Fig. 2: Transcriptional landscape of the neonatal ileum and the circulating immune compartment is influenced by vaginal community state type.

A Schematic of experimental outline to determine the influence of the human microbiome on transcriptional profiles of the neonatal ileum and the circulating immune compartment. Created with BioRender.com. B Principal component analysis plots of gene expression data demonstrating the distribution of P1 male ileum samples colored by treatment group, revealing clustering is driven by developmental maturity, mode of delivery, and colonization (C-section Amies inoculated vs. vaginally delivered) and the C-section pups inoculated with human vaginal microbiota as an intermediate between the Amies and vaginally delivered pups. P1 corresponds to 24 hrs post-inoculation. N = 3–4 males per treatment. C Heatmap depicting mean expression of genes in the ileum P1 males, showing differences between CST I and CST IV males (linear fit model, FDR < 0.1, log(FC) = 1.5). Unbiased hierarchical clustering showing similarity in transcriptional patterns between CST IV and VD males, and CST I and Amies males. P1 corresponds to 24 h post-inoculation. Color based on row Z-scores for each gene. Three clusters of differentially regulated genes in each treatment group are indicated. N = 3–4 males per treatment. D Cluster-based functional enrichment analysis of differentially expressed genes in the ileum of P1 males showing significant enrichment of pathways involved in defense response to bacteria, innate immune activation, chemotaxis, and mucus secretion in the ileum of CST IV relative to CST I inoculated males (FDR < 0.05). Bubble plot size denotes enrichment. N = 4–6 males per treatment. E t-SNE visualization demonstrating CyTOF phenotyping of CD45+ immune cells in whole blood of CST I, CST IV, and vaginally delivered males showing neutrophils as the major immune subset in the circulating immune compartment at P1 (equal sampling across treatment groups, total 10,000 events). P1 corresponds to 24 h post-inoculation. N = 3 males per treatment. F Average frequencies of major leukocytes within whole blood in P1 CST I, CST IV, and VD males. N = 3 males per treatment. G–L Frequencies of circulating (G) neutrophils, (H) monocytes, (I) MHC II⁺ B cells, (J) MHC II^- B cells, K CD8 + T cells, and L CD4⁺ T cells within whole blood in P1 CST I, CST IV, and VD males. (1) Frequency of CD4⁺ T cells were significantly increased in VD males compared with CST I and CST IV males (One-way ANOVA, F_2,6 = 15.97, P = 0.0040; Tukey’s post-hoc CST I vs. VD P = 0.0072; CST IV vs. VD P = 0.0059). N = 3 males per treatment. Data represented as mean ± SEM with individual data points overlaid. **P < 0.01.