Maintaining Gut Homeostasis Research Articles

Gut microbiota-derived short chain fatty acids are potential mediators in gut inflammation.

Gut inflammation is a challenging concern in humans and animals, which disturbs normal growth and leads to severe bowel diseases. Short chain fatty acids (SCFA) are the gut microbiota metabolites produced from fermentation of non-digestible carbohydrates, and have been reported to modulate gut inflammation. SCFA have been implicated as the potential therapeutic bioactive molecules for gut inflammatory diseases, and could be an alternative to antibiotic growth promoters (AGP). In this review, the existing knowledge about the types of SCFA, the related gut microbes producing SCFA, the roles of SCFA in maintaining gut homeostasis, and how SCFA modulate gut inflammation is summarized. The therapeutic application of SCFA in the treatment of inflammatory bowel disease (IBD) is also highlighted.

The Multifaceted Effects of Gut Microbiota on the Immune System of the Intestinal Mucosa

The gut microbiota has diverse microbial components, including bacteria, viruses, and fungi. The interaction between gut microbiome components and immune responses has been studied extensively over the last decade. Several studies have reported the potential role of the gut microbiome in maintaining gut homeostasis and the development of disease. The commensal microbiome can preserve the integrity of the mucosal barrier by acting on the host immune system. Contrastingly, dysbiosis-induced inflammation can lead to the initiation and progression of several diseases through inflammatory processes and oxidative stress. In this review, we describe the multifaceted effects of the gut microbiota on several diseases from the perspective of mucosal immunological responses.

Enterococcus faecium Alleviates Gut Barrier Injury in C57BL/6 Mice with Dextran Sulfate Sodium-Induced Ulcerative Colitis.

The involvement of gut microbiota composition in ulcerative colitis is strongly supported by previous research. Growing evidence suggests that probiotic therapy protects against inflammatory bowel disease in animal models and patients. However, as a probiotic, the role of Enterococcus faecium (E. faecium) in UC remains unclear. Nevertheless, the potential mechanism of the protective effect of E. faecium remains unknown. In this study, a dextran sulphate sodium-induced (DSS-induced) colitis model was used to detect the underlying mechanism of E. faecium in maintaining gut homeostasis. ELISA was performed to detect the levels of cytokines (TNF-α, IL-1β, IL-6, and IL-10). Furthermore, 454 pyrosequencing was used to investigate the microbiota composition in fecal samples. The results illustrate that E. faecium administration could prevent DSS-induced gut inflammation and intestinal flora imbalance. At the same time, the damage to intestinal mucosal barrier and tight junctions was partially repaired. These results demonstrate the preventive effect of E. faecium in DSS-induced intestinal injury. The present study provides new insights into the medicinal value of E. faecium for UC.

SPAK Deficiency Attenuates Chemotherapy-Induced Intestinal Mucositis.

IntroductionSte20-related protein proline/alanine-rich kinase (SPAK) affects cell proliferation, differentiation, and transformation, and sodium and chloride transport in the gut. However, its role in gut injury pathogenesis is unclear.ObjectiveWe determined the role of SPAK in chemotherapy-induced intestinal mucositis using in vivo and in vitro models.MethodsUsing SPAK-knockout (KO) mice, we evaluated the severity of intestinal mucositis induced by 5-fluorouracil (5-FU) by assessing body weight loss, histological changes in the intestinal mucosa, length of villi in the small intestine, pro-inflammatory cytokine levels, proliferative indices, and apoptotic indices. We also evaluated changes in gut permeability and tight junction-associated protein expression. Changes in cell permeability, proliferation, and apoptosis were assessed in SPAK siRNA-transfected 5FU-treated IEC-6 cells.Results5-FU-treated SPAK-KO mice exhibited milder intestinal mucositis, reduced pro-inflammatory cytokine expression, increased villus length, good maintenance of proliferative indices of villus cells, decreased apoptotic index of enterocytes, reduced gut permeability, and restoration of tight junction protein expression (vs. 5-FU-treated wild-type mice). Under in vitro conditions, siRNA-mediated SPAK-knockdown in IEC-6 cells decreased cell permeability and maintained homeostasis following 5-FU treatment.ConclusionSPAK deficiency attenuated chemotherapy-induced intestinal mucositis by modulating gut permeability and tight junction-associated protein expression and maintaining gut homeostasis in murine small intestinal tissues following gut injury. The expression of SPAK may influence the pathogenesis of chemotherapy-induced intestinal mucositis.

