Intracellular glycogen accumulation by human gut commensals as a niche adaptation trait.

The human gut microbiota is a key contributor to host metabolism and physiology, thereby impacting in various ways on host health. This complex microbial community has developed many metabolic strategies to colonize, persist and survive in the gastrointestinal environment. In this regard, intracellular glycogen accumulation has been associated with important physiological functions in several bacterial species, including gut commensals. However, the role of glycogen storage in shaping the composition and functionality of the gut microbiota offers a novel perspective in gut microbiome research. Here, we review what is known about the enzymatic machinery and regulation of glycogen metabolism in selected enteric bacteria, while we also discuss its potential impact on colonization and adaptation to the gastrointestinal tract. Furthermore, we survey the presence of such glycogen biosynthesis pathways in gut metagenomic data to highlight the relevance of this metabolic trait in enhancing survival in the highly competitive and dynamic gut ecosystem.

Intestinal group 1 innate lymphoid cells drive macrophage-induced inflammation and endocrine defects in obesity and promote insulinemia.

Hypercaloric diets overactivate the intestinal immune system and disrupt the microbiome and epithelial cell functions, impairing glucose metabolism. The origins of this inflammatory cascade are poorly characterized. We investigated the involvement of intestinal proinflammatory group 1 innate lymphoid cells (ILC1s) in obesity progression and metabolic disruption. In obese mice, we studied longitudinally the ILC1s response to the diet and ILC1s depletion to address its role in obesity. ILC1s are required for the expansion of pro-inflammatory macrophages and ILC2s. ILC1s depletion induced the ILC3-IL-22 pathway, increasing mucin production, antimicrobial peptides, and neuroendocrine cells. These changes were translated into higher gut hormones and reduced insulinemia and adiposity. ILC1s depletion was also associated with a bloom in Akkermansia muciniphila and decreases in Bilophila spp. Intestinal-ILC1s are upstream activators of inflammatory signals, connecting immunity with the microbiome, the enteroendocrine system, and the intestinal barrier in the control of glucose metabolism and adiposity.

An IBD-associated pathobiont synergises with NSAID to promote colitis which is blocked by NLRP3 inflammasome and Caspase-8 inhibitors.

Conflicting evidence exists on the association between consumption of non-steroidal anti-inflammatory drugs (NSAIDs) and symptomatic worsening of inflammatory bowel disease (IBD). We hypothesized that the heterogeneous prevalence of pathobionts [e.g., adherent-invasive Escherichia coli (AIEC)], might explain this inconsistent NSAIDs/IBD correlation. Using IL10-/- mice, we found that NSAID aggravated colitis in AIEC-colonized animals. This was accompanied by activation of the NLRP3 inflammasome, Caspase-8, apoptosis, and pyroptosis, features not seen in mice exposed to AIEC or NSAID alone, revealing an AIEC/NSAID synergistic effect. Inhibition of NLRP3 or Caspase-8 activity ameliorated colitis, with reduction in NLRP3 inflammasome activation, cell death markers, activated T-cells and macrophages, improved histology, and increased abundance of Clostridium cluster XIVa species. Our findings provide new insights into how NSAIDs and an opportunistic gut-pathobiont can synergize to worsen IBD symptoms. Targeting the NLRP3 inflammasome or Caspase-8 could be a potential therapeutic strategy in IBD patients with gut inflammation, which is worsened by NSAIDs.

Microbial bile salt hydrolase activity influences gene expression profiles and gastrointestinal maturation in infant mice.

The mechanisms by which early microbial colonizers of the neonate influence gut development are poorly understood. Bacterial bile salt hydrolase (BSH) acts as a putative colonization factor that influences bile acid signatures and microbe-host signaling pathways and we considered whether this activity can influence infant gut development. In silico analysis of the human neonatal gut metagenome confirmed that BSH enzyme sequences are present as early as one day postpartum. Gastrointestinal delivery of cloned BSH to immature gnotobiotic mice accelerated shortening of the colon and regularized gene expression profiles, with monocolonised mice more closely resembling conventionally raised animals. In situ expression of BSH decreased markers of cell proliferation (Ki67, Hes2 and Ascl2) and strongly increased expression of ALPI, a marker of cell differentiation and barrier function. These data suggest an evolutionary paradigm whereby microbial BSH activity potentially influences bacterial colonization and in-turn benefits host gastrointestinal maturation.

Identification of Gut Bacteria such as Lactobacillus johnsonii that Disseminate to Systemic Tissues of Wild Type and MyD88-/- Mice.

