Crosstalk between muscularis macrophages and enteric neurons regulates gastrointestinal motility

Abstract

Intestinal peristalsis is a dynamic physiologic process influenced by dietary and microbial changes. It is tightly regulated by complex cellular interactions; however, our understanding of these controls is incomplete. A distinct population of macrophages is distributed in the intestinal muscularis externa. We demonstrate that, in the steady state, muscularis macrophages regulate peristaltic activity of the colon. They change the pattern of smooth muscle contractions by secreting bone morphogenetic protein 2 (BMP2), which activates BMP receptor (BMPR) expressed by enteric neurons. Enteric neurons, in turn, secrete colony stimulatory factor 1 (CSF1), a growth factor required for macrophage development. Finally, stimuli from microbial commensals regulate BMP2 expression by macrophages and CSF1 expression by enteric neurons. Our findings identify a plastic, microbiota-driven crosstalk between muscularis macrophages and enteric neurons that controls gastrointestinal motility. PAPERFLICK:

Figure 1. MHCII⁺CX₃CR1⁺ MMs require CSF1R signaling for their development

(A–B) Distribution of CX₃CR1⁺ MMs shown in cross-section (A) or muscularis sheet (B) of large bowel (LB) from Cx₃cr1-GFP^+/– mice. Scale bar – 100 nm. (C) Phenotype of MMs in small bowel (SB) muscularis from Cx₃cr1-GFP^+/– mice by FACS analysis (gated on DAPI^– cells). (D) Flt3 and Csf1r gene expression in sorted MMs measured by whole mouse genome microarray and presented as relative units (RU). (E) FACS plots of whole bowel suspensions from WT or Cscf1r^–/– mice show % of CD11c^loMHCII^hi MMs (oval gate, solid line) and CD11c^hiMHCII^hi LP phagocytes (oval gate, dashed line). Gated on CD45⁺CD11c^lo/hiCD11b^lo/hi cells as demonstrated in Figure S1. (F) Absolute numbers of MMs in total gut of WT and Csf1r^–/– mice quantified by FACS. (G) Relative reduction of MMs and CD103⁺CD11b⁺ and CD103^–CD11b⁺ LP phagocytes in Csf1r^–/– mice as compared to WT littermates (quantified by FACS). (H) % of WT CD45.1⁺ and Csf1r^–/– CD45.2⁺ cells among CD45⁺ LP CD103⁺CD11b⁺ and LP CD103^–CD11b⁺ phagocytes and MMs (Muscularis) in SB from 10% WT + 90% Csf1r^–/– mixed BM chimeras.

Figure 2. Model of MM depletion

(A–B) FACS plots of separated SB muscularis (A) and whole SB (B) single cell suspensions from WT mice 2 days after i.p. injection of isotype IgG or αCSF1R mAb show % of CD11c^loMHCII^hi MMs (oval gate, solid line) and CD11c^hiMHCII^hi LP phagocytes (oval gate, dashed line). A – gated on total viable cells. B – gated on CD45⁺CD11c^lo/hiCD11b^lo/hi cells using the gating strategy as in Figure S1. (C) Absolute numbers of LP CD103⁺CD11b⁺ and LP CD103^–CD11b⁺ phagocyte and MMs (Muscularis) in SB of WT mice 2 days after i.p. injection with isotype IgG or αCSF1R mAb quantified by FACS. (D) Relative reduction of LP phagocyte and MM numbers quantified by FACS in WT mice treated with αCSF1R mAb (Day 2) as compared to isotype IgG treated mice. (E–F) Distribution of MHCII⁺ macrophages (E) and CD3⁺ T cells (F) in LB muscularis from WT mice 2 days after i.p. injection of isotype IgG or αCSF1R mAb analyzed by immunofluorescence (IF). Scale bars – 100 nm.