Mimicking the Gastrointestinal Mucus Barrier: Laboratory-Based Approaches to Facilitate an Enhanced Understanding of Mucus Permeation.

The gastrointestinal mucus layer plays a significant role in maintaining gut homeostasis and health, offering protective capacities against the absorption of harmful pathogens as well as commensal gut bacteria and buffering stomach acid to protect the underlying epithelium. Despite this, the mucus barrier is often overlooked during preclinical pharmaceutical development and may pose a significant absorption barrier to high molecular weight or lipophilic drug species. The complex chemical and physical nature of the dynamic mucus layer has proven problematic to reliably replicate in a laboratory setting, leading to the development of multiple mucus models with varying complexity and predictive capacity. This, coupled with the wide range of analysis methods available, has led to a plethora of possible approaches to quantifying mucus permeation; however, the field remains significantly under-represented in biomedical research. For this reason, the development of a concise collation of the available approaches to mucus permeation is essential. In this review, we explore widely utilized mucus mimics ranging in complexity from simple mucin solutions to native mucus preparations for their predictive capacity in mucus permeation analysis. Furthermore, we highlight the diverse range of laboratory-based models available for the analysis of mucus interaction and permeability with a specific focus on in vitro, ex vivo, and in situ models. Finally, we highlight the predictive capacity of these models in correlation with in vivo pharmacokinetic data. This review provides a comprehensive and critical overview of the available technologies to analyze mucus permeation, facilitating the efficient selection of appropriate tools for further advancement in oral drug delivery.

Attributes of intestinal microbiota composition and their correlation with clinical primary non-response to anti-TNF-α agents in inflammatory bowel disease patients.

The largest microbial aggregation in the human body exists in the gastrointestinal tract. The microbiota in the host gastrointestinal tract comprises a diverse ecosystem, and the intestinal microbiota plays a key role in maintaining gut homeostasis. This study aims to examine whether the gut microbiota influence unresponsiveness to anti-TNF-α treatments in primary non-responder patients and consequently identify the responsible microbes as biomarkers of unresponsiveness. Stool samples were collected from a cohort of patients with an established diagnosis of IBD, either ulcerative colitis or Crohn’s disease, following completion of the induction phase of anti-TNF therapy. 16S rRNA sequencing analysis was used to examine the pattern of microbiota communities in fecal samples. The quality and quantity of fecal microbiota were compared in responder and primary non-responder IBD patients following anti-TNF-α therapy. As per our hypothesis, a difference in gut microbiome composition between the two patient subgroups was observed. A decreased abundance of short-chain fatty acid (SCFA)-producing bacteria, including Anaerostipes, Coprococcus, Lachnospira, Roseburia, and Ruminococcus, was detected in non-responsive patients, which was the hallmark of dysbiosis. Biomarkers of dysbiosis that was identified as predictors of clinical non-response included Klebsiella, Eubacteriaceae, RF32, Bifidobacterium animalis, and Muribaculaceae previously known as S24-7. Signature biomarkers showed dramatic alteration in the composition of gut microbiota in patients who demonstrated primary non-response to anti-TNF-α agents. Dysbiosis, with features including a dropped biodiversity, augmentation in opportunistic pathogenic microbiota, and a lack of SCFA-producing bacteria, is a prominent feature of the microbiome of primary non-responders to anti-TNF-α therapy.

