IL-27 Is Essential for Suppression of Experimental Allergic Asthma by the TLR7/8 Agonist R848 (Resiquimod)

Abstract

Different models of experimental allergic asthma have shown that the TLR7/8 agonist resiquimod (R848) is a potential inhibitor of type 2 helper cell-driven inflammatory responses. However, the mechanisms mediating its therapeutic effects are not fully understood. Using a model of experimental allergic asthma, we show that induction of IL-27 by R848 is critical for the observed ameliorative effects. R848 significantly inhibited all hallmarks of experimental allergic asthma, including airway hyperreactivity, eosinophilic airway inflammation, mucus hypersecretion, and Ag-specific Ig production. Whereas R848 significantly reduced IL-5, IL-13, and IL-17, it induced IFN-γ and IL-27. Neutralization of IL-27 completely reversed the therapeutic effect of R848 in the experimental asthma model, demonstrating dependence of R848-mediated suppression on IL-27. In vitro, R848 induced production of IL-27 by murine alveolar macrophages and dendritic cells and enhanced expression of programmed death-ligand 1, whose expression on monocytes and dendritic cells has been shown to regulate peripheral tolerance in both murine and human studies. Moreover, in vitro IL-27 enhanced secretion of IFN-γ whereas it inhibited IL-5 and IL-13, demonstrating its direct effect on attenuating Th2 responses. Taken together, our study proves that R848-mediated suppression of experimental asthma is dependent on IL-27. These data provide evidence of a central role of IL-27 for the control of Th2-mediated allergic diseases.

FIGURE 1.

Treatment with R848 alleviates airway inflammation and mucus hypersecretion in a model of experimental allergic asthma. Wild-type animals were subjected to the experimental asthma model and challenged with OVA alone (OVA), treated additionally with R848 before the first OVA challenge (R848), or treated with control vehicle only (Alum) (A). Total cell counts and eosinophilia in BALF (B and C) are shown. Reduced inflammation and mucus production in the lungs of treated animals, at original magnification ×20 (D) and attenuated levels of OVA-specific IgG1 and IgE in serum of control versus treated mice (E) are shown. Airway hyperresponsiveness after methacholine challenge was measured in an invasive lung function assay (F). Data are expressed as means ± SEM of three independent experiments with n ≥ 6 animals per group except for IgE measurements, in which two independent experiments were analyzed with n = 3–4 animals per group. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

R848 reduces Th2 and Th17 cytokines but enhances production of IL-27 and IFN-γ. On restimulation with the allergen, BLN cells from the R848 group displayed a decreased Th2 cytokine production and increased expression of IFN-γ and IL-27 (A and B) compared with those from allergic mice (OVA). Ex vivo proliferation of BLN cells after restimulation with OVA (C) and frequencies of regulatory T cells in BLN (D) are shown. PD-L1 expression on lung DCs in mice subjected to the asthma protocol after application of R848 (E) is shown. Data are expressed as means ± SEM of two to three independent experiments with n ≥ 6 animals per group. **p < 0.01, ***p < 0.001.

FIGURE 3.

The therapeutic effect of R848 on experimental allergic asthma is dependent on IL-27. Mice subjected to the asthma protocol were either pretreated with an IL-27–neutralizing Ab or isotype control or left untreated before application of R848 (A). Neutralization of IL-27 abrogated the beneficial effects of R848 as shown by increased AHR (B) and elevation of total BALF cell counts and eosinophilia (C and D). Increased eosinophilic airway inflammation and mucus secretion are shown in H&E- and PAS-stained lung sections, at original magnification ×20 (E). Effects of antagonizing IL-27 on Th2 cytokine production by BLN cells and IgE in serum of experimental animals (F and G) or activation/proliferation (H) of BLN cells stimulated ex vivo using OVA are shown. Data are expressed as means ± SEM of two to three independent experiments with n ≥ 4 animals per group.*p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

Lung APCs produce IL-27 in response to R848, which impacts Th2/Th1 cytokine production and tolerogenic marker expression in murine and human APCs. Gating strategy for AMs and DCs in murine lungs (A) and staining strategy used for identification of human monocytes and DCs in peripheral blood (B) are shown. Murine AMs and DCs sorted from lungs of wild-type mice as well as human DCs and monocytes display increased expression of PD-L1 on stimulation with R848 (C). Additionally, murine AMs and DCs produce IL-27 on exposure to R848 (D). The dose-dependent influence of in vitro stimulation with recombinant murine IL-27 (rmIL-27) on IFN-γ production by murine DCs and AMs (E) and expression of PD-L1 (F) are shown. Reduced Th2 cytokine and IFN-γ production by BLN cells of allergic mice restimulated in presence of rmIL-27 (G). Enhanced expression of PD-L1 on human DCs and monocytes after in vitro stimulation with recombinant human IL-27 (rhIL-27) (H) is shown. Flow cytometry histograms are representative of one of two murine experiments or of DCs and monocytes from five human donors. Graphs display a summary of two to three independent experiments with means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001. rhIL-27, recombinant human IL-27; rmIL-27, recombinant murine IL-27.