Allergen-Induced C5a/C5aR1 Axis Activation in Pulmonary CD11b+ cDCs Promotes Pulmonary Tolerance through Downregulation of CD40

Activation of the C5/C5a/C5a receptor 1 (C5aR1) axis during allergen sensitization protects from maladaptive T cell activation. To explore the underlying regulatory mechanisms, we analyzed the impact of C5aR1 activation on pulmonary CD11b+ conventional dendritic cells (cDCs) in the context of house-dust-mite (HDM) exposure. BALB/c mice were intratracheally immunized with an HDM/ovalbumin (OVA) mixture. After 24 h, we detected two CD11b+ cDC populations that could be distinguished on the basis of C5aR1 expression. C5aR1− but not C5aR1+ cDCs strongly induced T cell proliferation of OVA-reactive transgenic CD4+ T cells after re-exposure to antigen in vitro. C5aR1− cDCs expressed higher levels of MHC-II and CD40 than their C5aR1+ counterparts, which correlated directly with a higher frequency of interactions with cognate CD4+ T cells. Priming of OVA-specific T cells by C5aR1+ cDCs could be markedly increased by in vitro blockade of C5aR1 and this was associated with increased CD40 expression. Simultaneous blockade of C5aR1 and CD40L on C5aR1+ cDCs decreased T cell proliferation. Finally, pulsing with OVA-induced C5 production and its cleavage into C5a by both populations of CD11b+ cDCs. Thus, we propose a model in which allergen-induced autocrine C5a generation and subsequent C5aR1 activation in pulmonary CD11b+ cDCs promotes tolerance towards aeroallergens through downregulation of CD40

The C5a/C5aR1 axis controls the development of experimental allergic asthma independent of LysM-expressing pulmonary immune cells.

C5a regulates the development of maladaptive immune responses in allergic asthma mainly through the activation of C5a receptor 1 (C5aR1). Yet, the cell types and the mechanisms underlying this regulation are ill-defined. Recently, we described increased C5aR1 expression in lung tissue eosinophils but decreased expression in airway and pulmonary macrophages as well as in pulmonary CD11b+ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs) during the allergic effector phase using a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we determined the role of C5aR1 signaling in neutrophils, moDCs and macrophages for the pulmonary recruitment of such cells and the importance of C5aR1-mediated activation of LysM-expressing cells for the development of allergic asthma. We used LysM-C5aR1 KO mice with a specific deletion of C5aR1 in LysMCre-expressing cells and confirmed the specific deletion of C5aR1 in neutrophils, macrophages and moDCs in the airways and/or the lung tissue. We found that alveolar macrophage numbers were significantly increased in LysM-C5aR1 KO mice. Induction of ovalbumin (OVA)-driven experimental allergic asthma in GFP-C5aR1fl/fl and LysM-C5aR1 KO mice resulted in strong but similar airway resistance, mucus production and Th2/Th17 cytokine production. In contrast, the number of airway but not of pulmonary neutrophils was lower in LysM-C5aR1 KO as compared with GFP-C5aR1fl/fl mice. The recruitment of macrophages, cDCs, moDCs, T cells and type 2 innate lymphoid cells was not altered in LysM-C5aR1 KO mice. Our findings demonstrate that C5aR1 is critical for steady state control of alveolar macrophage numbers and the transition of neutrophils from the lung into the airways in OVA-driven allergic asthma. However, C5aR1 activation of LysM-expressing cells plays a surprisingly minor role in the recruitment and activation of such cells and the development of the allergic phenotype in OVA-driven experimental allergic asthma

Differential regulation of C5a receptor 1 in innate immune cells during the allergic asthma effector phase

<div><p>C5a drives airway constriction and inflammation during the effector phase of allergic asthma, mainly through the activation of C5a receptor 1 (C5aR1). Yet, C5aR1 expression on myeloid and lymphoid cells during the allergic effector phase is ill-defined. Recently, we generated and characterized a floxed green fluorescent protein (GFP)-C5aR1 knock-in mouse. Here, we used this reporter strain to monitor C5aR1 expression in airway, pulmonary and lymph node cells during the effector phase of OVA-driven allergic asthma. C5aR1 reporter and wildtype mice developed a similar allergic phenotype with comparable airway resistance, mucus production, eosinophilic/neutrophilic airway inflammation and Th2/Th17 cytokine production. During the allergic effector phase, C5aR1 expression increased in lung tissue eosinophils but decreased in airway and pulmonary macrophages as well as in pulmonary CD11b⁺ conventional dendritic cells (cDCs) and monocyte-derived DCs (moDCs). Surprisingly, expression in neutrophils was not affected. Of note, moDCs but not CD11b⁺ cDCs from mediastinal lymph nodes (mLN) expressed less C5aR1 than DCs residing in the lung after OVA challenge. Finally, neither CD103⁺ cDCs nor cells of the lymphoid lineage such as Th2 or Th17-differentiated CD4⁺ T cells, B cells or type 2 innate lymphoid cells (ILC2) expressed C5aR1 under allergic conditions. Our findings demonstrate a complex regulation pattern of C5aR1 in the airways, lung tissue and mLN of mice, suggesting that the C5a/C5aR1 axis controls airway constriction and inflammation through activation of myeloid cells in all three compartments in an experimental model of allergic asthma.</p></div

WT and GFP-C5aR1^flox/flox mice show a strong and similar pulmonary recruitment of inflammatory cells during the allergic effector phase.

