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. 2023 May 19:14:1197709.
doi: 10.3389/fimmu.2023.1197709. eCollection 2023.

The complement receptor C5aR2 regulates neutrophil activation and function contributing to neutrophil-driven epidermolysis bullosa acquisita

Daniel L Seiler  1 , Katja H Kähler  1 , Marie Kleingarn  1 , Christian D Sadik  2   3 , Katja Bieber  2   3   4 , Jörg Köhl  1   5 , Ralf J Ludwig  2   3   4 , Christian M Karsten  1
Affiliations

The complement receptor C5aR2 regulates neutrophil activation and function contributing to neutrophil-driven epidermolysis bullosa acquisita

Daniel L Seiler et al. Front Immunol. .

Abstract

Introduction: The function of the second receptor for the complement cleavage product C5a, C5aR2, is poorly understood and often neglected in the immunological context. Using mice with a global deficiency of C5aR2, we have previously reported an important role of this receptor in the pathogenesis of the neutrophil-driven autoimmune disease epidermolysis bullosa acquisita (EBA). Based on in vitro analyses, we hypothesized that the absence of C5aR2 specifically on neutrophils is the cause of the observed differences. Here, we report the generation of a new mouse line with a LysM-specific deficiency of C5aR2.

Methods: LysM-specific deletion of C5aR2 was achieved by crossing LysMcre mice with tdTomato-C5ar2fl/fl mice in which the tdTomato-C5ar2 gene is flanked by loxP sites. Passive EBA was induced by subcutaneous injection of rabbit anti-mouse collagen type VII IgG. The effects of targeted deletion of C5ar2 on C5a-induced effector functions of neutrophils were examined in in vitro assays.

Results: We confirm the successful deletion of C5aR2 at both the genetic and protein levels in neutrophils. The mice appeared healthy and the expression of C5aR1 in bone marrow and blood neutrophils was not negatively affected by LysM-specific deletion of C5aR2. Using the antibody transfer mouse model of EBA, we found that the absence of C5aR2 in LysM-positive cells resulted in an overall amelioration of disease progression, similar to what we had previously found in mice with global deficiency of C5aR2. Neutrophils lacking C5aR2 showed decreased activation after C5a stimulation and increased expression of the inhibitory Fcγ receptor FcγRIIb.

Discussion: Overall, with the data presented here, we confirm and extend our previous findings and show that C5aR2 in neutrophils regulates their activation and function in response to C5a by potentially affecting the expression of Fcγ receptors and CD11b. Thus, C5aR2 regulates the finely tuned interaction network between immune complexes, Fcγ receptors, CD11b, and C5aR1 that is important for neutrophil recruitment and sustained activation. This underscores the importance of C5aR2 in the pathogenesis of neutrophil-mediated autoimmune diseases.

Keywords: C5a; C5aR2; CD11b; EBA; FcgRIIB; Fcγ receptor (FcγR); Mac-1; neutrophil.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Characterization of the cell-specific C5aR2 knock-out mouse line. (A) PCR-based detection of the floxed tdTomato-C5ar2 gene cassette in BM neutrophils (N), BM Ly6G-/CD11b+ cells (C), splenic B (B) and T cells (T) from LysM cre -C5ar2 –/– mice. Positive control from C5ar2-tdTomato fl/fl mice (PC) and No Template Control (NTC) are shown. M: GeneRuler 50 bp DNA Ladder. (B) Histogram overlay of tdTomato reporter signal indicating C5aR2 expression in BM and blood neutrophils (Ly6G+/CD11b+) and Ly6G-/CD11b+ cells from LysM cre -C5ar2 –/– (purple) and C5ar2-tdTomato fl/fl (red) mice. The tdTomato signal of corresponding cells from WT (grey) mice served as a negative control. (C) Histogram overlay indicating C5aR1 expression in BM and blood neutrophils from LysM cre -C5ar2 –/– (purple), C5ar2-tdTomato fl/fl (red), and WT (grey) mice. The signal corresponding to the FMO control is shown in black. (D) C5aR1 expression in BM and blood neutrophils (Ly6G+ cells) from LysM cre -C5ar2 –/–, C5ar2-tdTomato fl/fl, and WT mice quantified by the gMFI. (E) Frequency (of parent) of neutrophil subsets in the BM and blood of naïve LysM cre -C5ar2 –/– and C5ar2-tdTomato fl/fl control mice (n = 6/group). * p < 0.05; ns, not significant.
Figure 2
Figure 2
C5aR2 deficiency in neutrophils ameliorates pathogenesis in an experimental model of EBA. (A) Schematic representation of the antibody transfer mouse model of EBA. Subepidermal blistering is induced by subcutaneous injection of affinity-purified rabbit anti-mCOL7 IgG on days (d) 0, 2, and 4. Cutaneous lesions are scored every other day. Mice are sacrificed on d-12 (figure created with BioRender.com). (B) Development of the clinical disease phenotype as indicated by the percentage of body surface area affected by lesions, erosions, and blisters in LysMcre-C5ar2–/– and C5ar2-tdTomatofl/fl control mice (n = 15 per group). Representative pictures of clinical lesions found in (C) C5ar2-tdTomatofl/fl and (E) LysMcre-C5ar2–/– mice. Example photographs of histopathologically examined skin sections from (D) C5ar2-tdTomatofl/fl and (F) LysMcre-C5ar2–/– mice. **** p < 0.0001.
Figure 3
Figure 3
C5a-mediated in vitro activation of neutrophils from LysMcre-C5ar2–/– mice. (A) In vitro chemotaxis of BM cells of LysMcre-C5ar2–/– , C5ar2-tdTomatofl/fl and WT control mice towards C5a (n ≥ 5/group). (B) Neutrophil frequency in the blood of diseased (EBA) LysMcre-C5ar2–/– and C5ar2-tdTomatofl/fl control mice (n = 15/group). (C) C5a-induced increase in CD11b surface expression of LysMcre-C5ar2–/– , C5ar2-tdTomatofl/fl and WT control mice (n ≥ 6/group). * p < 0.05; ns, not significant.
Figure 4
Figure 4
Fcγ receptor expression on BM and blood neutrophils. Histogram overlays of surface expression of FcγRs (FcγRI, FcγRIIb, FcγRIII, FcγRIV) on neutrophils from (A) BM and (B) blood of WT, C5ar2-tdTomatofl/fl , and LysMcre-C5ar2–/– mice. The signal corresponding to the FMO control is shown in black. Relative gMFI of FcγRs corresponding to surface expression on (C) BM and (D) blood neutrophils of WT, C5ar2-tdTomatofl/fl , and LysMcre-C5ar2–/– mice. * p < 0.05, ** p < 0.01; ns, not significant.
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This work was funded by Deutsche Forschungsgemeinschaft through CRC 1526 Pathomechanisms of Antibody-mediated Autoimmunity (PANTAU), project 413535489 (A04).
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