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

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

Deletion of C5aR1 in LysM-expressing cells does not affect the pulmonary accumulation of innate immune cells in response to OVA.

<p><b>(A)</b> Histological examination of airway inflammation in GFP-C5aR1^fl/fl and LysM-C5aR1 KO mice. Sections from PBS or OVA-immunized mice were stained with H&E (original magnification x 200). Pictures shown are representative of n = 4–6 lungs per group. Scale bar represents 200μm. <b>(B)</b> Differential cell counts of lung eosinophils, neutrophils and macrophages of PBS-treated or OVA-immunized GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Values shown are the mean ± SEM; n = 6–9 per group. <b>(C)</b> Cell counts of DC subsets in the lungs of PBS-treated or OVA-immunized GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Values shown are the mean ± SEM; n = 6–9 per group. Asterisks indicate significant differences between the PBS and OVA treatment groups; * p<0.05, ** p<0.01, and *** p <0.001.</p

C5aR1 expression in pulmonary cells.

<p><b>(A)</b> GFP-C5aR1 expression in BAL alveolar macrophages, lung eosinophils, macrophages, and neutrophils of GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Grey histogram: GFP signal in WT cells; dashed line: GFP signal in cells from LysM-C5aR1 KO mice; solid line: GFP signal in cells from GFP-C5aR1^fl/fl mice. Data are representative of at least 3 independent experiments. <b>(B)</b> Scatter plot showing the number of AMs present at steady state in the airways of GFP-C5aR1^fl/fl (open triangle) or LysM-C5aR1 KO mice (open square). <b>(C)</b> Scatter plot showing the number of tissue-associated AMs and neutrophils in GFP-C5aR1^fl/fl (open triangle) or LysM-C5aR1 KO mice (open square). <b>(D)</b> GFP-C5aR1 expression in lung DCs of GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Grey histogram: GFP signal in WT cells; dashed line: GFP signal in cells from LysM-C5aR1 KO mice; solid line: GFP signal in cells from GFP-C5aR1^fl/fl mice. Data are representative of at least 3 independent experiments. <b>(E)</b> Scatter plot showing the number of DC subsets present at steady state in the lungs of GFP-C5aR1^fl/fl (open triangle) or LysM-C5aR1 KO (open square) mice. For all plots, values shown are the mean ± SEM; n = 7 per group. The asterisk indicates significant differences between PBS-treated GFP-C5aR1^fl/fl and LysM-C5aR1 KO mice. *p<0.05.</p

Similar accumulation of pulmonary CD4⁺ T and ILC2 in GFP-C5aR1^fl/fl and LysM-C5aR1 KO mice upon OVA treatment.

<p><b>(A)</b> Number of CD4⁺ T cells in the lungs of PBS-treated or OVA-immunized GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Values shown are the mean ± SEM; n = 6–8 per group. <b>(B)</b> Expression of <i>FoxP3</i> (Treg), <i>GATA3</i> (Th2), <i>RORγT</i> (Th17), and <i>TBX21</i> (Th1) transcripts in sorted CD4⁺CD44⁺CD62L^- T cells. The abundance of transcripts was evaluated after reverse transcription by real-time PCR. Values shown are the mean abundance of target mRNA as compared to actin; n = 6–8 per group. <b>(C)</b> Expression of the Th2 and Th17 cytokines IL-13 and IL-17A, respectively, in sorted CD4⁺CD44⁺CD62L^- T cells as determined by real-time PCR. Values shown are the mean abundance of target mRNA as compared to actin; n = 6–8 per group. <b>(D)</b> ILC2 cell numbers in the lungs of PBS-treated or OVA-immunized GFP-C5aR1^fl/fl or LysM-C5aR1 KO mice. Values shown are the mean ± SEM; n = 6–8 per group. Asterisks indicate significant differences between PBS and OVA treatment groups; * p<0.05, ** p < 0.01, and *** p <0.001.</p

Monitoring C5AR2 expression using a floxed tdtomato-C5AR2 knock-in mouse

The biological significance of C5a receptor [(C5aR)2/C5L2], a seven-transmembrane receptor binding C5a and C5adesArg, remains ill-defined. Specific ligation of C5aR2 inhibits C5a-induced ERK1/2 activation, strengthening the view that C5aR2 regulates C5aR1- mediated effector functions. Although C5aR2 and C5aR1 are often coexpressed, a detailed picture of C5aR2 expression in murine cells and tissues is still lacking. To close this gap, we generated a floxed tandem dye (td)Tomato-C5aR2 knock-in mouse that we used to track C5aR2 expression in tissue-residing and circulating immune cells. We found the strongest C5aR2 expression in the brain, bone marrow, and airways. All myeloid-derived cells expressed C5aR2, although with different intensities. C5aR2 expression in blood and tissue neutrophils was strong and homogeneous. Specific ligation of C5aR2 in neutrophils from tdTomato-C5aR2 mice blocked C5a-driven ERK1/2 phosphorylation, demonstrating functionality of C5aR2 in the reporter mice. In contrast to neutrophils, we found tissue-specific differences in C5aR2 expression in eosinophils, macrophages, and dendritic cell subsets. Naive and activated T cells stained negative for C5aR2, whereas B cells from different tissues homogeneously expressed C5aR2. Also, NK cell subsets in blood and spleen strongly expressed C5aR2. Activation of C5aR2 in NK cells suppressed IL-12/IL-18-induced IFN-g production. Intratracheal IL-33 challenge resulted in decreased C5aR2 expression in pulmonary eosinophils and monocytederived dendritic cells. In summary, we provide a detailed map of murine C5aR2 immune cell expression in different tissues under steady-state conditions and upon pulmonary inflammation. The C5aR2 knock-in mouse will help to reliably track and conditionally delete C5aR2 expression in experimental models of inflammation