In healthy hosts the gut microbiota is restricted to gut tissues by several barriers some of which require MyD88-dependent innate immune sensor pathways. Nevertheless, some gut taxa have been reported to disseminate to systemic tissues. However, the extent to which this normally occurs during homeostasis in healthy organisms is still unknown. In this study, we recovered viable gut bacteria from systemic tissues of healthy wild type (WT) and MyD88-/- mice. Shotgun metagenomic-sequencing revealed a marked increase in the relative abundance of L. johnsonii in intestinal tissues of MyD88-/- mice compared to WT mice. Lactobacillus johnsonii was detected most frequently from multiple systemic tissues and at higher levels in MyD88-/- mice compared to WT mice. Viable L. johnsonii strains were recovered from different cell types sorted from intestinal and systemic tissues of WT and MyD88-/- mice. L. johnsonii could persist in dendritic cells and may represent murine immunomodulatory endosymbionts.

Regulation of CEACAM Family Members by IBD-Associated Triggers in Intestinal Epithelial Cells, Their Correlation to Inflammation and Relevance to IBD Pathogenesis.

Carcinoembryogenic antigen cellular adhesion molecules (CEACAMs) are intercellular adhesion molecules highly expressed in intestinal epithelial cells. CEACAM1, -3, -5, -6, -7 are altered in patients suffering from colon cancer and inflammatory bowel diseases (IBD), but their role in the onset and pathogenesis of IBD is not well known. Herein, we aim to correlate CEACAM1, -3, -5, -6, -7 expression to the degree of inflammation in pediatric and adult IBD colon biopsies and to examine the regulation of CEACAMs on human intestinal epithelial cell lines (C2BBe1/HT29) by different IBD-associated triggers (cytokines, bacteria/metabolites, emulsifiers) and IBD-drugs (6-Mercaptopurine, Prednisolone, Tofacitinib). Biopsies from patients with pediatric Crohn's disease (CD) and adult ulcerative colitis (UC, active/inactive disease) showed a significant increase in CEACAM3, -5, -6 expression, while CEACAM5 expression was reduced in adult CD patients (active/inactive disease). Intestinal epithelial cells cultured with a pro-inflammatory cytokine cocktail and Adherent-invasive Escherichia coli (AIEC) showed a rapid induction of CEACAM1, -5, -7 followed by a reduced RNA and protein expression overtime and a constant expression of CEACAM3, correlating with IL-8 expression. Cells cultured with the emulsifier polysorbate-80 resulted in a significant induction of CEACAM3, -5, -6, -7 at a late time point, while SCFA treatment reduced CEACAM1, -5, -7 expression. No major alterations in expression of CEACAMs were noted on cells cultured with the commensal Escherichia coli K12 or the pathogen Salmonella typhimurium. IBD drugs, particularly Tofacitinib, significantly reduced cytokine-induced CEACAM1, -3, -5, -6, -7 expression associated with a reduced IL-8 secretion. In conclusion, we provide new evidence on the regulation of CEACAMs by different IBD-associated triggers, identifying a role of CEACAMs in IBD pathogenesis.

Inflammasome Signaling Regulates the Microbial-Neuroimmune Axis and Visceral Pain in Mice.

Interactions between the intestinal microbiota, immune system and nervous system are essential for homeostasis in the gut. Inflammasomes contribute to innate immunity and brain-gut interactions, but their role in microbiota-neuro-immune interactions is not clear. Therefore, we investigated the effect of the inflammasome on visceral pain and local and systemic neuroimmune responses after antibiotic-induced changes to the microbiota. Wild-type (WT) and caspase-1/11 deficient (Casp1 KO) mice were orally treated for 2 weeks with an antibiotic cocktail (Abx, Bacitracin A and Neomycin), followed by quantification of representative fecal commensals (by qPCR), cecal short chain fatty acids (by HPLC), pathways implicated in the gut-neuro-immune axis (by RT-qPCR, immunofluorescence staining, and flow cytometry) in addition to capsaicin-induced visceral pain responses. Abx-treatment in WT-mice resulted in an increase in colonic macrophages, central neuro-immune interactions, colonic inflammasome and nociceptive receptor gene expression and a reduction in capsaicin-induced visceral pain. In contrast, these responses were attenuated in Abx-treated Casp1 KO mice. Collectively, the data indicate an important role for the inflammasome pathway in functional and inflammatory gastrointestinal conditions where pain and alterations in microbiota composition are prominent.

