Fig 2: Numbers of enteric neurons are similar in C1qafl/fl and C1qa?Mf mice.(A) Immunofluorescence analysis of enteric neuronal ganglia marked with HuC/D (red) and neuronal fibers marked with TUBB3 (green) in LMMP wholemounts of small intestines and colons from C1qafl/fl and C1qa?Mf mice. Anti-mouse IgG AlexaFluor 594 and anti-rabbit IgG AlexaFluor 488 were used as secondary stains for HuC/D and TUBB3, respectively. Images are representative of three independent experiments. Scale bars = 50 µm. (B) Quantification of total enteric neurons per unit area (mm2) from the images shown in panel (A). Data are pooled from two independent experiments. Each data point represents one mouse. (C) Visualization of specific neuronal subsets in the LMMP from C1qafl/fl and C1qa?Mf mice by RNAscope detection. Inhibitory neurons were marked by Nos1 (green) and excitatory neurons were marked by Chat (red). Neuronal nuclei marked by HuC/D (blue) were detected by immunofluorescence. Images are representative of two independent experiments. Scale bars = 50 µm. (D) Immunofluorescence detection of enteric glial cells marked by S100B (green) in LMMP wholemounts from the small intestines and colons of C1qafl/fl and C1qa?Mf mice. Images are representative of two independent experiments. Scale bars = 50 µm. (E) qPCR analysis of Nos1, Chat, and S100b in the LMMP of small intestines and colons from C1qafl/fl and C1qa?Mf mice. Each data point represents one mouse. Error bars represent SEM. ns, not significant by the two-tailed Student’s t-test. LMMP, longitudinal muscle-myenteric plexus.

Fig 3: Macrophages are the primary source of complement component 1q (C1q) in the mouse gastrointestinal tract.(A) Macrophages were selectively depleted in C57BL/6 mice by intraperitoneal injection of anti-CSF1R antibody. Control mice were injected with isotype-matched non-specific antibodies. Mice were analyzed 72 hr after antibody injection. Panel was generated at Biorender.com. (B) Representative flow cytometry analysis of mouse small intestines after intraperitoneal injection of anti-CSF1R or isotype control antibody. All cells were gated as live CD45+. Macrophages were MHCII+ F4/80hi; B cells were CD19+; T cells were CD3+. Total small intestinal cell yields were 1.5 × 106 ± 4.9 × 105 cells. (C) Quantitative PCR (qPCR) measurement of C1qa, C1qb, and C1qc transcript abundance in mouse small intestines after intraperitoneal injection of anti-CSF1R or rat IgG2a (isotype control). Each data point represents one mouse and results are pooled from two independent experiments. (D) C1qafl/fl mice were crossed with LysM-Cre transgenic mice to generate mice having a macrophage-selective deletion of C1qa (C1qa?Mf mice). Panel was generated at Biorender.com. (E) Representative flow cytometry analysis of intracellular C1q expression in small intestinal macrophages from C1qafl/fl and C1qa?Mf mice. Mice were littermates from heterozygous crosses that remained co-housed. Cells were gated on live CD45+CD11b+MHCII+. (F) Quantitation of the flow cytometry analysis in (E). Each data point represents one mouse. Results are representative of two independent experiments. (G) qPCR measurement of C1qa transcript abundance in the small intestines (sm. int.) and colons of C1qafl/fl and C1qa?Mf littermates. Each data point represents one mouse. Error bars represent SEM. **p<0.01; ***p<0.001; ****p<0.0001; ns, not significant by the two-tailed Student’s t-test.

