IFN-γ protects from lethal IL-17 mediated viral encephalomyelitis independent of neutrophils

Abstract

Background: The interplay between IFN-γ, IL-17 and neutrophils during CNS inflammatory disease is complex due to cross-regulatory factors affecting both positive and negative feedback loops. These interactions have hindered the ability to distinguish the relative contributions of neutrophils, Th1 and Th17 cell-derived effector molecules from secondary mediators to tissue damage and morbidity.

Methods: Encephalitis induced by a gliatropic murine coronavirus was used as a model to assess the direct contributions of neutrophils, IFN-γ and IL-17 to virus-induced mortality. CNS inflammatory conditions were selectively manipulated by adoptive transfer of virus-primed wild-type (WT) or IFN-γ deficient (GKO) memory CD4+ T cells into infected SCID mice, coupled with antibody-mediated neutrophil depletion and cytokine blockade.

Results: Transfer of GKO memory CD4+ T cells into infected SCID mice induced rapid mortality compared to recipients of WT memory CD4+ T cells, despite similar virus control and demyelination. In contrast to recipients of WT CD4+ T cells, extensive neutrophil infiltration and IL-17 expression within the CNS in recipients of GKO CD4+ T cells provided a model to directly assess their contribution(s) to disease. Recipients of WT CD4+ T cells depleted of IFN-γ did not express IL-17 and were spared from mortality despite abundant CNS neutrophil infiltration, indicating that mortality was not mediated by excessive CNS neutrophil accumulation. By contrast, IL-17 depletion rescued recipients of GKO CD4+ T cells from rapid mortality without diminishing neutrophils or reducing GM-CSF, associated with pathogenic Th17 cells in CNS autoimmune models. Furthermore, co-transfer of WT and GKO CD4+ T cells prolonged survival in an IFN-γ dependent manner, although IL-17 transcription was not reduced.

Conclusions: These data demonstrate that IL-17 mediates detrimental clinical consequences in an IFN-γ-deprived environment, independent of extensive neutrophil accumulation or GM-CSF upregulation. The results also suggest that IFN-γ overrides the detrimental IL-17 effector responses via a mechanism downstream of transcriptional regulation.

Figure 1

Neutrophil depletion does not prevent early mortality. (A) Neutrophil infiltration was characterized by flow cytometry based on CD45^hi Ly6G⁺ expression (R4 region) in the CNS of controls (control (ctr); infected SCID mice without CD4⁺ T cell transfer), infected SCID recipients of WT (WT) or GKO CD4⁺ T cells (GKO) eight days p.i. (B) Mean expression (±SEM) of IFN-γ and CXCL1 mRNA analyzed in brains of naïve (n = 4), controls (n = 3), SCID recipients of WT (n = 4) or GKO (n = 4) CD4⁺ T cells at day eight p.i. Data are representative of two experiments. (C) Survival rates of SCID recipients of WT or GKO CD4⁺ T cells (with and without neutrophil depletion) observed daily following JHMV infection. Data represent the mean of sixteen mice per group combined from two separate experiments with n = 8 per group in each experiment. Efficiency of neutrophil (CD45^hi Ly6G⁺, R4 region) depletion after antibody (Ab) treatment analyzed by flow cytometry in the CNS of SCID recipients of GKO CD4⁺ T cells at day eight p.i. Density plots are representative of eight animals per group.

Figure 2

Anti-IFN-γ induced CNS neutrophil infiltration but did not increase mortality in SCID recipients of WT CD4⁺T cells. (A) Survival of infected SCID recipients of WT CD4⁺ T cells in the absence or presence of anti-IFN-γ mAb and recipients of GKO CD4⁺ T cells assessed daily until day ten p.i. Data are mean of at least six mice per group combined from two separate experiments (B) Anti-IFN-γ mAb efficiency assessed by flow cytometry by measuring MHC class II expression on microglia (CD45^lo F4/80⁺) in infected SCID recipients of WT (n = 6), WT + anti-IFN-γ mAb (n = 8) or GKO (n = 8) CD4⁺ T cells. Data are representative of two experiments. (C) Numbers of infiltrating neutrophils in the brain of infected SCID recipients of WT, WT + anti-IFN-γ mAb or GKO CD4⁺ T cells determined by flow cytometry at day eight p.i. Data represent the mean (±SD) from two independent experiments (n = 6, untreated group and n = 8, anti-IFN-γ treated group).