The Absence of NLRP6 in Donor T Cells Exacerbates Gvhd

The Nlrp6 (NOD-like receptor family pyrin domain containing 6) inflammasome is important for intestinal epithelial cell innate immune responses and for maintaining gut homeostasis by preventing microbial dysbiosis. Contrary to its role in epithelial cell inflammasome-mediated responses, we recently showed that Nlrp6 in gut epithelial cells exacerbates GVHD in a manner independent of the inflammasome or gut microbiota. However, donor allogeneic T cells are also critical for GVHD development, yet, the function of Nlrp6 in allogeneic T cells is unknown. We hypothesized that Nlrp6 deficient donor T cells would ameliorate experimental GVHD.To test our hypothesis, WT-BALB/crecipients were lethally irradiated and transplanted on day 0 with 5x10 6 bone marrow and 1.0x10 6 splenic CD90 +T cells from either syngeneic WT-BALB/c, allogeneic MHC-mismatched WT-B6 or Nlrp6 -/- donors. Contrary to our hypothesis, the survival of allogeneic recipients of Nlrp6 -/- donor T cells was significantly worse than those receiving WT-B6 T cells (p<0.05). Nlrp6 -/- donor T cells also caused greater GVHD mortality and morbidity in an MHC mismatched haploidentical B6 into B6D2F1 model (p<0.05) and an MHC mismatched B10.BR into B6 model. Similar results were obtained using B6 into BALB/c and B6 into B6D2F1 models performed at the University of Michigan, suggesting our results were not unique to local environmental factors. By contrast, GVHD severity and mortality were similar in an MHC matched multiple minor antigen mismatched B6 into C3H.sw model. Because the B6 into C3H.sw model is largely driven by CD8+ T cells whereas the previous models are mediated by both CD4+ and CD8+ T cells, we examined whether Nlrp6 separately regulates CD4+ and CD8+ T cell-mediated GVHD. In order to test this, we transplanted C3H.sw recipients as above except we infused either 1x10 6 CD4+ or CD8+ T cells from B6-WT or Nlrp6 -/- animals. GVHD severity and mortality (P<0.05) were enhanced only when Nlrp6 -/- CD4+ T cells were transplanted. These data suggested that Nlrp6 regulates allogeneic T cell responses in a subset-specific manner.To explore how Nlrp6 regulates intrinsic responses in donor T cell subsets, we tested naïve T cell proliferation in vitro after allogeneic or non-specific TCR stimulation. Consistent with the lack of increased GVHD induced by CD8+ Nlrp6 -/- donor T cells in the B6 into C3H.sw model, Nlrp6 -/- CD4+ but not CD8+ T cells proliferated more than WT-B6 CD4+ or CD8+ T cells, respectively, when stimulated with either anti-CD3/CD28 antibodies or lethally irradiated allogeneic antigen presenting cells in a mixed lymphocyte reaction. In addition, activation-induced apoptosis was decreased in Nlrp6 -/- CD4+ T cells compared to WT T cells. Importantly, Treg suppressive function was not altered in Nlrp6 -/- T cells. Therefore, increased proliferative responses and resistance to activation-induced apoptosis may have contributed to the enhanced GVHD caused by Nlrp6 -/- donor T cells.Increased Th1 and Th17 polarization is associated with worse GVHD. Because only CD4+ Nlrp6 -/- T cells enhanced GVHD, we tested whether Nlrp6 influenced T helper cell differentiation into Th1, Th17, and Th2 subsets. Consistent with our in vivo data, Th1 in vitro differentiation was enhanced in Nlrp6 -/- CD4+ T cells. To determine the molecular signaling events altered by Nlrp6 deficiency, we tested various T cell activation signaling pathways and found that phosphorylation of ZAP-70 was increased in Nlrp6 -/- T cells. These data suggested that Nlrp6 in donor T cells may regulate allo-immune responses via ZAP-70 pathway.GVH and graft-versus-tumor (GVT) responses are intricately linked. Because CD8+ responses were not affected by Nlrp6 deficiency, we hypothesized that GVT responses would be unaltered in Nlrp6 -/- donor T cells. Indeed, Nlrp6 -/- T cells showed equivalent in vivo GVL responses to MLL-AF4 leukemia cells as WT-T cells. Hence Nlrp6in donor T cells is not required for GVT responses.Altogether our data suggested that Nlrp6 negatively-regulates allogeneic donor CD4+ T cell responses, possibly via negative regulation of ZAP-70 signaling, resulting in mitigation of GVHD and maintenance of robust GVT responses. DisclosuresIshizawa: AbbVie: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; Bristol Myers Squibb: Speakers Bureau; Pfizer: Research Funding; Kyowa Kirin: Consultancy; SymBio: Honoraria, Research Funding; Otsuka: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Research Funding; IQVIA: Research Funding.

Butyric and Citric Acids and Their Salts in Poultry Nutrition: Effects on Gut Health and Intestinal Microbiota.

