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

Presentation_1_Distribution and Interaction of Murine Pulmonary Phagocytes in the Naive and Allergic Lung.PDF

<p>The division of labor between pulmonary phagocytic subsets [macrophage/monocyte and dendritic cell (DC) subpopulations] has been described at the functional level. However, whether these lung phagocytes also display unique spatial distribution remains unclear. Here, to analyze cellular distribution in lung compartments and contacts between phagocyte subpopulations, we established an immunohistochemistry (IHC)-based method to clearly identify murine lung phagocyte subsets in situ based on differential expression of CD11c, CD11b, MHC-II, Langerin and mPDCA-1. Furthermore, we investigated subset-specific functional differences in antigen uptake and spatial changes upon allergic sensitization. Our staining allowed the distinction between alveolar macrophages (AMs), interstitial macrophage (IM) subpopulations, CD11b⁺ DC subpopulations, CD103⁺ DCs, and plasmacytoid DCs (pDCs). We identified interstitial regions between airways and around airways as regions of IM/CD11b⁺ DC/CD103⁺ DC clusters, where a subset of IMs (IM2) and CD103⁺ DCs formed intense contacts that decreased upon allergic sensitization. These data indicate functional interactions between both cell types either in steady state or after antigen encounter affecting the development of allergies or tolerance. Furthermore, we observed major antigen uptake in AMs and IMs rather than DC subpopulations that was not restricted to airways and adjacent areas. This will enable to focus future studies to immunologically relevant cellular interactions and to unravel which cells are tipping the balance between pro-inflammatory immune responses or tolerance.</p

<i>GFP-C5aR1</i> expression in innate and adaptive immune cells recruited into the lungs in response to OVA challenge.

<p><b>(A)</b> Histograms showing the expression levels of GFP, used as surrogate marker for C5aR1 expression, in lung eosinophils, BAL-derived alveolar and lung macrophages in PBS-treated or OVA-challenged WT and GFP-C5aR1^flox/flox animals. (<b>B</b>) The corresponding graphs show the relative mean fluorescence intensity (MFI) of the GFP signal in the indicated cell types. Values shown are the mean ± SEM; n = 8–18 per group. <b>(C)</b> Histograms showing the expression levels of GFP in CD11b⁺ cDCs and moDCs in PBS-treated or OVA-challenged WT or GFP-C5aR1^flox/flox animals. (<b>D</b>) The corresponding graphs show the mean fluorescence intensity (MFI) of the GFP signal in the indicated cell types. Values shown are the mean ± SEM; n = 13–16 per group. (<b>E</b>) Histograms showing the expression levels of GFP in lung CD4⁺ T cells, CD44⁺CD62L^- effector T cells, CD19⁺B220⁺ B cells and ILC2. Histograms are representative of 8–11 animals per group. Grey histogram: GFP signal of WT cells; solid line: GFP signal in cells from PBS-treated GFP-C5aR1^flox/flox mice; dashed line: GFP signal in cells from OVA-immunized GFP-C5aR1^flox/flox mice.</p

Comparison of C5aR1 expression in different myeloid cells in response to PBS.

<p>Comparison of C5aR1 expression in different myeloid cells in response to PBS.</p

Similar accumulation of DCs and CD4⁺ T cells in mLNs of GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice following OVA-immunization.

<p><b>(A)</b> Numbers of different DC subsets in mLNs of PBS-treated or OVA-immunized GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Values shown are the mean ± SEM; n = 7–9 per group. <b>(B)</b> Numbers CD4⁺, and CD44⁺CD62L^- or CD44^-CD62L⁺ T cells in mLNs of PBS-treated or OVA-immunized GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Values shown are the mean ± SEM; n = 7–9 per group. Asterisks indicate significant differences between the PBS and OVA treatment groups; * p<0.05.</p

Deletion of C5aR1 in LysM-expressing cells controls recruitment of airway neutrophils but is dispensable for the development of strong AHR, airway inflammation and mucus production.

<p><b>(A)</b> AHR in response to increased doses of nebulized methacholine in the airways, expressed as airway resistance. Shown are dose response curves in PBS-treated controls or OVA-immunized mice from the GFP-C5aR1^fl/fl and LysM-C5aR1 KO mice. Values shown are the mean ± SEM; n = 7–9 per group. <b>(B)</b> Total and differential cell counts in BAL fluid of GFP-C5aR1^fl/fl and LysM-C5aR1 KO mice in response to PBS treatment or OVA-immunization. Values shown are the mean ± SEM; n = 7–9 per group. <b>(C)</b> Histological examination of mucus production in airways of PBS-treated or OVA-immunized GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Sections were stained with PAS for mucus production (original magnification x 200). Pictures shown are representative of n = 4–6 lungs per group. Scale bar represents 200μm. (<b>D)</b> Frequency of PAS-positive bronchi in PBS-treated or OVA immunized mice. Values shown are the mean ± SEM; n = 4–6 per group. Asterisks indicate significant differences between the PBS and OVA treatment groups, The § symbol indicates significant differences between OVA-treated GFP-C5aR1^fl/fl and LysM-C5aR1 KOmice. * or § p<0.05, ** p<0.01, and *** p <0.001.</p