IL-12 can alleviate Th17-mediated allergic lung inflammation through induction of pulmonary IL-10 expression

Abstract

Interleukin (IL)-12 has been shown to suppress T helper type 2 (Th2)-induced pathogenesis that is associated with allergic asthma, largely through interferon (IFN)-gamma production. We have recently shown that in the absence of T-bet, the major regulator of IFN-gamma expression, allergic lung inflammation is primarily associated with IL-17-associated recruitment of neutrophils into the pulmonary tract of mice. In the absence of T-bet, exogenous IL-12 was still able to suppress neutrophilic infiltration and to diminish levels of IL-17, IL-23, and IL-23R, as well as retinoic acid-related orphan receptor gamma t, the transcriptional regulator of the Th17 pathway. The same effects were observed in T-bet(-/-) IFN-gamma(-/-) double knockout mice, showing an IFN-gamma-independent effect of IL-12 in this model. IL-10 expression in the lungs of T-bet-deficient mice was significantly increased after IL-12 treatment, and inoculation of anti-IL-10R mAb completely reversed the ability of IL-12 to suppress histological inflammation, recruitment of inflammatory cell subsets into the lung, bronchiole hyperresponsiveness, and IL-17 production. We conclude that Th17-mediated allergic lung inflammation that becomes dominant in the absence of effective IFN-gamma signaling can be effectively suppressed by IL-12 through an IL-10-dependent mechanism.

Figure 1

Interleukin (IL)-12 suppresses pulmonary Th17 activity. Cytokine levels in lung homogenates obtained from unchallenged, ovalbumin (OVA)-challenged, and OVA-challenged plus IL-12-treated BALB/c T-bet^+/+ (wild type, WT) and T-bet^−/− mice were measured 24 h after a 5-day OVA challenge. Protein levels were normalized to lung tissue total protein. (a) IL-4, IL-5, and interferon (IFN)-γ were measured by murine cytometric bead array and (b) IL-17 levels were measured by enzyme-linked immunosorbent assay (ELISA). The data are shown as means±s.e.m.; 8 mice per group. (c) IL-17, retinoic acid-related orphan receptor (ROR)γt, and RORα transcripts were measured by quantitative real-time (RT)-PCR. Transcript expression was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and used to calculate expression relative to untreated mice. (d, e) Expression of IL-23 protein and IL-23R transcripts, respectively, in the lungs of sensitized mice upon antigen challenge relative to non-sensitized mice after antigen challenge. Lungs were collected 24 h after a 5-day OVA challenge from both groups of mice. Levels of IL-23 were measured by ELISA, and levels of IL-23R transcripts were determined by quantitative RT-PCR. The data represent means±s.e.m.; 8 mice per group. (f) Flow cytometric detection of IFN-γ and IL-17-producing cells in lung lymphocyte preparations from OVA-challenged and OVA-challenged plus IL-12-treated T-bet^+/+ (WT) and T-bet^−/− mice. The data represent mean percentages of positively stained cells within the lymphocyte gate. *P < 0.05. All results are representative of at least three independent experiments with 6–8 mice per group.