Intestinal dysfunction of farm animals, such as intestinal inflammation and altered gut microbiota, is the critical problem affecting animal welfare, performance and farm profitability. China has prohibited the use of antibiotics to improve feed efficiency and growth performance for farm animals, including poultry, in 2020. With the advantages of maintaining gut homeostasis, enhancing digestion, and absorption and modulating gut microbiota, organic acids are regarded as promising antibiotic alternatives. Butyric and citric acids as presentative organic acids positively impact growth performance, welfare, and intestinal health of livestock mainly by reducing pathogenic bacteria and maintaining the gastrointestinal tract (GIT) pH. This review summarizes the discovery of butyric acid (BA), citric acid (CA) and their salt forms, molecular structure and properties, metabolism, biological functions and their applications in poultry nutrition. The research findings about BA, CA and their salts on rats, pigs and humans are also briefly reviewed. Therefore, this review will fill the knowledge gaps of the scientific community and may be of great interest for poultry nutritionists, researchers and feed manufacturers about these two weak organic acids and their effects on intestinal health and gut microbiota community, with the hope of providing safe, healthy and nutrient-rich poultry products to consumers.

Anchor maintains gut homeostasis by restricting the JNK and Notch pathways in Drosophila

The adult Drosophila intestinal epithelium must be tightly regulated to maintain regeneration and homeostasis. The dysregulation of the regenerative capacity is frequently associated with intestinal diseases such as inflammation and tumorigenesis. Here, we showed that the G protein-coupled receptor Anchor maintains Drosophila adult midgut homeostasis by restricting Jun-N-terminal kinase (JNK) and Notch pathway activity. anchor inactivation resulted in aberrant JNK pathway activation, which led to excessive enteroblast (EB) production and premature enterocyte (EC) differentiation. In addition, increased Notch levels promoted premature EC differentiation following the loss of anchor. This defect induced by the loss of anchor ultimately caused sensitivity to stress or environmental challenge in adult flies. Taken together, our results demonstrate that the activity of anchor is essential to coordinate stem cell differentiation and proliferation to maintain intestinal homeostasis.

Tryptophan-derived serotonin-kynurenine balance in immune activation and intestinal inflammation.

Endogenous tryptophan metabolism pathways lead to the production of serotonin (5‐hydroxytryptamine; 5‐HT), kynurenine, and several downstream metabolites which are involved in a multitude of immunological functions in both health and disease states. Ingested tryptophan is largely shunted to the kynurenine pathway (95%) while only minor portions (1%–2%) are sequestered for 5‐HT production. Though often associated with the functioning of the central nervous system, significant production of 5‐HT, kynurenine and their downstream metabolites takes place within the gut. Accumulating evidence suggests that these metabolites have essential roles in regulating immune cell function, intestinal inflammation, as well as in altering the production and suppression of inflammatory cytokines. In addition, both 5‐HT and kynurenine have a considerable influence on gut microbiota suggesting that these metabolites impact host physiology both directly and indirectly via compositional changes. It is also now evident that complex interactions exist between the two pathways to maintain gut homeostasis. Alterations in 5‐HT and kynurenine are implicated in the pathogenesis of many gastrointestinal dysfunctions, including inflammatory bowel disease. Thus, these pathways present numerous potential therapeutic targets, manipulation of which may aid those suffering from gastrointestinal disorders. This review aims to update both the role of 5‐HT and kynurenine in immune regulation and intestinal inflammation, and analyze the current knowledge of the relationship and interactions between 5‐HT and kynurenine pathways.

PD-L1+ and XCR1+ dendritic cells are region-specific regulators of gut homeostasis

The intestinal mucosa constitutes an environment of closely regulated immune cells. Dendritic cells (DC) interact with the gut microbiome and antigens and are important in maintaining gut homeostasis. Here, we investigate DC transcriptome, phenotype and function in five anatomical locations of the gut lamina propria (LP) which constitute different antigenic environments. We show that DC from distinct gut LP compartments induce distinct T cell differentiation and cytokine secretion. We also find that PD-L1+ DC in the duodenal LP and XCR1+ DC in the colonic LP comprise distinct tolerogenic DC subsets that are crucial for gut homeostasis. Mice lacking PD-L1+ and XCR1+ DC have a proinflammatory gut milieu associated with an increase in Th1/Th17 cells and a decrease in Treg cells and have exacerbated disease in the models of 5-FU-induced mucositis and DSS-induced colitis. Our findings identify PD-L1+ and XCR1+ DC as region-specific physiologic regulators of intestinal homeostasis.

Generating Enteric Nervous System Progenitors from Human Pluripotent Stem Cells.

