Complement modulates the cutaneous microbiome and inflammatory milieu

Abstract

The skin is colonized by a plethora of microbes that include commensals and potential pathogens, but it is currently unknown how cutaneous host immune mechanisms influence the composition, diversity, and quantity of the skin microbiota. Here we reveal an interactive role for complement in cutaneous host-microbiome interactions. Inhibiting signaling of the complement component C5a receptor (C5aR) altered the composition and diversity of the skin microbiota as revealed by deep sequencing of the bacterial 16S rRNA gene. In parallel, we demonstrate that C5aR inhibition results in down-regulation of genes encoding cutaneous antimicrobial peptides, pattern recognition receptors, and proinflammatory mediators. Immunohistochemistry of inflammatory cell infiltrates in the skin showed reduced numbers of macrophages and lymphocytes with C5aR inhibition. Further, comparing cutaneous gene expression in germ-free mice vs. conventionally raised mice suggests that the commensal microbiota regulates expression of complement genes in the skin. These findings demonstrate a component of host immunity that impacts colonization of the skin by the commensal microbiota and vice versa, a critical step toward understanding host-microbe immune mutualism of the skin and its implications for health and disease. Additionally, we reveal a role for complement in homeostatic host-microbiome interactions of the skin.

Fig. 1.

Antagonism of complement C5aR results in an overall change in the skin microbiota. Skin microbiota samples were collected from C57BL/6J mice before treatment to determine BL microbiota (purple dots), and then treated with C5aRA (red dots) or iC5aRA (green dots). Following a 2-wk treatment course, skin microbiota were collected again to compare with BL microbiota. Depicted are PCoA plots of the weighted UniFrac metric comparing skin microbiota of (A) C5aRA-treated mice and their BL, and (B) iC5aRA-treated mice and their BL. Percentage of variation explained by the principal coordinates is indicated in parentheses on the axes. Depicted is one of two independent experiments (n = 9 mice per treatment group). Table S1 shows R² and P values of the Adonis test, which assigns strength and statistical significance to the sample groupings visually observed. Fig. S1 illustrates experiment-dependent microbiome effects.

Fig. 2.

Impact of C5aR signaling on the skin microbiota taxonomical composition. Mean relative abundance (y axis) of the six most abundant phyla (x axis) is depicted. Paired t tests were calculated for each mouse comparing the phylum-level taxa of its BL skin microbiota and its posttreatment (C5aRA or iC5aRA) skin microbiota. Only those phyla that significantly changed following C5aRA treatment (P < 0.05), but not iC5aRA treatment, were selected as significant. Error bars represent SEM (n = 14 mice for each group over two independent experiments; *P = 0.008 and **P = 0.007).

Fig. 3.

Diversity and richness of skin microbiota decreases when C5aR signaling is inhibited. (A) The mean Shannon diversity index, a measure of α-diversity that takes into account OTU richness and evenness, was used to compare skin microbiota before treatment (BL) to C5aRA- and iC5aRA-treated skin microbiota. Higher Shannon diversity index indicates higher diversity. (B) Mean number of species-level OTUs observed before treatment (BL) and following treatment with C5aRA or iC5aRA. Error bars represent SEM (n = 14 mice for each group over two independent experiments; *P = 0.037 and **P = 0.005).

Fig. 4.

Inhibition of complement C5aR decreases cutaneous expression of innate immune and inflammatory mediators and skin inflammatory cell infiltration. (A) After 2-wk treatment with C5aRA or iC5aRA, skin was collected and RNA extracted for gene expression analysis. Skin mRNA levels were determined by quantitative real-time PCR (normalized to B2m) and expressed as fold change in C5aRA-treated transcript levels relative to iC5aRA-treated transcript levels, which were assigned an average value of 1. Data are means ± SD (n = 9 C5aRA-treated mice and n = 8 iC5aRA-treated mice from two independent experiments; *P < 0.05 and **P < 0.01). Table S2 shows all genes assayed and primer/probe sets. (B and C) Skin sections (6 µM thick) of mice treated with C5aRA and iC5aRA for 2 wk were stained with antibodies specific for (B) CD3 and (C) F4/80 to identify infiltrating lymphocytes and macrophages, respectively. Depicted are representative images at magnifications of 300× (Left) and the same image at a magnification of 600× (Right). Fig. S2 shows control and H&E staining. (D) For all staining, three to five fields per section were analyzed at magnification of 400× and positive cells were counted. Data are expressed as average number of immunoreactive cells in the field and are representative of one experiment consisting of three mice for each treatment and two skin biopsies per mouse. Error bars represent SEM (*P < 0.05). (Scale bars: Left, 100 µm; Right, 200 µm.)

Fig. 5.

Mice colonized with commensal microbiota have higher cutaneous expression of complement genes compared with GF mice. Cutaneous gene expression of GF mice was compared with CONV mice. Depicted on the x axis are all genes categorized under GO terms “complement activation” and “complement binding” that were expressed in skin above the threshold FPKM >1 in at least two of the eight skin samples subjected to RNA sequencing (SI Materials and Methods). Data are expressed as mean expression level (y axis), as measured by FPKM normalized transcripts, of CONV mouse skin relative to GF mouse skin. Error bars represent propagated SE of the ratio CONV/GF (n = 4 GF mice and CONV mice each; *P < 0.05).