Abstract Summary Monocytes restrict Yersinia infection within intestinal granulomas. Here, we report that monocyte-intrinsic TNF signaling drives production of IL-1 that signals to non-hematopoietic cells to control intestinal Yersinia infection within granulomas. Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas that control the bacterial infection. Inflammatory monocytes are essential for control and clearance of Yersinia within intestinal pyogranulomas, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptor on non-hematopoietic cells to enable pyogranuloma-mediated control of Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative circuit as a crucial driver of intestinal granuloma function, and defines the cellular target of TNF signaling that restricts intestinal Yersinia infection.
Endoplasmic reticulum (ER) stress compromises the secretion of MUC2 from goblet cells and has been linked with inflammatory bowel disease (IBD). Although Bifidobacterium can beneficially modulate mucin production, little work has been done investigating the effects of Bifidobacterium on goblet cell ER stress. We hypothesized that secreted factors from Bifidobacterium dentium downregulate ER stress genes and modulates the unfolded protein response (UPR) to promote MUC2 secretion. We identified by mass spectrometry that B. dentium secretes the antioxidant γ-glutamylcysteine, which we speculate dampens ER stress-mediated ROS and minimizes ER stress phenotypes. B. dentium cell-free supernatant and γ-glutamylcysteine were taken up by human colonic T84 cells, increased glutathione levels, and reduced ROS generated by the ER-stressors thapsigargin and tunicamycin. Moreover, B. dentium supernatant and γ-glutamylcysteine were able to suppress NF-kB activation and IL-8 secretion. We found that B. dentium supernatant, γ-glutamylcysteine, and the positive control IL-10 attenuated the induction of UPR genes GRP78, CHOP, and sXBP1. To examine ER stress in vivo, we first examined mono-association of B. dentium in germ-free mice which increased MUC2 and IL-10 levels compared to germ-free controls. However, no changes were observed in ER stress-related genes, indicating that B. dentium can promote mucus secretion without inducing ER stress. In a TNBS-mediated ER stress model, we observed increased levels of UPR genes and pro-inflammatory cytokines in TNBS treated mice, which were reduced with addition of live B. dentium or γ-glutamylcysteine. We also observed increased colonic and serum levels of IL-10 in B. dentium- and γ-glutamylcysteine-treated mice compared to vehicle control. Immunostaining revealed retention of goblet cells and mucus secretion in both B. dentium- and γ-glutamylcysteine-treated animals. Collectively, these data demonstrate positive modulation of the UPR and MUC2 production by B. dentium-secreted compounds.
Metabolites have emerged as the quintessential effectors mediating the impact of the commensal microbiome on human physiology, both locally at the sites of microbial colonization and systemically. The endocrine activity of the microbiome and its involvement in a multitude of complex diseases has made microbiome-modulated metabolites an attractive target for the development of new therapies. Several properties make metabolites uniquely suited for interventional strategies: natural occurrence in a broad range of concentrations, functional pleiotropy, ease of administration, and tissue bioavailability. Here, we provide an overview of recently discovered physiological effects of microbiome-associated small molecules that may serve as the first examples of metabolite-based therapies. We also highlight challenges and obstacles that the field needs to overcome on the path toward successful clinical trials of microbial metabolites for human disease.
Abstract The histamine H2 receptor (H2R) is a G protein‐coupled receptor that mediates cyclic AMP production, protein kinase A activation, and MAP kinase signaling. In order to explore the multifaceted effects of histamine signaling on immune cells, phagocytosis was evaluated using primary mouse‐derived macrophages. Phagocytosis is initiated by signaling via surface‐bound scavenger receptors and can be regulated by autophagy. Absence of H2R signaling resulted in diminished phagocytosis of live bacteria and synthetic microspheres by primary macrophages from histamine H2 receptor gene ( Hrh2 )‐deficient mice. Flow cytometry and immunofluorescence microscopy were used to quantify phagocytosis of phylogenetically diverse bacteria as well as microspheres of defined chemical composition. Autophagy and scavenger receptor gene expression were quantified in macrophages after exposure to Escherichia coli . Expression of the autophagy genes, Becn1 and Atg12, was increased in Hrh2 −/− macrophages, indicating upregulation of autophagy pathways. Expression of the Macrophage Scavenger Receptor 1 gene ( Msr1 ) was diminished in Hrh2 ‐deficient macrophages, supporting the possible importance of histamine signaling in scavenger receptor abundance and macrophage function. Flow cytometry confirmed diminished MSR1 surface abundance in Hrh2 −/− macrophages. These data suggest that H2R signaling is required for effective phagocytosis by regulating the process of autophagy and scavenger receptor MSR1 abundance in macrophages.