The intrinsic innervation of the gastrointestinal (GI) tract is comprised of enteric neurons and glia, which are buried within the wall of the bowel and organized into two concentric plexuses that run along the length of the gut forming the enteric nervous system (ENS). The ENS regulates vital GI functions including gut motility, blood flow, fluid secretion, and absorption and thus maintains gut homeostasis. During vertebrate development it originates predominantly from the vagal neural crest (NC), a multipotent cell population that emerges from the caudal hindbrain region, migrates to and within the gut to ultimately generate neurons and glia in response to gut-derived signals. Loss of GI innervation due to congenital or acquired defects in ENS development causes enteric neuropathies which lack curative treatment. Human pluripotent stem cells (hPSCs) offer a promising in vitro source of enteric neurons for modeling human ENS development and pathology and potential use in cell therapy applications. Here we describe in detail a differentiation strategy for the derivation of enteric neural progenitors and neurons from hPSCs through a vagal NC intermediate. Using a combination of instructive signals and retinoic acid in a dose/time dependent manner, vagal NC cells commit into the ENS lineage and develop into enteric neurons and glia upon culture in neurotrophic media. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of vagal neural crest/early ENS progenitors from hPSCs Basic Protocol 2: Differentiation of hPSC-derived vagal NC/early ENS progenitors to enteric neurons and glia.

Oroxylin A maintains the colonic mucus barrier to reduce disease susceptibility by reconstituting a dietary fiber-deprived gut microbiota.

Dietary fiber intake helps to maintain gut homeostasis. Fiber deficiency causes commensals to utilize mucins as an energy source to destroy mucus layer, thus promoting susceptibility to inflammatory bowel disease. Here, we reported that oroxylin A, a natural flavonoid, ameliorated low-grade colonic inflammation caused by fiber deficiency, alleviated colitis, and further prevented colitis-associated colon cancer in mice. The anti-inflammatory effect of oroxylin A was due to its alteration of gut microbiota. We found that the levels of Eubacterium coprostanoligenes was significantly increased by oroxylin A and the colonized Eubacterium coprostanoligenes significantly protected against colitis and carcinogenesis in colon of mice. Together, our results in this study suggest that oroxylin A may reduce the susceptibility to intestinal diseases by increasing the level of Eubacterium coprostanoligenes which could provide a therapeutic alternation for the treatment of intestinal diseases.

Indole-3-propionic Acid-aggravated CCl4-induced Liver Fibrosis via the TGF-β1/Smads Signaling Pathway.

Background and AimsThe pathogenesis of liver fibrosis involves liver damage, inflammation, oxidative stress, and intestinal dysfunction. Indole-3-propionic acid (IPA) has been demonstrated to have antioxidant, anti-inflammatory and anticancer activities, and a role in maintaining gut homeostasis. The current study aimed to investigate the role of IPA in carbon tetrachloride (CCl4)-induced liver fibrosis and explore the underlying mechanisms.MethodsThe liver fibrosis model was established in male C57BL/6 mice by intraperitoneal injection of CCl4 twice weekly. IPA intervention was made orally (20 mg/kg daily). The degree of liver injury and fibrosis were assessed by serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and histopathology. Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction (qPCR) were used to detect the inflammatory cytokines. The malondialdehyde (MDA), glutathione, glutathione peroxidase, superoxide dismutase, and catalase were determined via commercial kits. Hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. The expression of mRNA and protein was assayed by qPCR, Western blotting, or immunohistochemical staining.ResultsAfter IPA treatment, the ALT and AST, apoptotic cells, and pro-inflammatory factor levels were enhanced significantly. Moreover, IPA intervention up-regulated the expression of collagen I, α-smooth muscle actin, tissue inhibitor of matrix metalloproteinase-1, matrix metalloproteinase-2, transforming growth factor-β1 (TGF-β1), Smad3, and phosphorylated-Smad2/3. Additionally, IPA intervention did not affect the MDA level. Attractively, the administration of IPA remodeled the gut flora structure.ConclusionsIPA aggravated CCl4-induced liver damage and fibrosis by activating HSCs via the TGF-β1/Smads signaling pathway.