Figure 3

IL-17 expression in SCID recipients of GKO CD4⁺T cells. IL-6 and IL-1β (A), IL-17 (B), and IL-22 and IL-21 (C) mRNA expression analyzed by quantitative real-time PCR in brains of naïve (n = 4), infected control (n = 3), infected SCID recipients of WT (n = 4) or GKO (n = 8) CD4⁺ T cells at day eight p.i. IL-17 mRNA expression was measured in infected SCID recipients of WT CD4⁺ T cells + anti-IFN-γ mAb (n = 8). Data represent the mean (±SEM) from two separate experiments. (D) IL-17 (green) and CD3 (red) expression in the brains of SCID recipients of GKO CD4⁺ T cells. CD3⁺ (arrows) and IL-17-producing T cells (arrow heads) detected at day eight p.i. (E) IL-17-expressing CD4⁺ T cells from cervical lymph nodes of SCID recipients of GKO CD4⁺ T cells at day eight p.i. Dot plots are representative of four individuals. (F) Expression of IFN-γ and IL-17 by donor-derived CD4⁺ T cells prior to transfer analyzed by flow cytometry after in vitro stimulation. Intracellular cytokine expression measured using FITC-IFN-γ, PE-IL-17 and the corresponding isotype controls. Dot plots are representative of duplicates from two separate experiments. N.D= Not Detected.

Figure 4

IL-17 mediates mortality, independent of CNS neutrophil infiltration. (A) Survival was assessed daily in infected SCID recipients of WT (n = 8), WT + anti-IL-17 mAb (n = 12), GKO (n = 8) and GKO + anti-IL-17 mAb (n = 12) CD4⁺ T cells until day 18 p.i. Data are mean from two separate experiments (B) Total numbers of neutrophils determined by flow cytometry at day eight p.i. in infected SCID recipients of WT, WT + anti-IL-17 mAb, GKO, GKO + anti-IL-17 mAb CD4⁺ T cells. Data represent the mean from two independent experiments (n = 4, untreated mice and n = 8, anti-IL-17-treated mice per experiment).

Figure 5

WT CD4⁺T cell co-transfer prevents GKO CD4⁺T cell induced mortality. (A) Relative MHC I-A^d class II mRNA expression in the CNS of infected SCID recipients of WT, WT/GKO or GKO CD4⁺ T cells determined by quantitative real-time PCR. Data represent the mean ± SEM of two independent experiments with n = 4 per experiment. Microglial MHC class II expression quantified by flow cytometry in infected SCID recipients of WT, WT/GKO or GKO CD4⁺ T cells. Data represent the mean ± SD of three separate experiments with n = 4 per group per experiment. (B) Survival was assessed following infection of SCID recipients of WT (n = 20), WT/GKO (n = 20) or GKO (n = 12) CD4⁺ T cells. Data represent four separate experiments. (C) Control of virus replication analyzed by plaque assay in infected control, SCID recipients of WT, WT/GKO or GKO CD4⁺ T cells at day eight p.i. Data represent the average (±SD) of eight mice per group combined from two independent experiments. (D) Total number of bone-marrow-derived leukocytes (CD45^hi) and neutrophils (Ly6G⁺) analyzed by flow cytometry at day eight p.i. in brains of infected SCID recipients of WT, WT/GKO and GKO CD4⁺ T cells. Data represent the mean of twelve mice per group combined from three independent experiments.

Figure 6

IFN-γ-mediated protection prevents IL-17-mediated mortality. (A) Number of CD4⁺ T cells in the infiltrating population and distribution of Thy1.1 positive cells measured by flow cytometry at day eight p.i. Data represent means (±SD) of twelve mice per group combined from three separate experiments. (B) IL-17 mRNA expression determined by quantitative real-time PCR in infected SCID recipients of WT, WT/GKO or GKO CD4⁺ T cells. Data represent the mean of two experiments with n = 4 in each group per experiment. (C) Splenocytes of immunized GKO donors cultured in the presence of JHMV with or without recombinant IFN-γ (10 ng/ml) for six days and restimulated four hours with PMA/Ionomycin. Intracellular cytokine expression on CD4⁺ T cells analyzed by flow cytometry using FITC-IFN-γ, PE-IL-17 and the corresponding isotype controls. Dot plots are representative of duplicates from two separate experiments. (D) Survival of infected SCID recipients of WT/GKO (n = 6), WT/GKO + anti-IFN-γ mAb (n = 8) and GKO (n = 4) CD4⁺ T cells assessed daily. Data are representative of two separate experiments.

Figure 7

Alterations in chemokines, MMPs and GM-CSF mRNA do not correlate with IL-17-mediated mortality. (A) mRNA expression analyzed in the CNS of naïve mice, infected control mice, and SCID recipients of WT or GKO CD4⁺ T cells at day eight p.i. Data represent the mean (±SEM) of four individual mice per group (B) MMP9, MMP3, MMP12 and GM-CSF mRNA expression measured in the CNS of naïve, controls, infected SCID recipients of WT, GKO or GKO + anti-IL-17 CD4⁺ T cells at day eight p.i.. Data are representative of the mean (±SEM) of four individual mice per group.