Figure 3. Depletion of MMs results in intestinal dysmotility

(A) Illustration of ex vivo method to measure stretch-induced peristaltic contractions of colonic rings using a myograph. (B) Five hour recording of stretch-induced contractions of colonic rings during repeated application of 0.25 mm long stretching up to 5.00 mm total stretch length. 3 mm colonic rings were obtained from WT mice 2 days after i.p. injection of isotype IgG or αCSF1R mAb. (C) Number of peaks (left) or Root Mean Square (RMS) normalized to baseline in % (right) during 10 min recordings of colonic contraction at stretch distance 2.75 mm. (D) RMS normalized to baseline (%) during 10 min recordings of colonic contractions at each stretching step from 1.5 to 3.5 mm. (E) Colonic transit time measured by bead expulsion assay in WT mice 1, 3 and 5 days after i.p. injection of isotype IgG or αCSF1R mAb.

Figure 4. MMs regulate intestinal peristalsis by secreting BMP2

(A–B) Bmp2 gene expression levels in MMs (Muscularis) compared to LP CD103^–CD11b⁺ phagocytes (A) and LP CD103⁺CD11b⁺ and LP CD103^–CD11b⁺ phagocytes (B) measured by whole mouse genome microarray. (C) Bmp2-7 gene expression levels in MMs measured by whole mouse genome microarray. (D) Bmp2 relative gene expression levels measured by qPCR in intact SB muscularis (whole tissue) or in SSC^loCD45⁺CD11b^– lymphocytes and macrophages sorted from separated SB muscularis. FI – fold increase as compared to “whole tissue”. (E) IF analysis of LB muscularis from WT mice stained with anti-BMP2 and anti-MHCII mAbs and counterstained with DAPI. Scale bar – 100 nm. (F) RMS of colonic contraction recordings normalized to baseline (%) at stretch intervals 1.5–3.5 mm. 3 mm colonic rings were obtained from WT mice treated with BMP receptor inhibitor dorsomorphin or control vehicle. (G) WT mice were injected i.p. with isotype IgG (top) or αCSF1R mAb (bottom) and analyzed 2 days later. Panels show ex vivo recordings of stretch-induced contractions of colonic rings from these mice before and after adding 1, 5 and 10 ng of human recombinant BMP2 or control vehicle (performed at 2.75 mm “optimal” stretch distance). (H) RMS of 10 min recordings described in F (bottom panel, αCSF1R mAb treated mice) before and after adding 5 ng of BMP2. (I) RMS normalized to baseline (%) of recordings described in F (bottom panel, αCSF1R mAb treated mice) after adding BMP2 (1, 5, 10 ng) or control vehicle. Baseline here is the recording at the same stretch distance (2.75 mm) prior to adding BMP2 or control vehicle. (J) Colonic transit time measured by bead expulsion assay in WT mice prior receiving αCSF1R mAb (day −1) and on days 3 and 4 after receiving αCSF1R mAb; 18 and 3 hours prior the last assessment (day 4) mice received 1 µg of BMP2 i.p.

Figure 5. MMs activate BMP2 receptor on enteric neurons

(A) Distribution of CX₃CR1⁺ MMs in colon (left) and ileum (middle and right) muscularis from Cx₃cr1-GFP^+/– mice stained with anti- βIII Tubulin Ab and analyzed by IF. Scale bars – 500 nm (left), 100 nm (middle) and 10 nm (right). (B) IF analysis of LB muscularis from WT mice stained with anti-BMPRII and anti-βIII Tubulin Abs and counterstained with DAPI. Scale bars – 500 nm. (C) IF analysis of LB muscularis from WT mice 2 days after i.p. injection of isotype IgG (top), αCSF1R mAb (middle and bottom) stained with anti-pSMAD1/5/8 and anti-BMPRII Abs and counterstained with DAPI. The bottom panel shows pSMAD1/5/8 distribution in the muscularis from αCSF1R mAb injected mouse that was incubated with BMP2 as described in D. Scale bars – 100 nm. (D) Quantitative summary of the distribution of pSMAD1/5/8⁺BMPRII⁺ neurons in the muscularis from WT mice 2 days after i.p. injection of isotype IgG or αCSF1R mAb. In all cases, the muscularis was incubated for 30 min at 37°C in complete medium in the presence or absence of BMP2 (10 ng/ml) as indicated. Each data point represents % of pSMAD1/5/8⁺ neurons among total BMPRII⁺ neurons in each visual field; each column summarizes the results from three animals.

