The intestinal microbiome plays a critical role in the development of the immune system, including the development of specific CD4+ T cell subtypes, T helper (Th)1, Th2, Th17 and T regulatory (Treg) cells (each with a characteristic cytokine profile), which can have numerous long-term health consequences. Certain cytokines, including IL-4, IL-13, and IL-17A, are linked to the development of allergic and autoimmune diseases, whereas IFN-β has been shown to reduce the induction of Th1 and Th17 cells while increasing IL-10-producing Treg cells. Given the pleiotropic activities of these cytokines, including the coordination of tissue inflammation, infiltration, and destruction, we evaluated fecal cytokines from breastfed infants fed B. infantis EVC001 (EVC001) during the first 60 days of life. Stool samples were collected at Day 6 (baseline) and day 60 of life from exclusively breastfed infants (n=40) randomly selected to receive either 1.8 x 1010 CFU B. infantis EVC001 daily for 21 days starting Day 7 postnatal (EVC001) or breast milk alone (controls). Samples were analyzed for microbiome composition using 16S ribosomal RNA, qPCR, metagenomics, and for fecal proinflammatory cytokines using multiplexed immunoassay. Pairwise correlation tests were performed at day 60 of life between the fecal microbial taxonomic composition and specific enteric cytokine concentrations (Spearman correlation with Benjamini-Hochberg false discovery rate correction a < 0.02). Clostridiaceae correlated with increased IL-21, IL-33, and IL-4, Enterobacteriaceae correlated with IL-13, IL-17A, IL-21, IL-33, and IL-4, while Staphylococceae correlated with IL-21 production. Clostridiaceae and Enterobacteriaceae negatively correlated with IFNβ levels. Conversely, Bifidobacteriaceae abundance was the only taxa that correlated with increased levels of IFNβ and negatively with proinflammatory cytokines IL-13, IL-21, IL-4, MIP1α. Comparison between Bifidobacteriaceae abundance and IFNβ show a direct correlation (P = 9.8e-06, rho = 0.65), while Enterobacteriaceae and Clostridiaceae abundance negatively correlated with IFNβ concentrations (P = 0.0013, 0.0034 rho = -0.5, -0.46, respectively). At a species level, B. longum abundance at day 21 correlated with IFNβ levels at day 60 postnatal (P = 0.0028, rho = 0.48). No other Bifidobacterium species correlated with increased IFNβ levels. Importantly, infants fed B. infantis EVC001 produced significantly decreased levels of IL-4, IL-13, IL21, IL-31, and IL-33 (P =0.038, 0.0065, 0.03, and 0.0003, respectively), while IFNβ levels were significantly increased (P =0.047) compared to the controls day 60 postnatal. These findings suggest a novel immunomodulatory function of B. infantis in breastfed infants, presumably through its unique ability to metabolize human milk oligosaccharides and further imply this strain of bacteria may induce CD4+ T cell development, specifically proliferation of Treg cell subset, critically important in the reduction of Th2 and Th17-mediated autoimmune and allergic diseases.

Correlations between specific gut taxa and enteric inflammatory cytokine production.

A. Heatmap shows correlations between bacterial families and specific cytokines computed via Spearman correlation. P values are corrected using Benjamini-Hochberg procedure (false discovery rate) to estimate significant correlations between microbial taxonomic compositions and specific cytokine concentration detected in the feces of exclusively breastfed infants at day 60 postnatal. B. Total Bifidobacterium abundance was correlated with IFNb concentration at day 60 postnatal. C. Total Clostridiaceae abundance was correlated with IFNb concentration at day 60 postnatal. D. Total Enterobacteriaceae abundance was correlated with IFNb concentration at day 60 postnatal. Each cytokine was tested in duplicate three different times. P values were adjusted and considered to be statistically significant is *P < 0.05, **P < 0.01,*** P < 0.001.

Infants fed B. infantis EVC001 had significantly modulated fecal cytokine levels compared to controls

A. Radar plot representations of median cytokine concentrations [pg/mg] detected in fecal samples from the controls (n = 20) and infants fed B. infantis EVC001 (n = 20; EVC001) on day 6 (baseline). B. Controls infants at day 60 postnatal. C. EVC001 fed infants at day 60 postnatal. Median values were adjusted to log scale then normalized within each cytokine from 0 to 1. Statistical analysis was completed using between controls and EVC001-fed infant fecal samples at day 60 postnatal using Wilcoxon rank-sum test. P values were adjusted using Bonferonni-Holm method and considered statistically significant if *P < 0.05, **P < 0.01, *** P < 0.001.