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. 2022:2:831084.
doi: 10.3389/fopht.2022.831084. Epub 2022 Mar 7.

Bidirectional Effect of IFN-γ on Th17 Responses in Experimental Autoimmune Uveitis

Hui Shao  1 , Henry J Kaplan  2 , Deming Sun  3
Affiliations

Bidirectional Effect of IFN-γ on Th17 Responses in Experimental Autoimmune Uveitis

Hui Shao et al. Front Ophthalmol (Lausanne). 2022.

Abstract

Pro- and ant-inflammatory effects of IFN-γ have been repeatedly found in various immune responses, including cancer and autoimmune diseases. In a previous study we showed that the timing of treatment determines the effect of adenosine-based immunotherapy. In this study we examined the role of IFN-γ in pathogenic Th17 responses in experimental autoimmune uveitis (EAU). We observed that IFN-γ has a bidirectional effect on Th17 responses, when tested both in vitro and in vivo. Anti-IFN-γ antibody inhibits Th17 responses when applied in the initial phase of the immune response; however, it enhances the Th17 response if administered in a later phase of EAU. In the current study we showed that IFN-γ is an important immunomodulatory molecule in γδ T cell activation, as well as in Th17 responses. These results should advance our understanding of the regulation of Th17 responses in autoimmunity.

Keywords: IFN-gamma; Th17; autoimmunity; experimental autoimmune uveitis; gamma delta T cell.

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Conflict of interest statement

