Distribution and Interaction of Murine Pulmonary Phagocytes in the Naive and Allergic Lung

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

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

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

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

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

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