Additional file 1 of CD71 + erythroid cells promote intestinal symbiotic microbial communities in pregnancy and neonatal period

Supplementary Material 1: Supplementary Figures S Fig 1. A) The gating strategy for the identification of CECs in intestinal tissues. B) Representative flow plots and cumulative data of the % of CECs in the spleen, small intestines, and colon tissues of 4-day-old control mice vs those treated with the anti-CD71 antibody a day earlier. C) Gene expression assay for detection of α4β7 integrin, and D) its ligand MAdCAM-1 in small intestinal tissues of control or CECs depleted animals. E) Correlation analysis of % CECs with the intensity of α4β7 in splenic CECs in 3-day-old mice. F) Representative flow cytometry plots showing the gating strategy for central macrophages (CD11b-CD169+F4-80+) in the small intestine of a 3-day-old mouse. G) Representative flow plots of central macrophages in a 3-day-old germ-free mouse. Results are presented as SD and P values were calculated using two tailed, Mann–Whitney t test (B-D) or the Spearmen correlation analysis (E). P < 0.05 (*). Anti-CD71 (aCD71). not significant (ns). S Fig. 2. Representative immunofluorescence staining (IF) plots at different indicated magnifications illustrating the presence of erythropoiesis niches in submucosal tissues of small intestine. DAPI (blue), TER119 (red), F4/80 (green). S Fig. 3. Representative IF plots at different indicated magnifications illustrating the presence of erythropoiesis niches in the villus of small intestine. DAPI (blue), TER119 (red), F4/80 (green). S Fig. 4. A) Representative IF plots illustrating the presence of mature red blood cells, without nuclei, in venules of small intestine. B) Representative IF plot illustrating the presence of erythropoiesis niches in the spleen of a neonatal mouse. DAPI (blue), TER119 (red), F4/80 (green). S Fig. 5. A) Representative flow cytometry plot of central macrophages in a germ-free mouse. B) Representative histogram and cumulative data of α4β7 expression in intestinal CECs of 3-day old conventional and germ-free mice. C) Representative flow cytometry plots, and D) cumulative data of % of CECs in the small intestine of conventional (con.) vs. germ-free mice. E) Representative flow cytometry plots, and F) cumulative data showing TNF-α production by intestinal CD11b+ and CD11c+ cells among treated mice (3-day old) with the anti-CD71 antibody or the isotype control antibody 24 hr later quantified by intracellular cytokine staining ex-vivo. G) Cumulative data of fold change in TNF-a expression in either CD11b+ or CD11c+ cells from germ-free mice treated with the anti-CD71 antibody compared to control animals. H) Quantified expression levels of CD71 obtained from the anti-CD71 treated and control mice (2-day post-treatment). I) Comparison of the mean number of cleaved caspase-3-positive (CC3+) intestinal epithelial cells (CC3+ IEC) per high power field (HPF) between the anti-CD71-treated versus isotype control in conventional or germ-free mice (treated at age 3 and examined at day 5). The immunofluorescence staining for CC3 was used as a marker of apoptosis and the experiment was performed with 10 mice per group with 10 fields per mouse. Results are presented as SD andP values were calculated using two tailed, Mann–Whitney t test (B, F-H) and One-way ANOVA (D, I).P < 0.05 (*), **P < 0.01, *** P <0.001, **** P < 0.0001. Anti-CD71 (aCD71), not significant (ns). S Fig. 6. A) Showing the relative frequency of dominant bacterial communities at the phylum level in control female (Con. F), control male (Con. M), or anti-CD71-treated (male (M) or female (F) mice. Mice were treated at day-3 and their gut contents were collected at day 35. B) The qPCR data showing the correlation between total gut CECs (%) with the gene copies of Lactobacillus or C) the Enterobacteriaceae family in the gut of adult mice. D) Cumulative data of the gene copies of bacterial taxa in nonporous vs pregnant mice (controls or treated with the anti-CD71) in the cecum, E) Ileum, and F) Colon. Results are presented as SD and P values were calculated using One-way ANOVA (D-F). (P < 0.05 (*), P £ 0.01 (**), and P £ 0.00001 (****). Anti-CD71 (aCD71). S. Fig. 7. Cumulative data showing fold regulation of different indicated genes as quantified by qPCR in the colonic tissues of non-pregnant, pregnant control, and pregnant mice treated with the anti-CD71 at E12.5-13.5 and collected one day later. Each dot represents an animal. Results are presented as SD and P values were calculated using Kruskal–Wallis analysis with Dunn’s multiple comparisons test. (P < 0.05 (*), P £ 0.01 (**), and P £ 0.0001 (***). Anti-CD71 (aCD71). not significant (ns). S Fig 8. A) Representative flow cytometry plots of the gating strategy for the cord blood CECs. B) Representative flow cytometry plots, and C) cumulative data of percentages of CECs in different gestational ages as indicated compared to an adult peripheral blood. D) Representative flow cytometry plot of CECs in a placental tissue from a full-term delivery. Each dot represents a cord blood. Results are presented as SD and P values were calculated using One-way ANOVA test. P £ 0.01 (**), and P £ 0.00001 (****), not significant (ns).

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