Conflict of Interest: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Scheme 1
Scheme 1
Schematic experimental procedure.
Figure 1
Figure 1
Effect of anti-IFN-γ on in vitro Th1 and Th17 responses. Purified CD3+ T cells were stimulated with the immunizing antigen and irradiated splenic APCs, under Th1- (culture medium containing IL-12) or Th17 (culture medium containing IL-23) polarized condition and in the absence or presence of anti-IFN-γ antibody (5 μg/ml). The abundance of IFN-γ+ and IL-17+ T cells among the responder T cells was estimated after intracellular staining with fluorescence labeled anti-IFN-γ or anti–IL-17 antibodies, 5 d after in vitro stimulation. Compare to untreated (A), anti-IFN-γ inhibits T cell activation when added on d 0 (B) but enhances T cell activation if added on d 8 (C). The results in (A–C) are from a single experiment and pooled results of three separate studies are shown in (D). **p < 0.01. The IL-17 and IFN-γ levels in the culture medium were measured by ELISA after anti-IFN-γ Ab added on d0 (E). The data are pooled from three independent experiments. **p < 0.01.
Figure 2
Figure 2
Anti-IFN-γ inhibited Th17 responses in vivo. Anti-IFN-γ on d0 (the day of immunization) preferentially inhibited Th17 cells. Two groups of B6 mice (n = 6) were immunized with IRBP1-20/CFA; one was injected with anti-IFN-γ (100cμg/mouse) via i.p. and the group received PBS. Thirteen days post-immunization, mice were sacrificed and the number of Th1 and Th17 cells among responder T cells were assessed 5 d after in vitro stimulation after intracellular staining with anti-IL-17 and anti-IFN-γ antibody. The results in (A) are from a single experiment and pooled results of three separate studies are shown in (B). Responder T cells of B6 mice administered with anti-IFN-γ produced significantly decreased levels of IL-17. The IL-17 and IFN-γ levels in the culture supernatants were measured by ELISA after stimulation of responder T cells with the immunizing antigen and splenic APCs for 2 (d) The data are pooled from three independent experiments. *P < 0.05 and **P < 0.01.
Figure 3
Figure 3
Effect of anti-IFN-γ administration is “timing dependent”. Th17 responses were significantly decreased in mice early treated with anti-IFN-γ but were enhanced in mice late treated with anti-IFN-γ. Groups of B6 mice (n=6) immunized with IRBP1-20/CFA received anti-IFN-γ on d0 or d8. Thirteen days post immunization CD3+ responder T cells were stimulated in vitro with the immunizing peptide and APCs, under Th1- or Th17-polarized conditions. 5 d after in vitro stimulation the number of IFN-γ+ and IL-17+ T cells was estimated after intracellular staining. The results from (A) are from a single experiment and pooled results of three separate studies are shown in (B). **P < 0.01. B) Cytokine production by Th17 cells was enhanced by late anti-IFN-γ treatment. The IL-17 and IFN-γ levels in the culture medium were measured by ELISA after stimulation of responder T cells with the immunizing antigen and splenic APCs for 2 d (C). **P < 0.01. IRBP-specific T cells were separated from IRBP-immunized mice with or without an anti-IFN-γ administration on day 13 of immunization. After 2 d in vitro stimulation with the immunizing antigen and splenic APCs, under Th17-polarizing conditions, the activated T cells were adoptively transferred to naive B6 mice (2 x 106/mouse) via i.p. injection and clinical expression of EAU was scored (D).
Figure 4
Figure 4
Altered γδ T cell responses in anti-IFN-γ treated mice. γδ T cell activation and expansion was inhibited in early anti-IFN-γ treated mice. Freshly isolated CD3+ cells from naïve and immunized B6 mice, with or without anti-IFN-γ administration (day 0), were stained with PE-anti-αβTCR and FITC-anti-γδTCR antibodies before they were subjected to FACS analysis (A). They were also dually stained with PE-anti-mouse CD44 and FITC-anti-γδTCR antibodies (C). A summary of multiple assays is shown in (B, D). *P < 0.05, **P < 0.01.
Figure 5
Figure 5
γδ T cells isolated from late anti-IFN-γ treated mice have enhanced pro-Th17 activity.CD3+ responder T cells (1 5 x 106/well) isolated from immunized TCR-δ-/- mice, with or without a prior anti-IFN-γ administration were stimulated with the immunizing antigen and irradiated splenic APCs, under Th17 polarized condition. Total γδ T cell numbers (A), as well as proportional number of IL-17+ versus total γδ T cells (B) were compared. **P < 0.01. In vitro Th17 response of TCR-δ-/- responder T cells were assessed with or without an addition of γδ T cells isolated from early or late treated anti-IFN-γ mice (C). The number of IL-17+ T cells among the gated CD4+ responder T cells was estimated after intracellular staining with anti–IL-17 antibody, 5 d after in vitro stimulation.
Figure 6
Figure 6
Augmented Th17 responses in IRBP1-20/CFA-immunized IFN-γ-/- mice. CD3+ responder T cells were purified from IRBP1-20/CFA-immunized B6 and IFN-γ-/- mice. They were stimulated in vitro with the immunizing peptide and APCs, under Th17 or Th1 polarized conditions as indicated. After 5 d of in vitro stimulation, Th1 and Th17 responses specific for the immunizing antigen were estimated by assessing IFN-γ+ and IL-17+ T cells intracellularly stained with fluorescence -labeled anti-IFN-γ or anti–IL-17 antibodies (A). Immunized IFN-γ-/- mice also produced increased IL-17 compared to B6 mice. Production of IFN-γ and IL-17 by the B6 or IFN-γ-/- responder T cells after 48 h antigen stimulation in vitro was assessed by ELISA. Data are pooled from three independent experiments are shown (B). **P < 0.01. Number of γδ T cells and IL-17 secretion from immunized IFN-γ-/- mice exceeded that of immunized wt B6 mice. CD3+ T cells isolated from naïve, immunized B6 and immunized IFN-γ-/- mice were stained with anti-IL-17 and anti-γδTCR, before FACS analysis (C). The data from three independent experiments is shown in (D). **P < 0.01. NS, not significant. Contribution of antigen-specific T cells and the effect of splenic APCs in augmented Th17 responses were examined. IFN-γ-/- and B6 mice responder T cells (1.5 x 106/well) from IFN-γ-/- or B6 mice were stimulated by B6 splenic APCs (E, left panels) or IFN-γ-/- splenic APCs (E, right panels). IL-17 in culture supernatants were assessed by ELISA harvested 48 h after in vitro stimulation. The data are pooled from three independent experiments. **P < 0.01.
Figure 7
Figure 7
Multiple aberrant immune responses in IFN-γ-/- mice. Foxp3+ T cell expansion is increased in IFN-γ-/- mice. In a 24-well plate, the CD3+ responder T cells (1.5 x 106/well) derived from B6 mice or IFN-γ-/- mice were cultured in medium containing a very low dose of IL-2 (1ng/ml) for 5 d, which preferentially favors Foxp3+ T cell expansion (37). The percentage of Foxp3+ T cells among αβTCR+ cells was determined by FACS analysis (A). Data are from one single experiment, which is representative of three independent experiments. IRBP-specific T cells isolated from immunized IFN-γ-/- mice have stronger pathogenic activity after adoptive transfer to naïve B6 recipients. IRBP-specific T cells were prepared from the responder T cells of IFN-γ-/- mice and B6 mice. After a 2-d in vitro stimulation with the immunizing antigen and splenic APCs. 2 x 106 cells were adoptively transferred to naïve B6 recipient mice via i.p. injection (B). Pathologic examination. H&E histologic sections from an eye in each group were obtained on day 30 post-immunization. Severe vitritis and chorioretinal folds occur in IFN-γ-/- mice compared to minimal vitreous inflammation and a normal retina in wt-B6 mice (C).
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