Macrophages participate in IL-17-mediated inflammation

Abstract

The involvement of macrophages (MΦs) in Th17-cell responses is still poorly understood. While neutrophils are thought to be the predominant effector of Th17-cell responses, IL-17 is also known to induce myelotropic chemokines and growth factors. Other T-cell-derived cytokines induce non-classical functions, suggesting that IL-17 sigxnaling may similarly elicit unique MΦ functions. Here, we characterized the expression of subunits of the IL-17 receptor on primary murine MΦs from different anatomical compartments. The greatest expression of IL-17 receptors was observed on mucosal Ly6C(hi) "inflammatory" MΦs. We further observed upregulation of IL-17 receptors in vitro on bone marrow-derived macrophages (BMMΦs) in response to peptidoglycan or CpG oligonucleotide stimuli, and in vivo, upon CFA administration. Macrophages expressing IL-17 receptors were observed infiltrating the hearts of mice with myocarditis, and genetic ablation of IL-17RA altered MΦ recruitment. Treating primary MΦs from a wide variety of different anatomic sources (as well as cell lines) with IL-17A induced the production of unique profiles of cytokines and chemokines, including GM-CSF, IL-3, IL-9, CCL4/MIP-1β and CCL5/RANTES. IL-17A also induced production of IL-12p70; IL-17-signaling-deficient MΦs elicited diminished IFN-γ production by responding DO11.10 CD4(+) T cells when used as APCs. These data indicate that MΦs from different anatomic locations direct IL-17-mediated responses.

Figure 1

Expression of IL17 receptors on primary murine macrophages. Quantitative analysis of fluorescence intensities of A) IL17RA and B) IL17RC staining on primary murine Ly6C^hi inflammatory (filled) and Ly6C^loGr1⁻ resident (open) macrophages, by compartment. Statistics are by x-immediate multiple linear regression of mean fluorescence intensity against tissue and subpopulation. Statistics denote comparisons to lymphoid tissues or comparison of Ly6C^hi to Ly6C^loGr1⁻ cells. Data indicate means of individual animals plus standard deviation, n = 5. Representative of three independent experiments.

Figure 2

Coexpression of IL17RA and IL17RC on primary murine macrophages. A) Representative bivariate psuedocolor plot of IL17RA and IL17RC staining on spleen, peritoneal, and lamina propria F4/80⁺-gated macrophages. IL17RA⁺IL17RC⁺ double-positive gate is based on negative staining for isotype control (not shown). B) Quantitative analysis of IL17RA⁺IL17RC⁺ double-positive staining on F4/80⁺Ly6C^hi inflammatory (left) and F4/80⁺Ly6C^loGr1⁻ resident (right) macrophages, by compartment. Statistics are by x-immediate multiple linear regression of MFI against tissue and subpopulation. Arrows denote comparisons to spleen; printed p values denote comparison of Ly6C^hi versus Ly6C^loGr1⁻ cells. Data indicate means of individual animals plus standard deviation, n = 5. Representative confocal immunofluorescent staining of IL17RA (green), IL17RC (red), and F4/80 (blue) in C) duodenum and D) spleen of naïve mice. Data are representative of two independent experiments.

Figure 3

IL17 receptor expression on murine BMMφ, in response to inflammation in vitro. A) Representative histograms of IL17RA (left) and IL17RC (right) expression on BMMφ in response to TLR ligands; isotype control (filled), no stimulus (dashed), 6.0 μg/mL CpG ODN1668 (solid, thick). Quantitative analysis of B, D) IL17RA and C, E) IL17RC expression on BMMφ in response to (B, C) TLR ligands alone, and (D, E) TLR ligands in combination with proinflammatory cytokines TNFα (filled) and IFNγ (diagonal hatching) after 24h of stimulation. Data indicate means of duplicate wells plus standard error. Asterisks denote (p < 0.05) by two-tailed Students’ t-test, compared to control. Representative of three independent experiments.

Figure 4

IL17 receptor expression on primary murine macrophages, in response to inflammation in vivo. A) Representative histograms of IL17RA expression on primary liver macrophages 48h following sc CFA administration (open, thick), compared to control naïve mice (filled). Quantitative analysis of IL17RA expression on B) liver macrophages and C) peripheral blood monocytes in response to CFA (filled). Control animals were given sc sterile PBS (open). D) Quantitative analysis of IL17RA and IL17RC expression on CD11b^hiF4/80⁺Ly6C^hi inflammatory (left) and CD11b^hiF4/80⁺Ly6C^loGr1⁻ resident (right) peritoneal macrophages in response to sterile elicitation by proteose peptone (filled). Control animals were administered vehicle PBS alone (open). Data represent individual animals (diamonds), and mean of each group (bars). Statistics are by two-tailed Students’ t-test. Representative of three independent experiments.

Figure 5

IL17 receptor expression and signaling in experimental murine autoimmune myocarditis. A) Enumerative FACS analysis of expression of IL17RA (left), IL17RC (middle) and double-positive (right) CD11b⁺F4/80⁺CD45⁺ intracardiac macrophages on days 0, 14, and 21 of EAM. B) Proportion and C) absolute enumerative cardiac macrophage infiltration in IL17RA^−/− (filled) and WT (open) mice at day 21 of EAM. Data represent individual animals (diamonds), and mean of each group (bars). Statistics are by two-tailed Students’ t-test. D) Cytometric analysis of M-CSF-differentiated bone marrow-derived macrophages from IL17RA^−/− (dotted pink) and WT (thick blue) mice. Isotype controls are tinted grey.

Figure 6

Physiologic responses of primary murine macrophages, in response to IL17A signaling. Production of A) CCL4/MIP1β, B) CCL5/RANTES, C) GM-CSF, D) IL3, and E) IL9 by resident peritoneal (Per), proteose peptone-elicited peritoneal (ePer), naïve resident spleen (Spl), CFA-elicited spleen (eSpl), bronchoalveolar lavage (BAL), peripheral blood (PB), and bone marrow-derived (BMMφ) macrophages, in dose-response to IL17A, determined by multiplex cytokine assay. Data indicate means of triplicate cultures, plus/minus standard error. Asterisks indicate p < 0.05 by two-tailed Students’ t-test, compared to unstimulated control.

Figure 7

Physiologic responses of primary murine macrophages, in response to IL17A signaling. A) Production of IL12p70 in dose-response to IL17A from multiple macrophage cell systems. B) IFNγ production by DO11.10 (KJ1-26⁺-gated) CD4⁺ cells responding to 10 μg/mL OVA_323–339-pulsed BMMφ. BMMφ were preconditioned with 10 μg/mL α-mouse IL17A mAb 50104, or derived from Act1-deficient mice. Data indicate means of triplicate cultures, plus/minus standard error. Asterisks indicate p < 0.05 by two-tailed Students’ t-test. Representative of three independent experiments. C) Representative bivariate plots of viable CD4⁺CD3ε⁺-gated cells from B, median well of each condition.