Human milk oligosaccharides differentially support gut barrier integrity and enhance Th1 and Th17 cell effector responses in vitro.

Human milk oligosaccharides (HMOs) can modulate the intestinal barrier and regulate immune cells to favor the maturation of the infant intestinal tract and immune system, but the precise functions of individual HMOs are unclear. To determine the structure-dependent effects of individual HMOs (representing different structural classes) on the intestinal epithelium as well as innate and adaptive immune cells, we assessed fucosylated (2'FL and 3FL), sialylated (3'SL and 6'SL) and neutral non-fucosylated (LNT and LNT2) HMOs for their ability to support intestinal barrier integrity, to stimulate the secretion of chemokines from intestinal epithelial cells, and to modulate cytokine release from LPS-activated dendritic cells (DCs), M1 macrophages (MØs), and co-cultures with naïve CD4+ T cells. The fucosylated and neutral non-fucosylated HMOs increased barrier integrity and protected the barrier following an inflammatory insult but exerted minimal immunomodulatory activity. The sialylated HMOs enhanced the secretion of CXCL10, CCL20 and CXCL8 from intestinal epithelial cells, promoted the secretion of several cytokines (including IL-10, IL-12p70 and IL-23) from LPS-activated DCs and M1 MØs, and increased the secretion of IFN-γ and IL-17A from CD4+ T cells primed by LPS-activated DCs and MØs while reducing the secretion of IL-13. Thus, 3'SL and 6'SL supported Th1 and Th17 responses while reducing Th2 responses. Collectively, our data show that HMOs exert structure-dependent effects on the intestinal epithelium and possess immunomodulatory properties that confer benefits to infants and possibly also later in life.

Bacillus subtilis-597 induces changes in lung pathology and inflammation during influenza A virus infection in pigs.

In recent years, it has become apparent that imbalances in the gastrointestinal system can impact organs beyond the intestine such as the lungs. Given the established ability of probiotics to modulate the immune system by interacting with gastrointestinal cells, our research aimed to investigate whether administering the probiotic strain Bacillus subtilis-597 could mitigate the outcome of influenza virus infection in pigs. Pigs were fed a diet either with or without the probiotic strain B. subtilis-597 for 14 days before being intranasally inoculated with a swine influenza A H1N2 strain (1 C.2 lineage). Throughout the study, we collected fecal samples, blood samples, and nasal swabs to examine viral shedding and immune gene expression. After seven days of infection, the pigs were euthanized, and lung and ileum tissues were collected for gene expression analysis and pathological examination. Our findings indicate that the administration of B. subtilis-597 exhibit potential in reducing lung lesions, possibly attributable to a general suppression of the immune system as indicated by reduced C-reactive protein (CRP) levels in serum, decreased expression of interferon-stimulated genes (ISGs), and localized reduction of the inflammatory marker serum amyloid A (SAA) in ileum tissue. Notably, the immune-modulatory effects of B. subtilis-597 appeared to be unrelated to the gastrointestinal microbiota, as the composition remained unaltered by both the influenza infection and the administration of B. subtilis-597.

Ligilactobacillus animalis 506 Protects the Intestinal Barrier from the Damaging Effects of Enteric Pathogens and Deoxynivalenol.

This study investigated the impact of L. animalis 506 on gut barrier integrity and regulation of inflammation in vitro using intestinal epithelial cell lines. Caco-2 or HT29 cell monolayers were challenged with enterotoxigenic E. coli (ETEC) or a ruminant isolate of Salmonella Heidelberg in the presence or absence of one of six probiotic Lactobacillus spp. strains. Among these, L. animalis 506 excelled at exerting protective effects by significantly mitigating the decreased transepithelial electrical resistance (TEER) as assessed using area under the curve (AUC) (p < 0.0001) and increased apical-to-basolateral fluorescein isothiocyanate (FITC) dextran translocation (p < 0.0001) across Caco-2 cell monolayers caused by S. Heidelberg or ETEC, respectively. Similarly, L. animalis 506 and other probiotic strains significantly attenuated the S. Heidelberg- and ETEC-induced increase in IL-8 from HT29 cells (p < 0.0001). Moreover, L. animalis 506 significantly counteracted the TEER decrease (p < 0.0001) and FITC dextran translocation (p < 0.0001) upon challenge with Clostridium perfringens. Finally, L. animalis 506 significantly attenuated DON-induced TEER decrease (p < 0.01) and FITC dextran translocation (p < 0.05) and mitigated occludin and zona occludens (ZO)-1 redistribution in Caco-2 cells caused by the mycotoxin. Collectively, these results demonstrate the ability of L. animalis 506 to confer protective effects on the intestinal epithelium in vitro upon challenge with enteric pathogens and DON known to be of particular concern in farm animals.

Probiotic Bacillus spp. enhance TLR3-mediated TNF signalling in macrophages.

Probiotics have been reported to have immunomodulatory properties in the context of infectious disease and inflammation, although the underlying mechanisms are not fully understood. Here, we aimed to determine how different probiotic bacterial strains modulated macrophage function during TLR3 stimulation mimicking viral infection. We screened 14 different strains for their ability to modulate TNF-α, IL-6 IL-10, IFN-α, IFN-ß and IFN-γ secretion in RAW 264.7 macrophages with or without poly(I:C) stimulation. Seven strains were selected for further analysis using primary porcine alveolar macrophages. In-depth transcriptomic analysis on alveolar macrophages was conducted for two strains. Most strains induced a synergistic effect when co-incubated with poly(I:C) resulting in increased levels of IL-6 and TNF-α secretion from RAW 264.7 cells. This synergistic effect was found to be TLR2 independent. Only strains of Bacillus spp. could induce this effect in alveolar macrophages. Transcriptomic analysis indicated that the increased TNF-α secretion in alveolar macrophages after co-incubation with poly(I:C) correlated with significant upregulation of TNF and IL23A-related pathways. Collectively, our data show that probiotic bacteria possess strain-dependent immunomodulatory properties that may be harnessed to enhance innate immune responses to pathogens.

Enteroaggregative Escherichia coli disrupts epithelial cell tight junctions.

Enteroaggregative Escherichia coli (EAEC) is responsible for inflammatory diarrhea in diverse populations, but its mechanisms of pathogenesis have not been fully elucidated. We have used a previously characterized polarized intestinal T84 cell model to investigate the effects of infection with EAEC strain 042 on tight junction integrity. We find that infection with strain 042 induces a decrease in transepithelial electrical resistance (TER) compared to uninfected controls and to cells infected with commensal E. coli strain HS. When the infection was limited after 3 h by washing and application of gentamicin, we observed that the TER of EAEC-infected monolayers continued to decline, and they remained low even as long as 48 h after the infection. Cells infected with the afimbrial mutant strain 042aafA exhibited TER measurements similar to those seen in uninfected monolayers, implicating the aggregative adherence fimbriae II (AAF/II) as necessary for barrier dysfunction. Infection with wild-type strain 042 induced aberrant localization of the tight junction proteins claudin-1 and, to a lesser degree, occludin. EAEC-infected T84 cells exhibited irregular shapes, and some cells became elongated and/or enlarged; these effects were not observed after infection with commensal E. coli strain HS or 042aafA. The effects on tight junctions were also observed with AAF/I-producing strain JM221, and an afimbrial mutant was similarly deficient in inducing barrier dysfunction. Our results show that EAEC induces epithelial barrier dysfunction in vitro and implicates the AAF adhesins in this phenotype.