant was measured by ELISA after in vitro recall stimulation with p651–670. (E) WT and Il17a−/− mice on C57BL/6 background were treated i.p. with 300 μg of anti–IFN-γ Ab (R4–6A2) or isotype control (Rat IgG1) every other day, starting on day −1 relative to EAU immunization with p651–670. Shown are representative data of 2 (C, D), 3 (E, F) or 4 (A, B) independent experiments, and each group contains at least 3 mice. Data shown as mean ± SEM. *p < 0.05, ***p < 0.001, ****p < 0.0001, (A, B) Student’s t-test, (D, F) One way ANOVA with Bonferroni correction, (C, E) Mann-Whitney test.
Fig. 4.
Fig. 4.
Treatment with anti-GM-CSF antibody at either induction or effector phase of EAU suppresses disease in Ifng−/−Il17a−/− mice. WT and Ifng−/−Il17a−/− mice either on C57BL/6 (immunized with IRBP and p1–20) (B, D, E) or on B10.RIII background (immunized with p161–180) (G, H) were i.p. treated with anti-GMCSF mAb (MP1–22E9.11) or isotype Ig (Rat IgG2a) from the induction phase (starting on day −1) (A, B, G) or from the effector phase (starting on day 7) (C, D, H) of EAU induction every other day. (E) T cells were enriched from LNs and spleens of EAU-induced WT and Ifng−/−Il17a−/− mice, were restimulated in vitro with IRBP for 3 days and were adoptively transferred to naïve recipient mice that received treatment of 300 μg of anti-GM-CSF mAb or isotype control every other day starting from the day of cell transfer. (F) WT and Ifng−/−Il17a−/− mice were immunized for EAU. On day 10, 100 μg of anti-GM-CSF mAb in 2 μl of PBS was intravitreally injected into the right eye and the isotype control was injected into the left eye of each mouse. EAU scores were assessed by histology on day 12. Representative data from 2 (E, F) or 3 (B, D, G, H) independent experiments, and each group contains at least 3 mice. Data shown as mean ± SEM. *p < 0.05, ****p < 0.0001, (B, D, E, G, H) Mann-Whitney U test, (F) Wilcoxon matched-pairs signed rank test. NS, not significant.
Fig. 5.
Fig. 5.
GM-CSF produced by autoantigen-stimulated Ifng−/−Il17a−/− cells promotes eosinophil activation and migration (A) Bone-marrow derived eosinophils (bmEos) from WT B10.RIII mice were cultured with the supernatant of antigen (IRBP) re-stimulated splenocytes from EAU-induced WT or Ifng−/−Il17a−/− mice in the presence or absence of anti-GM-CSF antibody (10 μg/mL) or GM-CSF (100 ng/mL). After 2 days, the mean fluorescence intensity (MFI) of SiglecF and CD11b on bmEos was determined by FACS. Representative FACS plots with gates and histograms are shown on the left. (B) BmEos were placed into the upper chamber of a 96-well transwell chemotaxis assay plate with the supernatant of re-stimulated splenocytes in the lower chamber and incubated for 3 h. GM-CSF or eotaxin were used as positive controls. The total number of live cells that had migrated into the bottom chamber with the supernatant was counted and divided by the number of live cells that had migrated to medium alone to give the fold change over the medium control. Representative data from 2 independent experiments, each group contains 2 individual samples, and each sample was duplicated. Data shown as mean ± SEM. Significance was determined using unpaired t-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.
Fig. 6.
Fig. 6.
Blockade of GM-CSF decreases disease by suppressing migration and function of eosinophils. WT and Ifng−/−Il17a−/− mice on B10.RIII background were immunized with p161–180 and i.p. treated with anti-GM-CSF mAb (MP1–22E9.11) or isotype Ig (Rat IgG2a) from the effector phase (starting on day 7). (A) The number of eosinophils (SiglecF+CD11b+CD45+), neutrophils (Ly6G+Ly6C−CD11b+CD45+), monocytes (Ly6G−Ly6C+CD11b+CD45+) in eye-infiltrating cells was determined by FACS. (B) Representative FACS plots of eye-infiltrating eosinophils. (C) Eosinophil peroxidase (EPO) in the eye-supernatant and the serum were assessed by LEGENDPlex Representative data from 3 independent experiments, and each group contains at least 3 mice. Data shown as mean ± SEM. *p < 0.05, **p < 0.01, Student’s t-test.
Fig. 7.
Fig. 7.
IL-17F plays a pathogenic role in EAU-challenged Ifng−/−Il17a−/− mice by recruiting neutrophils in the eyes. (A) EAU scores were evaluated by histology of eyes from C57BL/6 WT, Il17a−/−, Il17f−/−, and Il17a−/−Il17f−/− mice 21 days after immunization with IRBP and p1–20. (B, C) Ifng−/−Il17a−/−, Il17a−/−Il17f−/−Ifng−/− on C57BL/6 background were immunized with 150 μg p651–670. (B) EAU scores were evaluated by fundoscopy on day 16 after immunization. (C) The number of SiglecF+, Ly6C−Ly6G+, or Ly6C+Ly6G− cells in the CD45+CD11b+CD11c− gates from eyes. Representative data from 3 independent experiments. Data shown as mean ± SEM. Significance was determined using Mann-Whitney U test (A, B) or Student’s t-test (C). *p < 0.05, **p < 0.01.
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