The alarmin Mrp8/14 as regulator of the adaptive immune response during allergic contact dermatitis

Abstract

Mrp8 and Mrp14 are endogenous alarmins amplifying inflammation via Toll-like receptor-4 (TLR-4) activation. Due to their pro-inflammatory properties, alarmins are supposed to enhance adaptive immunity via activation of dendritic cells (DCs). In contrast, analysing a model of allergic contact dermatitis (ACD) we observed a more severe disease outcome in Mrp8/14-deficient compared to wild-type mice. This unexpected phenotype was associated with an enhanced T-cell response due to an accelerated maturation of DCs in Mrp8/14-deficient mice. Accordingly, Mrp8, the active component of the heterocomplex, inhibits early DC maturation and antigen presentation in a TLR-4-dependent manner. Transfer of DCs purified from the local lymph nodes of sensitized Mrp8/14-deficient to wild-type mice determined the outcome of ACD. Our results link a pro-inflammatory role of the endogenous TLR-4 ligand Mrp8/14 to a regulatory function in adaptive immunity, which shows some similarities with the 'hygiene hypothesis' regarding continuous TLR-4 stimulation and decreased risk of allergy.

Figure 1

Mrp8/14-induced TLR-4-mediated activation of iBMDCs. (A) Expression of activation markers CD86 and MHC-II on in vitro generated iBMDCs from C57BL/6 mice. iBMDCs were activated for 48 h in the presence of Mrp8 or Mrp14 (8 μg/ml), Mrp8/14 (20 μg/ml), or LPS (300 ng/ml). Unstimulated iBMDCs served as control. M.f.i. shifts were determined by flow cytometry (three independent experiments; mean±s.e.m.). (B, C) Proliferation of CFSE-labelled CD4⁺ and CD8⁺ allogenic spleen T cells from BALB/c mice after 5 days of culture with unstimulated, Mrp8-, Mrp14-, Mrp8/14-, or LPS-stimulated C57BL/6 BMDCs (DC:T-cell ratio was 1:5). Panel (B) shows results from one of three independent experiments which are summarized in (C) (mean±s.e.m.). (D) Comparison of CD86 expression of unstimulated, LPS- (300 ng/ml) and Mrp8- (8 μg/ml) stimulated BMDCs from C57BL/6 wt, RAGE^−/−, and TLR-4^−/− mice. Following 48 h of stimulation, m.f.i. shifts were measured by flow cytometry and normalized to unstimulated control cells (three independent experiments; mean±s.e.m.). *P<0.05, **P<0.01, ***P<0.001, and NS=not significant.

Figure 2

(A–C) DNFB-induced ACD response in ears of C57BL/6 wt and Mrp14^−/− mice. (A) Ear swelling of wt and Mrp14^−/− mice after elicitation of ACD. Mice were sensitized by painting 0.4% DNFB in acetone/olive oil on the shaved abdomen. After 6 days, the right ears were challenged with 0.4% DNFB in acetone/olive oil. The left ears were treated with acetone/olive oil only and served as controls. Ear thickness was measured 24 and 48 h after challenge. Swelling was determined by calculating the difference between the control ear and the challenged ear (four independent experiments with each 8–15 mice per group; mean±s.e.m.) (***P<0.001). (B, C) Histological analyses of ear sections from DNFB-treated wt and Mrp14^−/− mice 48 h after challenge. Sections were stained for (B) immigrated immune cell (CD11b⁺ myeloid cells, Gr-1⁺ granulocytes, F4/80⁺ macrophages) and (C) expression of Mrp8 and Mrp14. (B, C) scale bar, 250 μm. (D) Croton oil-induced ICD response in ears of C57BL/6 wt and Mrp14^−/− mice. Mice were painted with 10 μl 3% croton oil in acetone/olive oil (4:1) onto the right ears. The left ears served as controls and were treated with acetone/olive oil only. Ear thickness was measured 24 and 48 h after the treatment. Ear swelling was determined by calculating the difference between the untreated ear and the treated ear (three independent experiments with five mice per group each; mean±s.e.m.).