Bifidobacterium breve Exopolysaccharide Blocks Dendritic Cell Maturation and Activation of CD4+ T Cells.

Exopolysaccharide (EPS) is a bacterial extracellular carbohydrate moiety which has been associated with immunomodulatory activity and host protective effects of several gut commensal bacteria. Bifidobacterium breve are early colonizers of the human gastrointestinal tract (GIT) but the role of EPS in mediating their effects on the host has not been investigated for many strains. Here, we characterized EPS production by a panel of human B. breve isolates and investigated the effect of EPS status on host immune responses using human and murine cell culture-based assay systems. We report that B. breve EPS production is heterogenous across strains and that immune responses in human THP-1 monocytes are strain-specific, but not EPS status-specific. Using wild type and isogenic EPS deficient mutants of B. breve strains UCC2003 and JCM7017 we show that EPS had strain-specific divergent effects on cytokine responses from murine bone marrow derived macrophages (BMDMs) and dendritic cells (BMDCs). The B. breve UCC2003 EPS negative (EPS-) strain increased expression of cytokine genes (Tnfa, Il6, Il12a, and Il23a) relative to untreated BMDCs and BMDCs treated with wild type strain. B. breve UCC2003 and JCM7017 EPS- strains increased expression of dendritic cell (DC) activation and maturation marker genes (Cd80, Cd83, and Cd86) relative to untreated BMDCs. Consistent with this, BMDCs co-cultured with B. breve UCC2003 and JCM7017 EPS- strains engineered to express OVA antigen activated OVA-specific OT-II CD4+ T-cells in a co-culture antigen-presentation assay while EPS proficient strains did not. Collectively, these data indicate that B. breve EPS proficient strains use EPS to prevent maturation of DCs and activation of antigen specific CD4+ T cells responses to B. breve. This study identifies a new immunomodulatory role for B. breve EPS and suggests it may be important for immune evasion of adaptive immunity by B. breve and contribute to host-microbe mutualism.

Macrophage cytokine responses to commensal Gram-positive Lactobacillus salivarius strains are TLR2-independent and Myd88-dependent.

The mechanisms through which cells of the host innate immune system distinguish commensal bacteria from pathogens are currently unclear. Toll-like receptors (TLRs) are a class of pattern recognition receptors (PRRs) expressed by host cells which recognize microbe-associated molecular patterns (MAMPs) common to both commensal and pathogenic bacteria. Of the different TLRs, TLR2/6 recognize bacterial lipopeptides and trigger cytokines responses, especially to Gram-positive and Gram-negative pathogens. We report here that TLR2 is dispensable for triggering macrophage cytokine responses to different strains of the Gram-positive commensal bacterial species Lactobacillus salivarius. The L. salivarius UCC118 strain strongly upregulated expression of the PRRs, Mincle (Clec4e), TLR1 and TLR2 in macrophages while downregulating other TLR pathways. Cytokine responses triggered by L. salivarius UCC118 were predominantly TLR2-independent but MyD88-dependent. However, macrophage cytokine responses triggered by another Gram-positive commensal bacteria, Bifidobacterium breve UCC2003 were predominantly TLR2-dependent. Thus, we report a differential requirement for TLR2-dependency in triggering macrophage cytokine responses to different commensal Gram-positive bacteria. Furthermore, TNF-α responses to the TLR2 ligand FSL-1 and L. salivarius UCC118 were partially Mincle-dependent suggesting that PRR pathways such as Mincle contribute to the recognition of MAMPs on distinct Gram-positive commensal bacteria. Ultimately, integration of signals from these different PRR pathways and other MyD88-dependent pathways may determine immune responses to commensal bacteria at the host-microbe interface.

Model for Murine Gut Colonization by Bifidobacteria.

Members of the Bifidobacterium genus are some of the earliest and most important colonizers of the human neonatal gastrointestinal tract (GIT), exerting wide-ranging effects on early development of the host. However, human isolates of bifidobacteria are very inefficient colonizers of specific-pathogen-free (SPF) mice creating a technical barrier to discovery and applied research in this area. We have developed a reproducible model to facilitate transient colonization of SPF mice with human isolates of this genus through prior depletion of the gut resident microbiota with antibiotics. This chapter outlines the technical details for performing efficient microbiota depletion with antibiotics and subsequent administration of bifidobacteria for colonization.

Diabetes influences cancer risk in patients with increased carotid atherosclerosis burden.