Tumor necrosis factor (TNF) is a pleiotropic inflammatory cytokine that mediates antimicrobial defense and granuloma formation in response to infection by numerous pathogens. We previously reported that Yersinia pseudotuberculosis colonizes the intestinal mucosa and induces the recruitment of neutrophils and inflammatory monocytes into organized immune structures termed pyogranulomas (PG) that control Yersinia infection. Inflammatory monocytes are essential for the control and clearance of Yersinia within intestinal PG, but how monocytes mediate Yersinia restriction is poorly understood. Here, we demonstrate that TNF signaling in monocytes is required for bacterial containment following enteric Yersinia infection. We further show that monocyte-intrinsic TNFR1 signaling drives the production of monocyte-derived interleukin-1 (IL-1), which signals through IL-1 receptors on non-hematopoietic cells to enable PG-mediated control of intestinal Yersinia infection. Altogether, our work reveals a monocyte-intrinsic TNF-IL-1 collaborative inflammatory circuit that restricts intestinal Yersinia infection.
Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.
Pathogen infection of host cells triggers an inflammatory cell death termed pyroptosis via activation of inflammatory caspases. However, blockade of immune signaling kinases by the
 scale(.12937)'%3e %3csvg width='12' viewBox='-120 -190.223125 240 309.188274' height='15.459'%3e %3cpath d='m110.26-19.478l9.74-143.75a280.22 280.22 0 0 0 -240 0l9.74 143.75a155.61 155.61 0 0 0 110.26 138.45 155.61 155.61 0 0 0 110.26 -138.45' fill='%23005da4'/%3e %3cpath d='m-90 0a138.29 138.29 0 0 0 90 100.77 138.29 138.29 0 0 0 90 -100.77l-45-60-18 24-27-54-27 54-18-24-45 60' fill='%23fff'/%3e %3cg id='wave' fill='%23005da4' transform='scale(5) translate(0 5.1962)'%3e %3cpath d='m-17.196-2.1962a6 6 0 0 0 8.1962 2.1962 6 6 0 0 1 6 0 6 6 0 0 0 6 0 6 6 0 0 1 6 0 6 6 0 0 0 8.1962 -2.1962v1.732a6 6 0 0 1 -8.1962 2.1962 6 6 0 0 0 -6 0 6 6 0 0 1 -6 0 6 6 0 0 0 -6 0 6 6 0 0 1 -8.1962 -2.1962z'/%3e %3c/g%3e %3cuse xlink:href='%23wave' transform='translate(0 17.321)'/%3e %3cg id='s' transform='translate(0,-120) scale(2.25)'%3e %3cpath stroke-width='.2' d='m0-5l1 3.2679 3.3301-0.7679-2.3301 2.5 2.3301 2.5-3.3301-0.7679-1 3.2679-1-3.2679-3.3301 0.7679 2.3301-2.5-2.3301-2.5 3.3301 0.7679z' fill='%23fd0'/%3e %3c/g%3e %3cuse xlink:href='%23s' transform='translate(-33.75,-45)'/%3e %3cuse xlink:href='%23s' transform='translate(33.75,-45)'/%3e %3cpath d='m-111.58-167.05l9.96 146.99a146.95 146.95 0 0 0 101.62 129.95 146.95 146.95 0 0 0 101.62 -129.95l9.96-146.99a280.22 280.22 0 0 0 8.42 3.82l-9.74 143.75a155.61 155.61 0 0 1 -110.26 138.45 155.61 155.61 0 0 1 -110.26 -138.45l-9.74-143.75a280.22 280.22 0 0 0 8.42 -3.82' fill='%23ed1c24'/%3e %3c/svg%3e %3c/g%3e %3c/svg%3e)