Investigation of the free fatty acid receptor 2 and its role in the treatment of type 2 diabetes mellitus

The free fatty acid receptor 2 (FFAR2) is a deorphanized G-protein coupled receptor (GPCR) that is widely expressed in various tissues in the body. Previous studies have shown that the short-chain fatty acids (SCFAs) acetate, butyrate and propionate activate FFAR2, leading to changes in cellular function. SCFAs are gut microbial metabolites important in shaping and maintaining gut homeostasis, as well as impacting other cell functions. FFAR2 is found in pancreatic islets and has led to investigational studies for the treatment of metabolic diseases. Our study focuses on FFAR2 as a potential treatment for type 2 diabetes mellitus (T2DM). FFAR2 contributes to glucose-stimulated insulin secretion (GSIS) and its potential in therapy has not been fully explored. This study focused on the effects of GPCR agonists targeting the deorphanized free fatty acid receptor 2 via intracellular calcium assays. We investigated the effects of numerous GPCR agonists on FFAR2, with a specific focus on identifying agonists or allosteric modulators. Further analysis and testing are underway to pinpoint specific agonists as potential treatment options for T2DM.

Defective Humoral Immunity Disrupts Bile Acid Homeostasis Which Promotes Inflammatory Disease of the Small Bowel

Abstract Mucosal antibodies maintain gut homeostasis by promoting spatial segregation between host tissues and luminal microbes. Whether and how mucosal antibody responses influence gut health through modulation of microbiota composition is unclear. One critical metabolic function carried out exclusively by bacteria in the gut is bio-transformation of host bile acids (BAs), and dysregulation of BAs metabolism has been linked to numerous inflammatory and metabolic diseases in humans. Here, we use a CD19−/− mouse model of antibody-deficiency to demonstrate that a relationship exists between dysbiosis, defects in BA homeostasis, and enteropathy of the small intestine (SI). SI enteropathy that develops in CD19−/− mice is associated with alterations to the luminal BA pool in the SI, marked by significant reductions in the abundance of conjugated BAs. Manipulation of BA availability, adoptive transfer of functional B cells, and ablation of bacterial bile salt hydrolase (bsh) activity all influence the severity of SI enteropathy in CD19−/− mice. Collectively, results from our experiments support a model whereby mucosal humoral immune responses limit inflammatory disease of the small bowel by regulating bacterial BA metabolism.

Sodium butyrate modulates chicken macrophage proteins essential for Salmonella Enteritidis invasion.

Salmonella Enteritidis is an intracellular foodborne pathogen that has developed multiple mechanisms to alter poultry intestinal physiology and infect the gut. Short chain fatty acid butyrate is derived from microbiota metabolic activities, and it maintains gut homeostasis. There is limited understanding on the interaction between S. Enteritidis infection, butyrate, and host intestinal response. To fill this knowledge gap, chicken macrophages (also known as HTC cells) were infected with S. Enteritidis, treated with sodium butyrate, and proteomic analysis was performed. A growth curve assay was conducted to determine sub-inhibitory concentration (SIC, concentration that do not affect bacterial growth compared to control) of sodium butyrate against S. Enteritidis. HTC cells were infected with S. Enteritidis in the presence and absence of SIC of sodium butyrate. The proteins were extracted and analyzed by tandem mass spectrometry. Our results showed that the SIC was 45 mM. Notably, S. Enteritidis-infected HTC cells upregulated macrophage proteins involved in ATP synthesis through oxidative phosphorylation such as ATP synthase subunit alpha (ATP5A1), ATP synthase subunit d, mitochondrial (ATP5PD) and cellular apoptosis such as Cytochrome-c (CYC). Furthermore, sodium butyrate influenced S. Enteritidis-infected HTC cells by reducing the expression of macrophage proteins mediating actin cytoskeletal rearrangements such as WD repeat-containing protein-1 (WDR1), Alpha actinin-1 (ACTN1), Vinculin (VCL) and Protein disulfide isomerase (P4HB) and intracellular S. Enteritidis growth and replication such as V-type proton ATPase catalytic subunit A (ATPV1A). Interestingly, sodium butyrate increased the expression of infected HTC cell protein involving in bacterial killing such as Vimentin (VIM). In conclusion, sodium butyrate modulates the expression of HTC cell proteins essential for S. Enteritidis invasion.