Fig 4: C1qa?Mf mice do not show altered microbiota composition, barrier function, or resistance to enteric infection.(A) Small intestinal C1qa expression is not induced by the intestinal microbiota. Quantitative PCR (qPCR) measurement of Reg3g and C1qa transcript abundances in the small intestines of germ-free (GF) and conventional (CV) C57BL/6 mice. Each data point represents one mouse and the results are representative of two independent experiments. (B) C1q is not detected in the mouse intestinal lumen or feces. Representative immunoblot of an ammonium sulfate precipitation of intestinal luminal contents and feces from germ-free and conventional mice with detection of C1q. C1q in small intestinal tissue is shown for comparison at right. REG3G was analyzed as a control, as it is secreted into the intestinal lumen of conventional mice (Cash et al., 2006). Each lane represents multiple mice pooled (n=5 and 9 for germ-free and conventional, respectively) and the immunoblot is representative of three independent experiments. (C) C1q gene expression is not altered by acute enteric infection with Salmonella typhimurium. qPCR measurement of C1qa transcript abundance in small intestinal tissue after oral inoculation of mice with 109 colony-forming units of S. typhimurium strain SL1344. Each data point represents one mouse, and the results are representative of two independent experiments. (D) Intestinal microbiota composition is not altered in C1qa?Mf mice. Phylogenetic analysis of 16S rRNA gene sequences from fecal pellets collected from C1qafl/fl and C1qa?Mf littermates. Operational taxonomic units with an average of 100 reads and populations greater than or equal to 1% were included in the graphical analysis. Each bar represents one mouse. Data are available from the Sequence Read Archive under BioProject ID PRJNA793870. (E) C1qa?Mf mice do not show altered translocation of bacteria to mesenteric lymph nodes (mLN). 16S rRNA gene copy numbers were measured by qPCR with reference to a standard curve. Each data point represents one mouse. (F) C1qa?Mf mice do not show altered susceptibility to dextran sulfate sodium (DSS)-induced colitis. Mice were provided with 3% DSS in drinking water and body weights were monitored for 7 days. n=4 and 6 for C1qafl/fl and C1qa?Mf littermates, respectively. Differences at each time point were not significant by the two-tailed Student’s t-test. (G) C1qa?Mf mice do not show altered intestinal permeability. To measure intestinal permeability, C1qafl/fl and C1qa?Mf littermates were gavaged with fluorescein isothiocyanate (FITC)-dextran (4 kDa), and serum FITC-dextran levels were determined by fluorescence microplate assay against a FITC-dextran standard curve. Indomethacin induces intestinal damage in mice and was used as a positive control. Each data point represents one mouse. (H) Time course of fecal Citrobacter rodentium burden following oral gavage of C1qafl/fl and C1qa?Mf mice with 5×108 colony forming units (CFU) of C. rodentium. n=5 and 5 for C1qafl/fl and C1qa?Mf littermates, respectively. Differences at each time point were not significant by the two-tailed Student’s t-test. (I) qPCR measurement of transcripts encoding secreted immune effectors in the small intestines of C1qafl/fl and C1qa?Mf littermates. Each data point represents one mouse. (J) Flow cytometry analysis of small intestinal immune cell subsets from C1qafl/fl and C1qa?Mf littermates. Gating strategies are shown in Figure 3—figure supplement 1 through 4. ILC, innate lymphoid cell. Total small intestinal cell yields were 8.8 × 106 ± 2.9 × 106 cells. Each data point represents one mouse. Sm. int., small intestine. Error bars represent SEM. **p<0.01; ns, not significant by the two-tailed Student’s t-test. Figure 3—source data 1.Unedited, uncropped immunoblot for Figure 3B.

Fig 5: Complement component 1q (C1q) is expressed by muscularis macrophages that are located near enteric neurons.(A) Graphic depicting the muscularis of the mouse small intestine. The lumen, epithelium (epi), lamina propria (LP), submucosal plexus (SP), and longitudinal muscle-myenteric plexus (LMMP) are indicated. Created at Biorender.com. (B) Immunofluorescence detection of C1q (violet) and macrophages marked with CD169 (green) in the small intestine and colon of C1qafl/fl and C1qa?Mf littermates. Nuclei were detected with 4’,6-diamidino-2-phenylindole (DAPI; blue). Detection with isotype control antibodies on C1qafl/fl small intestines is shown at right. Anti-rat IgG AlexaFluor 488 and streptavidin-Cy5 were used as secondary stains for CD169 and C1q, respectively. The intestinal surface is denoted with a red dotted line and the gut lumen, epithelium, and lamina propria are indicated. The approximate region encompassing the submucosal plexus and the muscularis is denoted with two white dotted lines. Examples of C1q+ areas are indicated with yellow arrows and examples of CD169+ macrophages are indicated with white arrowheads. Note that the violet staining near the bottom of the muscularis is non-specific, as indicated by its presence in the isotype control image. Images are representative of three independent experiments. Scale bars = 50 µm. (C) Immunofluorescence detection of C1q (violet), macrophages marked with CD169 (green), and neurons marked with TUBB3 (yellow) in the small intestines of wild-type C57BL/6 mice. Nuclei are detected with DAPI (blue). The epithelium and lamina propria are indicated. The approximate region encompassing the submucosal plexus and the muscularis is denoted with two white dotted lines. The expanded image area delineated by a yellow square shows an example of the close association between C1q and TUBB3+ neurons. Images are representative of images captured from three mice. Anti-rat IgG AlexaFluor 488, anti-rabbit IgG AlexaFluor 594, and streptavidin-Cy5 were used as secondary stains for CD169, TUBB3, and C1q, respectively, and an isotype control image is shown at upper right. Scale bars = 50 µm. (D) RNAscope detection of C1qa (green), muscularis macrophages marked by Csf1r (red), and immunofluorescence detection of enteric neuronal ganglia by HuC/D (blue) in LMMP wholemounts of small intestines and colons from C1qafl/fl and C1qa?Mf mice. The expanded area denoted by a yellow square shows a close association between C1qa-expresssing muscularis macrophages and enteric neurons. Images are representative of three independent experiments. Scale bars = 50 µm. (E) C1q+ intestinal macrophages express genes that are characteristic of nerve-adjacent macrophages. Flow cytometry analysis of CD169, Arginase 1, and TREM2 on C1q- and C1q+ macrophages recovered from the small intestines of wild-type C57BL/6 mice. Median fluorescence intensities from multiple mice are quantified in the panels at the right. Each data point represents one mouse (n=5–6 mice), and the results are representative of two independent experiments. Error bars represent SEM. **p<0.01; ****p<0.0001 by the two-tailed Student’s t-test. Epi, epithelium; LP, lamina propria; SP, submucosal plexus; Mf, macrophage; DAPI, 4',6-diamidino-2-phenylindole, LMMP, longitudinal muscle-myenteric plexus. Error bars represent SEM. ns, not significant by the two-tailed Student’s t-test.