Figure 2

Interleukin (IL)-12 alleviates allergen-induced asthma in the absence of T-bet. BALB/c T-bet^+/+ and T-bet^−/− mice (6–8 weeks old) were sensitized intraperitoneally (i.p.) on days 1 and 7 with 10 μg ovalbumin (OVA). Intranasal (i.n.) challenge with 100 μg OVA was performed for 5 consecutive days starting 1 week after the final sensitization. IL-12 (0.25 μg) was given i.n. during the first two days of the 5-day OVA challenge. Airway inflammation was assessed 24 h after the day 5 OVA challenge. (a) Representative hematoxylin and eosin (H&E)-stained lung sections from naive, OVA-challenged, and OVA-challenged plus IL-12-treated BALB/c T-bet^+/+ (wild type, WT) and T-bet^−/− mice (×200; 8 mice per group). (b) Scoring of H&E-stained lung sections based on the severity of peribronchial inflammation. (c) Numbers of total cellular infiltrates plus differential counts of eosinophils and neutrophils obtained from bronchoalveolar lavage (BAL). BAL cells were stained with Wright’s modified stain and enumerated according to standard morphological criteria. The data represent means±s.e.m., with at least 8 mice per group and are representative of at least three independent experiments. (d) Airway function was assessed using an invasive mechanical small animal ventilator. Newtonian resistance (R_N) and tissue elastance (H) were measured in response to increasing doses of methacholine. The data are shown as means of 6 mice per group±s.e.m. and are representative of two independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Interleukin (IL)-12 suppresses IL-17 in the absence of interferon (IFN)- γ. (a) Pooled splenocytes were isolated from 2–3 T-bet^+/+ IFN-γ^+/+ (wild type, WT), T-bet^−/− IFN-γ^+/+, and T-bet^−/− IFN-γ^−/− double knockout (DKO) mice, and 1×10⁶ cells per ml were cultured in the presence of 125 ng ml⁻¹ IL-12 and 100 ng ml⁻¹ lipopolysaccharide. Levels of IFN-γ in supernatant fluids were measured by enzyme-linked immunosorbent assay (ELISA) after 24, 48, 60, and 72 h. The data represent means of 4 wells per group±s.e.m. from one experiment. (b) Numbers of total cells, eosinophils, and neutrophils in bronchoalveolar lavage (BAL) obtained from WT and DKO mice were evaluated 24 h after OVA challenge. BAL cells were stained with Wright’s modified stain and counted according to standard morphological criteria. The data represent means of 6 mice per group±s.e.m. and are representative of at least three independent experiments. (c) Cytokine levels in WT and DKO lungs after induction of allergic lung inflammation and treatment with IL-12. Cytokine levels were measured by cytometric bead array (IL-4, IL-5, and IFN-γ) and by ELISA (IL-17). The data represent means of 6 mice per group±s.e.m. and are representative of at least three independent experiments. *P < 0.05.

Figure 4

Interleukin (IL)-12 induces expression of IL-10. BALB/c wild-type (WT), T-bet^−/−, and double knockout (DKO) mice were sensitized with ovalbumin (OVA) in alum, and 2 weeks later were challenged intranasally (i.n.) for 5 consecutive days with OVA. Twenty-four hours after the last OVA inoculation, homogenates were prepared from lung tissue, protein levels were normalized to lung total protein, and IL-10 was measured by enzyme-linked immunosorbent assay (ELISA). The data represent means of 6 mice per group±s.e.m. and are representative of at least three independent experiments *P <0.05.

Figure 5

Interleukin (IL)-10 is responsible for IL-12-induced suppression of IL-17-mediated inflammation. Ovalbumin (OVA)-sensitized and OVA-challenged BALB/c T-bet^−/− mice were left untreated or treated with IL-12, IL-12 in the presence of neutralizing anti-IL-10R mAb (α-IL-10R), IL-12 in the presence of isotype control mAb (IgG), or neutralizing anti-IL-10R mAb alone. (a) Representative hematoxylin and eosin (H&E)-stained lung sections from naive and OVA-challenged BALB/c T-bet^−/− mice (×200; 4 mice per group). Neutralizing anti-IL-10R mAb (α-IL-10R mAb) and IgG isotype control mAb were administered on days 1 through 3 during OVA intranasal (i.n.) challenge. (b) H&E-stained lung sections were scored for levels of peribronchial inflammation in each of the treatment groups. (c) Numbers of eosinophils and neutrophils in bronchoalveolar lavage (BAL) fluids were evaluated 24 h after OVA challenge. BAL cells were stained with Wright’s modified stain and counted according to standard morphological criteria. The data represent means±s.e.m.; 6 mice per group. (d) Methacholine dose–response curves 24 h after 5 days of i.n. OVA challenge are shown. The data represent means±s.e.m.; 6 mice per group. Levels of (e) IL-6, (f) IL-17, and (g) IL-10 in T-bet^−/− lungs after induction of allergic lung inflammation and treatment with neutralizing anti-IL-10R mAb. Cytokine levels were measured by ELISA. The data represent means of at least 6 mice per group±s.e.m. and are representative of at least two independent experiments. *P < 0.05.