Figure 3

Extracellular Mrp8 impairs DC differentiation. (A) C57BL/6 wt and Mrp14^−/−BMDCs generated in the presence of 8 μg/ml Mrp8 [Mrp8/LPS] were stimulated with 300 ng/ml LPS for 48 h on day 6. CD11c, CD86, and MHC-II surface expression was compared to conventionally LPS-stimulated BMDCs [-/LPS] (four independent experiments; mean±s.e.m.). (B) Proliferation of allogenic T cells (BALB/c) induced by C57BL/6 BMDCs generated as described in (A) (DC:T cells, 1:5) (four independent experiments; mean±s.e.m.). (C) CD86 and MHC-II expression on C57BL/6 wt and TLR-4^−/− BMDCs generated in the absence or presence of 8 μg/ml Mrp8 [Mrp8 (d0–d6)]. The right part shows one of three independent experiments that are summarized in the left part (mean±s.e.m.). (D) Phenotype of C57BL/6 wt BMDCs stimulated with 8 μg/ml Mrp8 on day 5 for 24 h [Mrp8 (d5–d6/LPS)]. On day 6, 300 ng/ml LPS was added for further 48 h. CD86 and MHC-II expression was compared to conventionally LPS-stimulated BMDCs [-/LPS] (three independent experiments; mean±s.e.m.). (E) Proliferation of allogenic T cells (BALB/c) induced by C57BL/6 wt BMDCs generated as described in (D) (DC:T cells, 1:5) (three independent experiments; mean±s.e.m.). (F) Accelerated BMDC differentiation due to the lack of Mrp8/14. CD86 and MHC-II expression on wt and Mrp14^−/− BMDCs stimulated for 48 h with LPS (300 ng/ml) (right) or left unstimulated (left). M.f.i. shifts were normalized to unstimulated wt cells (four independent experiments; mean±s.e.m.). (G) Proliferation of allogenic spleen T cells (BALB/c) induced by C57BL/6 wt or Mrp14^−/− BMDCs (DC:T cells, 1:5) (four independent experiments; mean±s.e.m.). (H) Improved ability of Mrp14^−/− BMDCs to contact T cells. Allogenic T cells (BALB/c) were embedded with unstimulated or LPS-stimulated wt and Mrp14^−/− BMDCs (C57BL/6) in 3-D collagen gels. Interactions were monitored by time-lapse microscopy. Left part: Number of DC–T-cell contacts per hour (two independent experiments; 40 DCs evaluated per condition). Right part: Embedded T cells were analysed for CD69 surface expression. (I) Mrp8/14 serum levels of DNFB-sensitized wt mice. Sera of 10 mice were analysed (two independent experiments, five mice each, mean±s.e.m.) *P<0.05, **P<0.01, ***P<0.001, and NS, not significant.

Figure 4

Increased DC-mediated T-cell response in Mrp14^−/− mice during ACD. (A) Proliferation of wt and Mrp14^−/− T cells during ACD. Wt and Mrp14^−/− spleen T cells isolated from DNFB-challenged C57BL/6 mice were cultured with DNBS-loaded wt or Mrp14^−/− BMDCs of naive C57BL/6 mice (left) or in vivo-primed EpCAM-negative dDCs from ear-draining LNs of the allergic wt or Mrp14^−/− C57BL/6 mice (right) (DC:T-cell ratio was 1:5). Proliferation was measured after 5 days of co-culture (four independent experiments; mean±s.e.m.). (B) Expression of pro-inflammatory cytokines IFN-γ and IL-17 by spleen T cells of DNFB-challenged wt and Mrp14^−/− C57BL/6 mice in response to stimulation with wt or Mrp14^−/− DNFB-primed EpCAM-negative dDCs from the same ACD mice, respectively (three independent experiments; mean±s.e.m.). (C) Proliferation of Mrp14^−/− T cells (left) or wt T cells (right) from spleens of DNFB-challenged C57BL/6 mice co-cultured with either DNBS-charged BMDCs of naive C57BL/6 mice or in vivo-primed EpCAM-negative dDCs from ear-draining LNs of the allergic wt or Mrp14^−/− C57BL/6 mice. T-cell proliferation was measured after 5 days of co-culture (three independent experiments; mean±s.e.m.). *P<0.05, ***P<0.001, and NS, not significant.

Figure 5

Increased T-cell response during ACD is partly dependent on the number of MDSCs. (A) Quantitative analysis of CD11b⁺Ly-6C⁺ MDSCs in LN suspensions from wt and Mrp14^−/− mice 48 h after ACD elicitation with DNFB (*P=0.04). Results from one of four independent experiments are shown. (B, C) Proliferation of T cells from allergic wt or Mrp14^−/− mice (C57BL/6) induced by MDSC-depleted EpCAM-negative dDC fractions (B, right and C white or grey hatched bars) in comparison to untreated EpCAM-negative dDCs (B, left and C white or grey bars) from the same DNFB-challenged wt and Mrp14^−/− mice. Proliferation was determined by CFSE dilution (four independent experiments). *P<0.05 and NS, not significant. (D) Release of the pro-inflammatory cytokine IL-17 by spleen T cells from DNFB-treated wt and Mrp14^−/− mice (C57BL/6) in response to MDSC-depleted EpCAM-negative dDCs or untreated EpCAM-negative dDCs of the same mice (three independent experiments; mean±s.e.m.). *P<0.05.

Figure 6

Wt mice develop an increased ear swelling after adoptive transfer of DNFB-primed Mrp14^−/− dDCs. EpCAM-negative dDCs were isolated from wt and Mrp14^−/− mice 48 h after challenge with DNFB. Subsequently, two identical groups of sensitized wt mice were injected intravenously with these freshly isolated wt or Mrp14^−/− dDCs (1.6 × 10⁶ dDCs per mouse) immediately before challenge with 0.4% DNFB. Ear swelling of both groups was evaluated 24 and 48 h after challenge. Data were compared to ear swelling of DNFB-challenged wt and Mrp14^−/− mice. Data are from five mice per group (mean±s.e.m.). *P<0.05.