Transcriptome Analysis Revealed Ameliorative Effects of Bacillus Based Probiotic on Immunity, Gut Barrier System, and Metabolism of Chicken under an Experimentally Induced Eimeria tenella Infection

In this study, we performed transcriptome analysis in the cecum tissues of negative control untreated non-challenged (NC), positive control untreated challenged (PC), and Bacillus subtilis (B. subtilis) fed challenged chickens (BS + ET) in order to examine the underlying potential therapeutic mechanisms of Bacillus based probiotic feeding under an experimental Eimeria tenella (E. tenella) infection. Our results for clinical parameters showed that birds in probiotic diet decreased the bloody diarrhea scores, oocyst shedding, and lesion scores compared to positive control birds. RNA-sequencing (RNA-seq) analysis revealed that in total, 2509 up-regulated and 2465 down-regulated differentially expressed genes (DEGs) were detected in the PC group versus NC group comparison. In the comparison of BS + ET group versus PC group, a total of 784 up-regulated and 493 down-regulated DEGs were found. Among them, several DEGs encoding proteins involved in immunity, gut barrier integrity, homeostasis, and metabolism were up-regulated by the treatment of probiotic. Functional analysis of DEGs also revealed that some gene ontology (GO) terms related with immunity, metabolism and cellular development were significantly affected by the exposure of probiotic. Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis showed that the DEGs in the cecum of B. subtilis-fed challenged group were mainly participated in the pathways related with immunity and gut barrier integrity, included mitogen-activated protein kinase (MAPK) signaling pathway, toll-like receptor (TLR) signaling pathway, extracellular matrix (ECM)–receptor interaction, tight junction, and so on. Taken together, these results suggest that Bacillus based probiotic modulate the immunity, maintain gut homeostasis as well as barrier system and improve chicken metabolism during E. tenella infection.

NOD2 drives early IL-33-dependent expansion of group 2 innate lymphoid cells during Crohn's disease-like ileitis.

Innate lymphoid cells (ILCs) are enriched at barrier surfaces, including the gastrointestinal tract. While most studies have focused on the balance between pathogenic group 1 ILCs (ILC1s) and protective ILC3s in maintaining gut homeostasis and during chronic intestinal inflammation, such as Crohn's disease (CD), less is known regarding ILC2s. Using an established murine model of CD-like ileitis, i.e., the SAMP1/YitFc (SAMP) mouse strain, we showed that ILC2s, compared with ILC1s and ILC3s, were increased within draining mesenteric lymph nodes and ilea of SAMP versus AKR (parental control) mice early, during the onset of disease. Gut-derived ILC2s from CD patients versus healthy controls were also increased and expanded, similarly to ILC1s, in greater proportion compared with ILC3s. Importantly, we report that the intracellular bacteria-sensing protein, nucleotide-binding oligomerization domaining-containing protein 2, encoded by Nod2, the first and strongest susceptibility gene identified for CD, promoted ILC2 expansion, which was dramatically reduced in SAMP mice lacking NOD2 and in SAMP mice raised under germ-free conditions. Furthermore, these effects occurred through a mechanism involving the IL-33/ST2 ligand-receptor pair. Collectively, our results indicate a functional link between NOD2 and ILC2s, regulated by the IL-33/ST2 axis, that mechanistically may contribute to early events leading to CD pathogenesis.

Dual oxidase enables insect gut symbiosis by mediating respiratory network formation

Most animals harbor a gut microbiota that consists of potentially pathogenic, commensal, and mutualistic microorganisms. Dual oxidase (Duox) is a well described enzyme involved in gut mucosal immunity by the production of reactive oxygen species (ROS) that antagonizes pathogenic bacteria and maintains gut homeostasis in insects. However, despite its nonspecific harmful activity on microorganisms, little is known about the role of Duox in the maintenance of mutualistic gut symbionts. Here we show that, in the bean bug Riptortus pedestris, Duox-dependent ROS did not directly contribute to epithelial immunity in the midgut in response to its mutualistic gut symbiont, Burkholderia insecticola Instead, we found that the expression of Duox is tracheae-specific and its down-regulation by RNAi results in the loss of dityrosine cross-links in the tracheal protein matrix and a collapse of the respiratory system. We further demonstrated that the establishment of symbiosis is a strong oxygen sink triggering the formation of an extensive network of tracheae enveloping the midgut symbiotic organ as well as other organs, and that tracheal breakdown by Duox RNAi provokes a disruption of the gut symbiosis. Down-regulation of the hypoxia-responsive transcription factor Sima or the regulators of tracheae formation Trachealess and Branchless produces similar phenotypes. Thus, in addition to known roles in immunity and in the formation of dityrosine networks in diverse extracellular matrices, Duox is also a crucial enzyme for tracheal integrity, which is crucial to sustain mutualistic symbionts and gut homeostasis. We expect that this is a conserved function in insects.