<p>(<b>A)</b> Histological examination of airway inflammation. Sections were stained with H&E (original magnification x 200). The pictures are representative of 5 histological sections per treatment group. (<b>B</b>) Gating strategies used to identify eosinophils (SiglecF⁺CD11c^-), macrophages (SiglecF⁺CD11c⁺) or neutrophils (Ly6G⁺SiglecF^-) in lung tissue. <b>(C)</b> Differential cell counts of PBS-treated or OVA-immunized WT or GFP-C5aR1^flox/flox animals. Values shown are the mean ± SEM; n = 9–17 per group. (<b>D</b>) Gating strategy to identify DC subsets in lung tissue. Data shown represent the pulmonary cell composition of OVA-treated mice. Cells were first gated on SiglecF^- cells. Then lineage negative cells were excluded. Subsequently DCs were identified as CD11c⁺MHCII⁺ cells. These cells were further subdivided into CD103⁺CD11b⁻ or CD103⁻CD11b⁺cDCs. Within the latter population, we identified CD11b⁺CD64^- cDCs and CD11b⁺CD64⁺moDCs. (<b>E</b>) DC counts in lung cell suspensions of PBS-treated or OVA-immunized WT or GFP-C5aR1^flox/flox animals. Values shown are the mean ± SEM; n = 7–18 per group. * indicates significant differences between PBS or OVA-treated groups. * p < 0.05, ** p < 0.01, *** p <0.001.</p

DC and CD4⁺ T cell accumulation and their C5aR1 expression in mLN of WT and GFP-C5aR1^flox/flox mice in the allergic effector phase.

<p><b>(A)</b> Cell counts of cDC and moDC subsets in mLN of WT or GFP-C5aR1^flox/flox mice in response to PBS treatment or OVA immunization; n = 7–15 per group. CD11b⁺ cDC data were analyzed by ANOVA on ranks (n = 7–15). <b>(B)</b> Counts of different CD4⁺ T cell subsets in mLN of PBS-treated or OVA-challenged WT or GFP-C5aR1^flox/flox animals. Values shown are the mean ± SEM; n = 7–15 per group. * indicates significant differences between PBS and OVA treatment groups; § indicates significant differences between WT and GFP-C5aR1^flox/flox OVA-treated groups. * or § p < 0.05; ** p < 0.001. <b>(C)</b> GFP signal in CD4⁺ or CD4⁺CD44⁺CD62L^- effector T cells from OVA-challenged WT (grey histogram) or GFP-C5aR1^flox/flox animals (black line).</p

WT and GFP-C5aR1^flox/flox mice develop a similar allergic asthma phenotype.

<p><b>(A)</b> AHR in response to i.t. administration of methacholine measured as airway resistance. Shown are dose response curves in PBS-treated controls or OVA-immunized mice from WT (++) or GFP-C5aR1^flox/flox (flfl) strains. Values shown are the mean ± SEM; n = 9–16 per group. <b>(B)</b> Gating strategy for the BAL fluid cell analysis. Cells were identified by flow cytometry using different markers to identify macrophages (SiglecF⁺autofluorescence⁺), eosinophils (SiglecF⁺autofluorescence^-) neutrophils (SiglecF^-Ly6G⁺CD4^-), and T cells (SiglecF^-Ly6G^-CD4⁺). <b>(C)</b> Total and differential cell counts in BAL fluid of PBS-treated or OVA-immunized WT or GFP-C5aR1^flox/flox animals. Values shown are the mean ± SEM; n = 9–17 per group. <b>(D)</b> GFP/C5aR1 expression in eosinophils, macrophages, neutrophils and CD4⁺ T cells from BAL fluid of GFP-C5aR1 reporter mice in response to OVA; grey histogram: WT controls. <b>(E)</b> Histological examination of mucus production in the airways of PBS-treated or OVA-immunized WT or GFP-C5aR1^flox/flox animals. Sections were stained with PAS for mucus production (original magnification x 200). (<b>F)</b> Frequency of PAS-positive bronchi in PBS-treated or OVA-immunized WT or GFP-C5aR1^flox/flox animals. Mucus producing airways are plotted relative to all analysed airways. Values shown are the mean ± SEM; n = 4–8 per group. * indicates significant differences between the PBS and OVA treatment groups; § indicates significant differences between OVA-treated WT and GFP-C5aR1^flox/flox mice. * or § p < 0.05, ** p < 0.01, *** p <0.001.</p