Dots represent common number per individual mouse; left: pooled data from seven impartial experiments; right: pooled data from another two impartial experiments. (H) Quantity of Ms in the proximal and distal colon, analyzed by imaging (F4/80+MHCII+CD103? cells per crypt; each dot represents common number per individual mouse; data pooled from three impartial experiments) and by circulation cytometry (offered as percentage of CD45+ cells; dots symbolize individual mouse; data pooled from four impartial experiments). (I) Quantity of extensions, normalized per M. mmc2.xlsx (26K) GUID:?BFCF7E6C-401D-4C99-A454-580C29D6BBC5 Table S3. Antibodies and Reagents, Related to STAR Methods mmc3.xlsx (13K) GUID:?F300FB90-450A-4C36-B379-12C05C9BB811 Video Abstract mmc6.mp4 (3.6M) GUID:?DF1A5509-C2BA-4CB3-B4CB-D2F06BD71D62 Data Availability StatementThe scRNaseq datasets generated in this study 18α-Glycyrrhetinic acid are available for download at the Gene Expression Omnibus (GEO): “type”:”entrez-geo”,”attrs”:”text”:”GSE146131″,”term_id”:”146131″GSE146131. Spectral un-mixing Fiji macro code is usually available on Mendeley Data: https://doi.org/10.17632/bdfyprfsv9.1 Initial/source data for figures in the paper is available on Mendeley Data: https://doi.org/10.17632/zwhhrht7bd.1. Rabbit Polyclonal to PEX3 Summary The colon is usually primarily responsible for absorbing fluids. It contains a large number of microorganisms including fungi, which are enriched in its distal segment. The colonic mucosa must therefore tightly regulate fluid influx to control absorption of fungal metabolites, which can be harmful to epithelial cells and lead to barrier dysfunction. How this is achieved remains unknown. Here, we describe a mechanism by which the innate immune system allows quick quality check of assimilated fluids to avoid intoxication of colonocytes. This mechanism relies on a populace of distal colon macrophages that are equipped with balloon-like protrusions (BLPs) inserted in the epithelium, which sample absorbed fluids. In the absence of macrophages or BLPs, epithelial cells keep absorbing fluids made up of fungal products, leading to their death and subsequent loss of epithelial barrier integrity. These results reveal an unexpected and essential role of macrophages in the maintenance of colon-microbiota interactions in homeostasis. Video Abstract Click here to view.(3.6M, mp4) remains unknown. To address this question, we depleted M?s using the CD64DTR mouse model (Baranska et?al., 2018) and evaluated the state of the epithelium (Physique?1A). We analyzed both proximal and distal colons as they are known to exhibit differences both in physiology (Dolman and Edmonds, 1975; Foster et?al., 1986; Hardin et?al., 1999) and microbiota composition (Flynn et?al., 2018; Leonardi et?al., 2018). The efficient depletion of colonic M?s was verified by circulation cytometry and immunostaining in both colon segments (Physique?S1A). Unexpectedly, we found that M? depletion led to massive apoptosis of epithelial cells in the distal but not the proximal colon (Figures 1B and 1C). Death of epithelial cells in 18α-Glycyrrhetinic acid the distal colon was also observed when M?s were depleted by injecting anti-CSF1 receptor antibodies in C57BL/6J mice (Figures S1B and S1C). Of notice, if the presence of apoptotic cells merely resulted from the lack of scavenging by M?s, equal numbers of dead cells should be detected in distal and proximal colons (Physique?1C). Our results, therefore, suggest that M?s facilitate the survival of epithelial cells in the distal colon specifically. Open in a separate window Physique?1 M?s Are Required for Epithelial Cell Survival in the Distal Colon and Form Balloon-like Protrusions Inserted in between Epithelial Cells (A) Plan of depletion. CD64WT or CD64DTR littermates received two injections of diphtheria toxin (DT) 24?h apart. (B) Maximum z-projection 18α-Glycyrrhetinic acid (30?m) of proximal and distal colon transversal sections 44?h after the first DT injection. Apoptotic cells were revealed with cleaved caspase 3 staining (reddish), F-actin (green). Level bar: 50?m. (C) Quantity of apoptotic epithelial cells per crypt in the distal or proximal colon. Pooled data from three impartial experiments; dots symbolize average number per individual mouse. Mean SEM, multiple comparison Kruskal-Wallis test, ?p? 0.05. (D) Serum fluorescence intensities 5C10?min after intra-rectal administration of hypotonic answer of hydrazide-AlexaFluor633. All mice were injected with DT. Pooled data from two impartial experiments; dots symbolize average number per individual mouse. Mean SEM, Mann-Whitney test, ?p? 0.05. (E) Morphological differences of s in the proximal and distal colon. Whole-mount staining of the distal and proximal colon of CD11c: Cre/R26mTmG mice. mGFP (green), CD11b (blue), CD103 (reddish), membrane tdTomato (gray). BLPs are indicated with arrows, the border between epithelium and 18α-Glycyrrhetinic acid the stroma is usually indicated with the dashed collection. Z-projections of 20C40?m; level bars: 50?m. (F) Single M forming BLPs (left) or thin extensions (right). Yellow star: cell body; green arrows: BLPs; green arrowheads: extensions. Maximum z-projection of 10C15?m; level bar: 2?m. (G) 18α-Glycyrrhetinic acid Quantity of BLPs, normalized per crypt (left) or per M (right). Dots symbolize average number per individual mouse; left: pooled data from seven impartial experiments; right: pooled data from another two impartial experiments. (H) Quantity of Ms.