C5a receptor-deficient dendritic cells promote induction of Treg and Th17 cells

Abstract

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen-derived DC results in increased production of TGF-beta leading to de novo differentiation of Foxp3(+) Treg within 12 h after co-incubation with CD4(+) T cells from DO11.10/RAG2(-/-) mice. Stimulation of C5aR(-/-) DC with OVA and TLR2 ligand Pam(3)CSK(4) increased TGF-beta production and induced high levels of IL-6 and IL-23 but only minor amounts of IL-12 leading to differentiation of Th cells producing IL-17A and IL-21. Th17 differentiation was also found in vivo after adoptive transfer of CD4(+) Th cell into C5aR(-/-) mice immunized with OVA and Pam(3)CSK(4). The altered cytokine production of C5aR(-/-) DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17-cell differentiation through regulation of DC function.

Figure 1. TLR2-challenge of C5aR^-/- sDC induces a cytokine milieu that drives Treg and Th17 differentiation

(A) sDC from WT, C5aR^-/-, and C5L2^-/- mice were co-cultured with naïve CD4⁺ T cells from OVA-TCR transgenic DO11.10/RAG2^-/- mice for 4 days in the absence or the presence of OVA ± Pam₃CSK₄ (PAM). OVA was added at a concentration of 1 μM. C5aR antagonist A8^Δ71-73 (C5aRA) was added to some cultures as indicated. Concentrations of IL-12, IFN-γ, IL-6, TGF-β, IL-23, IL-21, IL-17A and IL-10 in culture supernatants were determined by ELISA. Data show mean ± SD (n=3). The frequencies of (B) IL-17A⁺ and IFN-γ⁺ or (C) Foxp3⁺TCR⁺ among CD4⁺ cells were determined by intracellular staining and flow cytometry. Data are representative of 4 independent experiments.

Figure 2. C5aR signaling regulates early development of Foxp3⁺ Treg and subsequent Th17 differentiation

sDC from C5aR^-/- mice were co-cultured with naïve CD4⁺ T cells from DO11.10/RAG2^-/- mice in the absence or the presence of PAM. After the indicated incubation periods, the frequencies of (A) Foxp3⁺TCR⁺ T cells or (B) of IL-17A⁺IFN-γ⁺ cells among CD4⁺ T cells were determined by intracellular staining and flow cytometry. (C) Concentrations of IL-6, TGF-β, IL-23 and IL-17A in culture supernatants were determined by ELISA. Data show mean ± SD (n=3). Data are representative of 3 independent experiments.

Figure 3. IL-2 receptor alpha chain blockade abrogates C5aR^-/--induced Th17 differentiation

sDC from C5aR^-/- mice were co-cultured with naïve CD4⁺ T cells from DO11.10/RAG2^-/- mice. (A) Antibodies to CD25 or an appropriate isotype control (20 μg/ml) were added to the cell cultures and the frequencies of Foxp3⁺ (A) or IL-17A⁺/IFN-γ⁺ (B) CD4⁺ T cells were determined by intracellular staining using flow cytometry. (C) Concentrations of IL-12, IFN-γ, IL-6, TGF-β, IL-23, IL-21 and IL-17A in culture supernatants from (A) were determined by ELISA. Data show mean ± SD (n=4). Data are representative of 4 independent experiments.

Figure 4. Lack of C5aR in vivo promotes Th17 differentiation

(A) CD4⁺ T cells from DO11.10/RAG2^-/- mice mice were adoptively transferred into WT, C5L2^-/-, and C5aR^-/- mice. These mice were immunized i. p. with PBS or OVA + PAM. On day 4, splenocytes were isolated and the frequency of TCR transgenic CD4⁺ cells was assessed by flow cytometry. (B) Splenocytes from WT, C5L2^-/-, and C5aR^-/- mice from (A) were re-stimulated in vitro with medium or OVA + PAM and the frequency of IL-17A⁺ and IFN-γ⁺ cells among CD4⁺ T cells was determined by intracellular staining. (C, D) Cytokine levels in splenocyte culture supernatants re-stimulated with either medium (C) or OVA+PAM (D) as determined by ELISA. Data show mean ± SD (n=3). Data are representative of 3 independent experiments.

Figure 5. C5aR signaling has a direct regulatory impact on MHC-II expression, costimulatory molecule expression and cytokine production from sDC

(A) Left panel: Surface expression of MHC-II molecules as determined by flow cytometry. WT- black line, C5aR^-/-- dotted line, C5L2^-/-- gray line. Gray filled histogram depicts isotype control. Right panel: sDC from WT, C5aR^-/-, and C5L2^-/- mice were incubated in the absence and presence of PAM for 24h. Cells were gated on CD11c^hi cells; MFI: median fluorescence intensity. (B) Surface expression of CD80, CD86 and CD40 on sDC. (C and D) Cytokine production from PAM- or PAM + anti-CD40-challenged sDC. Cells were cultured in the absence or presence of PAM or PAM + anti-CD40 for 24 hours. Concentrations of IL-12, IL-6, IL-23, TGF-β and IL-1β were measured by ELISA in culture supernatants. Data show mean ± SD. Data are representative of 5-8 independent experiments.