Figure 6. Enteric neurons produce CSF1 required for MM development

(A) Csf1 gene expression levels measured by qPCR in intact muscularis (whole tissue), macrophages sorted from SB muscularis and cultured primary enteric neurons (FI compared to the “whole tissue”). (B) Il-34 and Csf1 relative gene expression levels (FI) measured by qPCR in cultured enteric neurons as compared to Il-34. (C) IF analysis of LB muscularis from WT mice stained with anti-BMPRII and anti-CSF1 Abs. Scale bar – 100 nm. (D) FACS plots of whole bowel single cell suspensions from WT mice and their Csf1^op/op littermates show % of CD11c^loMHCII^hi MMs (oval gate, solid line) and CD11c^hiMHCII^hi LP phagocytes (oval gate, dashed line). Gated on CD45⁺CD11c^lo/hiCD11b^lo/hi cells (E) Absolute MM numbers in the bowel of WT mice and Csf1^op/op mice quantified by FACS. (F) IF analysis of LB (cecum) muscularis from WT mice and their Csf1^op/op littermates stained with anti- βIII Tubulin and anti-MHCII Abs. Scale bars – 500 nm. (G) Quantitative summary of the distribution of pSMAD1/5/8⁺BMPRII⁺ neurons in the LB muscularis from WT and Csf1^op/op mice. Each data point represents % of pSMAD1/5/8⁺ neurons among total BMPRII⁺ neurons in each visual field; each column summarizes the results from three animals. (H) IF analysis of LB (colon) muscularis from WT and Csf1^op/op littermates stained with anti-BMPRII Ab. Scale bars – 500 nm. (I) Quantitative summary of the distribution of BMPRII⁺ neurons in the colon of WT and Csf1^op/op mice. Each data point represents the counts of BMPRII⁺ neurons in each visual field; each column summarizes the results from two animals.

Figure 7. Luminal microbiota regulates intestinal motility and MM-neuronal crosstalk

(A–D) WT mice received antibiotics with drinking water for 4 weeks and control age matched mice received only water. (A) Total GI transit time that represents the time required to expel feces containing carmine red dye measured in antibiotic-treated and control mice. (B) RMS of colonic contraction recordings normalized to baseline (%) at stretch intervals 1.5–3.5 mm. 3 mm colonic rings were obtained from antibiotic-treated and control mice. (C) Bmp2 relative gene expression levels measured by qPCR in macrophages sorted from separated SB muscularis of antibiotic-treated and control mice. FI – fold increase as compared to Bmp2 levels in the intact muscularis. Each data point represents qPCR results obtained from analyzing 50,000 cells after a single sort from 5 mice. Cell sorting from each group was performed in pairs, on the same day and under identical conditions. (D) Quantitative summary of the distribution of pSMAD1/5/8⁺BMPRII⁺ neurons in the muscularis from antibiotic-treated and control mice. Each data point represents % of pSMAD1/5/8⁺ neurons among total BMPRII⁺ neurons in each visual field; each column summarizes the results from three animals. (E) Csf1 relative gene expression levels in cultured primary enteric neurons measured by qPCR. Differentiated neurons were cultured with or without 10 ng/ml of LPS for 18 hrs prior to analyses. FI – fold increase as compared to Csf1 levels in the intact muscularis. Each data point represents qPCR results obtained from an independent neuronal culture. FI – fold increase as compared to Csf1 levels in the intact muscularis. (F) Csf1 relative gene expression levels quantified by qPCR in LB muscularis from WT mice were treated with antibiotics for 4 weeks (left), from WT mice treated with antibiotics and 50 µg/ml LPS in drinking water for 4 weeks (middle) and from WT mice 3 weeks after they received fecal transfer (FT) following a 4-week course of antibiotics (right). The data are compared to age matched control mice received only water. FI – fold increase as compared to average Csf1 levels in the control group received water. (G) Absolute numbers of MMs quantified by FACS in LB from WT mice received antibiotics (left), antibiotics with LPS (middle) and FT following antibiotic treatment (right) as in F. The data are compared to age matched control mice received only water. (H) Total GI transit time in WT mice received antibiotics and 50 µg/ml LPS with drinking water for 4 weeks and age matched mice that received only antibiotics for 4 weeks. (I) Total GI transit time in WT mice 3 weeks after they received FT following a 4-week course of antibiotics and control mice received water.