BACKGROUND AND AIMS: Atherosclerosis and cancer share several risk factors suggesting that at least in part their pathogenesis is sustained by common mechanisms. To investigate this relation we followed a group of subjects with carotid atherosclerosis at baseline up for malignancy development. METHODS AND RESULTS: we carried out an observational study exploring cancer incidence (study endpoint) in subjects with known carotid atherosclerosis at baseline (n = 766) without previous cancer or carotid vascular procedures. During the follow-up (160 ± 111 weeks) 24 cancer occurred, corresponding to an overall annual incidence rate of 0.11%. 10 diagnosis of cancer occurred in individuals with a carotid stenosis >50% (n = 90) whereas 14 in patients with a carotid stenosis <50% patients (n = 676) (p < 0.001). Respect to patients without cancer, diabetes was markedly more common in subjects with cancer diagnosis during the FU (37.3%vs75.0%, p < 0.001). After controlling for classic risk factors, carotid stenosis >50% (HR = 2.831, 95%CI = 1.034-5.714; p = 0.036) and diabetes (HR = 4.831, 95%CI = 1.506-15.501; p = 0.008) remained significantly associated with cancer diagnosis. CONCLUSIONS: to our knowledge this is the first study reporting a significant risk of cancer development in subjects with diabetes and high risk of cerebrovascular events, highlighting the need of a carefully clinical screening for cancer in diabetic patients with overt carotid atherosclerosis.

A role for the orphan nuclear receptor TLX in the interaction between neural precursor cells and microglia.

Microglia are an essential component of the neurogenic niche in the adult hippocampus and are involved in the control of neural precursor cell (NPC) proliferation, differentiation and the survival and integration of newborn neurons in hippocampal circuitry. Microglial and neuronal cross-talk is mediated in part by the chemokine fractalkine/chemokine (C-X3-C motif) ligand 1 (CX3CL1) released from neurons, and its receptor CX3C chemokine receptor 1 (CX3CR1) which is expressed on microglia. A disruption in this pathway has been associated with impaired neurogenesis yet the specific molecular mechanisms by which this interaction occurs remain unclear. The orphan nuclear receptor TLX (Nr2e1; homologue of the Drosophila tailless gene) is a key regulator of hippocampal neurogenesis, and we have shown that in its absence microglia exhibit a pro-inflammatory activation phenotype. However, it is unclear whether a disturbance in CX3CL1/CX3CR1 communication mediates an impairment in TLX-related pathways which may have subsequent effects on neurogenesis. To this end, we assessed miRNA expression of up- and down-stream signalling molecules of TLX in the hippocampus of mice lacking CX3CR1. Our results demonstrate that a lack of CX3CR1 is associated with altered expression of TLX and its downstream targets in the hippocampus without significantly affecting upstream regulators of TLX. Thus, TLX may be a potential participant in neural stem cell (NSC)-microglial cross-talk and may be an important target in understanding inflammatory-associated impairments in neurogenesis.

Development of an Antigen-driven Colitis Model to Study Presentation of Antigens by Antigen Presenting Cells to T Cells.

Inflammatory bowel disease (IBD) is a chronic inflammation which affects the gastrointestinal tract (GIT). One of the best ways to study the immunological mechanisms involved during the disease is the T cell transfer model of colitis. In this model, immunodeficient mice (RAG(-/-) recipients) are reconstituted with naive CD4(+) T cells from healthy wild type hosts. This model allows examination of the earliest immunological events leading to disease and chronic inflammation, when the gut inflammation perpetuates but does not depend on a defined antigen. To study the potential role of antigen presenting cells (APCs) in the disease process, it is helpful to have an antigen-driven disease model, in which a defined commensal-derived antigen leads to colitis. An antigen driven-colitis model has hence been developed. In this model OT-II CD4(+) T cells, that can recognize only specific epitopes in the OVA protein, are transferred into RAG(-/-) hosts challenged with CFP-OVA-expressing E. coli. This model allows the examination of interactions between APCs and T cells in the lamina propria.

The early activation marker CD69 regulates the expression of chemokines and CD4 T cell accumulation in intestine.

