Fig 2: BLP-Forming ?? Express Common Intestinal Macrophage Markers, Related to Figure 1(A) Cell bodies of M? are indicated with yellow stars, arrows indicate BLPs. CD11b, MHCII, F4/80 staining of distal colon ?? from CD11c: Cre/R26mTmG mouse, z-projection of 12 µm. Bottom panel (left): CD11b, CD103 staining of CX3CR1-GFP mouse, z-projection of 14 µm.(B) CD64 and MHCII staining of colon ?? from CD11c: Cre/R26mTmG mouse, z-projection of 11 µm, scale bars – 20 µm.

Fig 3: BLP+ M?s Have Distinct Transcriptomic Profile and Express CD11c as a Specific Marker(A) Single-cell RNA-seq experiment workflow. M?s were isolated from proximal/distal colon by FACS and sequenced. 2,106 M?s were identified, n = 1176 for proximal and n = 930 for distal colon, and shown on a t-distributed stochastic neighbor embedding (tSNE) representation; cells isolated from proximal (orange), cells from distal colon (blue) (top 22 PCs computed on the top 1,000 variable genes, vst method).(B) Selection of clustering parameters was based on the silhouette score and expected fraction of BLP+ M?s in proximal and distal colon obtained from IHC (mean fraction of BLP+ M?s out of all M?s; data pooled from two independent experiments). The selected clustering solution (graph-based clustering, 30 neighbors, resolution = 0.3) revealed the presence of two M? subpopulations, displayed on the same tSNE as in (A) and colored accordingly: cluster 0, containing 964 cells in proximal and 390 cells in distal colon, and cluster 1, containing 212 cells in proximal and 540 cells in distal colon. Cluster 1 expresses CD11c as a specific marker.(C) Co-staining of MHC class II and CD11c of the distal colon from C57B6J mouse. Maximum z-projection of 30 µm; scale bar: 20 µm.(D) Percentage of CD11c+ M?s out of all M?s in proximal and distal colon determined by imaging (top, % of CD11c+ M?s out of F4/80+ CD103- MHCII+ cells) or flow cytometry (bottom, % of CD11c+ ??s out of CD45+CD3-CD19-CD11b+CD103-CD64+F4/80+Ly6C-MHCII+ cells); dots represent average number per individual mice; three independent experiments. Mean ± SEM, Mann-Whitney test, **p < 0.01.(E) Heatmap of the top 20 significantly up- and downregulated genes between BLP+ M?s (cluster 1) and BLP- M?s (cluster 0), respectively, sorted by non-decreasing p value; entries represent the scaled (Z score) normalized expression, values <-2.5 or >2.5 are clipped; genes found respectively up- and downregulated in Kang et al. (2020) are highlighted in bold (see F).(F) (Top) Comparison of significantly upregulated genes in BLP+ M?s (cluster 1, 88 genes, in blue) and in cluster 4 from Kang et al. (2020) (166 genes, in gray). Fisher’s exact test using 10,768 detected genes in the 2,106 M?s as background. (Bottom) Comparison of significantly upregulated genes in BLP- M?s (cluster 0, 121 genes, in pink) and cluster 6 from Kang et al. (2020) (168 genes, in gray). See also Figures S3 and S4 and Tables S1 and S2.

Fig 4: The PILRa paired receptor and the actomyosin cytoskeleton are needed for epithelium colonization by CD103+CD11b+ cDC2s(A) Violin plot representing the expression of transendothelial migration-related genes among clusters of single-cell RNA-seq analysis: Pilra, Pilrb, and Amica1.(B) Plots of number of CD103+CD11b+ in live CD45+ CD11c+ MHCII+ CD64- cells from the epithelium and lamina propria of the whole small intestine in Pilra-/- and Pilra+/+ female mice. Data are pooled from three independent experiments, each symbol represents one mouse.(C) Pseudotime reconstruction showing the expression of the migratory related gene Abi3.(D) Plots of number of CD103+CD11b+ in live CD45+ CD11c+ MHCII+ CD64- cells from the epithelium and lamina propria of the whole small intestine in Arpc4-/- (Arpc4flox/flox × ItgaxCre+) and Arpc4+/+ (Arpc4flox/flox × ItgaxCre-) mice. Data are pooled from three independent experiments, each symbol represents one mouse.(E) Pseudotime reconstruction showing the expression of the migratory related gene Myh9 (MyoIIA).(F) Plots of number of CD103+CD11b+ in live CD45+ CD11c+ MHCII+ CD64- cells from the epithelium and lamina propria of the whole small intestine in MyoIIA deficient (Myh9flox/flox × ItgaxCre+) and WT (Myh9flox/flox × ItgaxCre-) mice. Data are pooled from three independent experiments, each symbol represents one mouse. In (B), (D), and (F), mean ± SEM, data were compared using Mann-Whitney test or t test, *p < 0,05, ***p < 0,001. Please also see Figures S2 and S3.

Fig 5: M?s Are Required for Epithelial Cell Survival in the Distal Colon and Form “Balloon-like” Protrusions Inserted in between Epithelial Cells(A) Scheme of ?? depletion. CD64WT or CD64DTR littermates received two injections of diphtheria toxin (DT) 24 h apart.(B) Maximum z-projection (30 µm) of proximal and distal colon transversal sections 44 h after the first DT injection. Apoptotic cells were revealed with cleaved caspase 3 staining (red), F-actin (green). Scale bar: 50 µm.(C) Number of apoptotic epithelial cells per crypt in the distal or proximal colon. Pooled data from three independent experiments; dots represent average number per individual mouse. Mean ± SEM, multiple comparison Kruskal-Wallis test, *p < 0.05.(D) Serum fluorescence intensities 5–10 min after intra-rectal administration of hypotonic solution of hydrazide-AlexaFluor633. All mice were injected with DT. Pooled data from two independent experiments; dots represent 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 (red), membrane tdTomato (gray). BLPs are indicated with arrows, the border between epithelium and the stroma is indicated with the dashed line. Z-projections of 20–40 µm; scale bars: 50 µm.(F) Single M? forming BLPs (left) or thin extensions (right). Yellow star: cell bodies; green arrows: BLPs; green arrowheads: extensions. Maximum z-projection of 10–15 µm; scale bar: 2 µm.(G) Number of BLPs, normalized per crypt (left) or per M? (right). Dots represent average number per individual mouse; left: pooled data from seven independent experiments; right: pooled data from another two independent experiments.(H) Number of M?s in the proximal and distal colon, analyzed by imaging (F4/80+MHCII+CD103- cells per crypt; each dot represents average number per individual mouse; data pooled from three independent experiments) and by flow cytometry (presented as percentage of CD45+ cells; dots represent individual mouse; data pooled from four independent experiments).(I) Number of extensions, normalized per M?. Dots represent average number per individual mouse; four independent experiments.In (G–I), mean ± SEM, Mann-Whitney test, *p < 0.05, **p < 0.01, ****p < 0.0001. See also Figure S1 and S2, Video S1.