Immune Complexes Indirectly Suppress the Generation of Th17 Responses In Vivo

Abstract

The precise context in which the innate immune system is activated plays a pivotal role in the subsequent instruction of CD4+ T helper (Th) cell responses. Th1 responses are downregulated when antigen is encountered in the presence of antigen-IgG immune complexes. To assess if Th17 responses to antigen are subject to similar influences in the presence of immune complexes we utilized an inflammatory airway disease model in which immunization of mice with Complete Freund's Adjuvant (CFA) and ovalbumin (Ova) induces a powerful Ova-specific Th1 and Th17 response. Here we show that modification of that immunization with CFA to include IgG-Ova immune complexes results in the suppression of CFA-induced Th17 responses and a concurrent enhancement of Ova-specific Th2 responses. Furthermore, we show the mechanism by which these immune complexes suppress Th17 responses is through the enhancement of IL-10 production. In addition, the generation of Th17 responses following immunization with CFA and Ova were dependent on IL-1α but independent of NLRP3 inflammasome activation. Together these data represent a novel mechanism by which the generation of Th17 responses is regulated.

Fig 1. Immune complexes suppress the development of neutrophilic airway responses in vivo.

(A) Schematic illustration of the inflammatory airway disease model; (B-D) WT mice were injected subcutaneously with either CFA/Ova or CFA/IgG-Ova on day 0; mice were then intranasally challenged with Ova on days 15, 16 and 17. 48 h after the final intranasal challenge hematoxylin and eosin lung histology sections (B) and differential cell counts in bronchoalveolar lavage fluid were analyzed (C). Representative sections from 4 mice per group are shown; upper panel bar = 50 μm; lower panel bar = 20 μm (B). Values represent the mean ± SEM of five separate experiments (n = 19 mice per group; C). (D) Ova-specific IgG1 and IgG2c levels in serum were measured by ELISA. Values represent the mean ± SEM of five separate experiments (n = 13–15 mice per group; D).

Fig 2. Immune complexes suppress Th1 and Th17 responses.

(A) Lung draining LN were collected and restimulated in vitro with or without Ova (10 μM) for 72 h and cytokine levels in the supernatants analyzed. Values represent the mean ± SD and are representative of five separate experiments each with a minimum of 3 mice per group. (B) Intracellular cytokine analysis of LN cells stimulated for 4 h with PMA and ionomycin in the presence of brefeldin A. Results are representative of two independent experiments. (C, D) WT mice were injected subcutaneously with either CFA/Ova, CFA/IgG-Ova, CFA/IgG^depl-Ova or CFA/Fab-Ova on day 0; mice were then intranasally challenged with Ova on days 15, 16 and 17. 48 h after the final intranasal challenge lung draining LN were collected and restimulated in vitro with or without Ova (10 μM) for 72 h and cytokine levels in the supernatants analyzed. Values represent the mean ± SD and are representative of two separate experiments each with a minimum of 2–3 mice per group. (E) CFSE labeled OT-II transgenic CD4⁺ cells were transferred into CD45.1 congenic mice, which were then immunized subcutaneously with either CFA/Ova or CFA/IgG-Ova and T cell proliferation assessed by flow cytometry 3 d later. Results are representative of two independent experiments.

Fig 3. Immune complexes modulate in vitro cytokine production by BMDC.

(A-H) BMDC from WT mice were stimulated with or without LPS (50 ng/ml) in the presence or absence of Ova or IgG-Ova immune complexes for 10 h; cytokine secretion into culture supernatants was measured by ELISA. (A-C) For the induction of IL-1α, IL1β and IL-18, 4 h after the addition of LPS cells were additionally stimulated with 5 mM ATP for 20 min; media was replaced with fresh media and cells were further incubated for another 6 h. Values represent the mean ± SEM of three independent experiments, each performed in triplicate.

Fig 4. Immune complex driven IL-10 production suppresses Th1 and Th17 responses.

(A) WT and Il10^-/- mice were injected subcutaneously with either CFA/Ova or CFA/IgG-Ova on day 0; mice were then intranasally challenged with Ova on days 15, 16 and 17. 24 h after the final intranasal challenge lung draining LN were collected and restimulated in vitro with or without Ova (10 μM) for 72 h and cytokine levels in the supernatants analyzed. Values represent the mean ± SD and are representative of three separate experiments each with a minimum of 3 mice per group. (B-D) BMDC from Il10^-/- mice were stimulated with or without LPS (50 ng/ml) in the presence or absence of Ova or IgG-Ova immune complexes for 10 h; cytokine secretion into culture supernatants was measured by ELISA. (B, C) For the induction of IL-1α and IL1β, 4 h after the addition of LPS cells were additionally stimulated with 5 mM ATP for 20 min; media was replaced with fresh media and cells were further incubated for another 6 h. Values represent the mean ± SEM of three independent experiments, each performed in triplicate. (E) WT and Il10^-/- mice were sensitized and challenged as described in (A), 24 h after the final intranasal challenge differential cell counts in bronchoalveolar lavage fluid were determined. Values represent the mean ± SEM of the three separate experiments (n = 6 mice per group).

Fig 5. CFA-induced Th17 responses are independent of the NLRP3 inflammasome.

WT, nlrp3^-/-, asc^-/-, and caspase-1^-/- mice were injected subcutaneously with CFA/Ova on day 0; mice were then intranasally challenged with Ova on days 15, 16 and 17. 24 h after the final intranasal differential cell counts in bronchoalveolar lavage fluid were determined (A, C, E). Lung draining LN were collected and restimulated in vitro with or without Ova (10 μM) for 72 h and cytokine levels in the supernatants analyzed (B, D, F). A, B, D, F; values represent the mean ± SEM of three separate experiments, n = 9–15 mice per group. C, E; values represent the mean ± SD and are representative of three separate experiments, n = 3–5 mice per group.

Fig 6. CFA-induced Th17 responses are dependent on IL-1R1 and IL-1α.

WT, Il1r1^-/-, Il1a^-/-, and Il1b^-/- mice were injected subcutaneously with CFA/Ova on day 0; mice were then intranasally challenged with Ova on days 15, 16 and 17. 24 h after the final intranasal differential cell counts in bronchoalveolar lavage fluid were determined (A, C, E). Lung draining LN were collected and restimulated in vitro with or without Ova (10 μM) for 72 h and cytokine levels in the supernatants analyzed (B, D, F). A, C, E, F; values represent the mean ± SEM of the three separate experiments, n = 9–15 mice per group. B, D; values represent the mean ± SD and are representative of three separate experiments, n = 3–5 mice per group.

Fig 7. Immune complexes suppress IL-1α production in vivo.

WT mice were injected subcutaneously with CFA/Ova or CFA/IgG-Ova; 24 h later skin at the injection site was harvested. IL-1α levels in tissue homogenates were measure by ELISA; each point represents an individual mouse.