Migration of naïve and activated lymphocytes is regulated by the expression of various molecules such as chemokine receptors and ligands. CD69, the early activation marker of C-type lectin domain family, is also shown to regulate the lymphocyte migration by affecting their egress from the thymus and secondary lymphoid organs. Here, we aimed to investigate the role of CD69 in accumulation of CD4 T cells in intestine using murine models of inflammatory bowel disease. We found that genetic deletion of CD69 in mice increases the expression of the chemokines CCL-1, CXCL-10 and CCL-19 in CD4(+) T cells and/or CD4(-) cells. Efficient in vitro migration of CD69-deficient CD4 T cells toward the chemokine stimuli was the result of increased expression and/or affinity of chemokine receptors. In vivo CD69(-/-) CD4 T cells accumulate in the intestine in higher numbers than B6 CD4 T cells as observed in competitive homing assay, dextran sodium sulphate (DSS)-induced colitis and antigen-specific transfer colitis. In DSS colitis CD69(-/-) CD4 T cell accumulation in colonic lamina propria (cLP) was associated with increased expression of CCL-1, CXCL-10 and CCL-19 genes. Furthermore, treatment of DSS-administrated CD69(-/-) mice with the mixture of CCL-1, CXCL-10 and CCL-19 neutralizing Abs significantly decreased the histopathological signs of colitis. Transfer of OT-II×CD69(-/-) CD45RB(high) CD4 T cells into RAG(-/-) hosts induced CD4 T cell accumulation in cLP. This study showed CD69 as negative regulator of inflammatory responses in intestine as it decreases the expression of chemotactic receptors and ligands and reduces the accumulation of CD4 T cells in cLP during colitis.

CD69 regulates type I IFN-induced tolerogenic signals to mucosal CD4 T cells that attenuate their colitogenic potential.

CD69 is highly expressed by lymphocytes at mucosal surfaces. We aimed to investigate the role of CD69 in mucosal immune responses. The expression of CD69 by CD4 T cells isolated from the spleen, mesenteric lymph nodes, small intestinal lamina propria, and colonic lamina propria was determined in specific pathogen-free B6 and TCR transgenic animals, as well as in germ-free B6 mice. Transfer colitis was induced by transplanting RAG(-/-) mice with B6 or CD69(-/-)CD45RB(high) CD4 T cells. CD69 expression by CD4 T cells is induced by the intestinal microflora, oral delivery of specific Ag, and type I IFN (IFN-I) signals. CD4 T cells from CD69(-/-) animals produce higher amounts of the proinflammatory cytokines IFN-γ, TNF-α, and IL-21, whereas the production of TGF-ß1 is decreased. CD69-deficient CD4 T cells showed reduced potential to differentiate into Foxp3(+) regulatory T cells in vivo and in vitro. The transfer of CD69(-/-)CD45RB(high) CD4 T cells into RAG(-/-) hosts induced an accelerated colitis. Oral tolerance was impaired in CD69(-/-) and IFN-I receptor 1-deficient mice when compared with B6 and OT-II × RAG(-/-) animals. Polyinosinic-polycytidylic acid treatment of RAG(-/-) mice transplanted with B6 but not CD69(-/-) or IFN-I receptor 1-deficient CD45RB(high) CD4 T cells attenuated transfer colitis. CD69 deficiency led to the increased production of proinflammatory cytokines, reduced Foxp3(+) regulatory T cell induction, impaired oral tolerance, and more severe colitis. Hence, the activation Ag CD69 plays an important role in regulating mucosal immune responses.

Effects of the polycyclic ketone tonalide (AHTN) on some cell viability parameters and transcription of P450 and immunoregulatory genes in rainbow trout RTG-2 cells.

To assess the potential effects of environmental pollutants belonging to the musk fragrances group in the physiology of aquatic animal species, in this work we treated rainbow trout RTG-2 cells with the polycyclic ketone tonalide (AHTN) at dilutions ranging from 3.5 to 500 ng/ml. The following parameters were monitored: intracellular ATP concentration (energy production), mitochondrial membrane potential (early apoptosis marker), cell viability (vital staining with DFP), quantitative expression of genes coding for the cytochrome P450 detoxifying enzymes CYP1A1 and CYP3A27, and of genes coding for the immunoregulatory peptides IL-1ß, IL-8, TNFα, Cox-2 and TGF-ß. Obtained results showed that incubation with tonalide induced in RTG-2 cells no effects on cell viability, a slight increase of mitochondrial membrane potential activity, and a significant increase in intracellular ATP concentration. However, dramatic effects were observed in transcription levels of some tested genes, with upregulation levels of 300 and 600 times measured for TGF-ß and TNFα, respectively and of 150 times for the CYP3A27 gene. Our results show for the first time the potent effects exerted by tonalide on immunoregulatory genes of RTG-2 cells and also indicate that the measured sensitivity of RTG-2 towards tonalide was in the same range of that currently available using chemical methods. A possible use of the panel of genes we employed as a tool for the monitoring of musk fragrances in biological samples is discussed.