Paneth Cell-Derived Lysozyme Defines the Composition of Mucolytic Microbiota and the Inflammatory Tone of the Intestine.

Paneth cells are the primary source of C-type lysozyme, a ß-1,4-N-acetylmuramoylhydrolase that enzymatically processes bacterial cell walls. Paneth cells are normally present in human cecum and ascending colon, but are rarely found in descending colon and rectum; Paneth cell metaplasia in this region and aberrant lysozyme production are hallmarks of inflammatory bowel disease (IBD) pathology. Here, we examined the impact of aberrant lysozyme production in colonic inflammation. Targeted disruption of Paneth cell lysozyme (Lyz1) protected mice from experimental colitis. Lyz1-deficiency diminished intestinal immune responses to bacterial molecular patterns and resulted in the expansion of lysozyme-sensitive mucolytic bacteria, including Ruminococcus gnavus, a Crohn's disease-associated pathobiont. Ectopic lysozyme production in colonic epithelium suppressed lysozyme-sensitive bacteria and exacerbated colitis. Transfer of R. gnavus into Lyz1-/- hosts elicited a type 2 immune response, causing epithelial reprograming and enhanced anti-colitogenic capacity. In contrast, in lysozyme-intact hosts, processed R. gnavus drove pro-inflammatory responses. Thus, Paneth cell lysozyme balances intestinal anti- and pro-inflammatory responses, with implications for IBD.

Intestinal Epithelial Expression of MHCII Determines Severity of Chemical, T-Cell-Induced, and Infectious Colitis in Mice.

BACKGROUND & AIMS: Intestinal epithelial cells (IECs) provide a barrier that separates the mucosal immune system from the luminal microbiota. IECs constitutively express low levels of major histocompatibility complex (MHC) class II proteins, which are upregulated upon exposure to interferon gamma. We investigated the effects of deleting MHCII proteins specifically in mice with infectious, dextran sodium sulfate (DSS)-, and T-cell-induced colitis. METHODS: We disrupted the histocompatibility 2, class II antigen A, beta 1 gene (H2-Ab1) in IECs of C57BL/6 mice (I-AbΔIEC) or Rag1-/- mice (Rag1-/-I-AbΔIEC); we used I-AbWT mice as controls. Colitis was induced by administration of DSS, transfer of CD4+CD45RBhi T cells, or infection with Citrobacter rodentium. Colon tissues were collected and analyzed by histology, immunofluorescence, xMAP, and reverse-transcription polymerase chain reaction and organoids were generated. Microbiota (total and immunoglobulin [Ig]A-coated) in intestinal samples were analyzed by16S amplicon profiling. IgA+CD138+ plasma cells from Peyer's patches and lamina propria were analyzed by flow cytometry and IgA repertoire was determined by next-generation sequencing. RESULTS: Mice with IEC-specific loss of MHCII (I-AbΔIEC mice) developed less severe DSS- or T-cell transfer-induced colitis than control mice. Intestinal tissues from I-AbΔIEC mice had a lower proportion of IgA-coated bacteria compared with control mice, and a reduced luminal concentration of secretory IgA (SIgA) following infection with C rodentium. There was no significant difference in the mucosal IgA repertoire of I-AbΔIEC vs control mice, but opsonization of cultured C rodentium by SIgA isolated from I-AbΔIEC mice was 50% lower than that of SIgA from mAbWT mice. Fifty percent of I-AbΔIEC mice died after infection with C rodentium, compared with none of the control mice. We observed a transient but significant expansion of the pathogen in the feces of I-AbΔIEC mice compared with I-AbWT mice. CONCLUSIONS: In mice with DSS or T-cell-induced colitis, loss of MHCII from IECs reduces but does not eliminate mucosal inflammation. However, in mice with C rodentium-induced colitis, loss of MHCII reduces bacterial clearance by decreasing binding of IgA to commensal and pathogenic bacteria.

An indisputable role of NHE8 in mucosal protection.

The loss of the intestinal Na+/H+ exchanger isoform 8 (NHE8) results in an ulcerative colitis-like condition with reduction of mucin production and dysbiosis, indicating that NHE8 plays an important role in intestinal mucosal protection. The aim of this study was to investigate the potential rebalance of the altered microbiota community of NHE8-deficient mice via fecal microbiota transplantation (FMT) and feeding probiotic VSL#3. We also aimed to stimulate mucin production by sodium butyrate administration via enema. Data from 16S rRNA sequencing showed that loss of NHE8 contributes to colonic microbial dysbiosis with reduction of butyrate-producing bacteria. FMT increased bacterial adhesion in the colon in NHE8 knockout (NHE8KO) mice. Periodic-acid Schiff reagent (PAS) stain and quantitative PCR showed no changes in mucin production during FMT. In mice treated with the probiotic VSL#3, a reduction of Lactobacillus and segmented filamentous bacteria (SFB) in NHE8KO mouse colon was detected and an increase in goblet cell theca was observed. In NHE8KO mice receiving sodium butyrate (NaB), 1 mM NaB stimulated Muc2 expression without changing goblet cell theca, but 10 mM NaB induced a significant reduction of goblet cell theca without altering Muc2 expression. Furthermore, 5 mM and 10 mM NaB-treated HT29-MTX cells displayed increased apoptosis, while 0.5 mM NaB stimulated Muc2 gene expression. These data showed that loss of NHE8 leads to dysbiosis with reduction of butyrate-producing bacteria and FMT and VSL#3 failed to rebalance the microbiota in NHE8KO mice. Therefore, FMT, VSL#3, and NaB are not able to restore mucin production in the absence of NHE8 in the intestine.NEW & NOTEWORTHY Loss of Na+/H+ exchanger isoform 8 (NHE8), a Slc9 family of exchanger that contributes to sodium uptake, cell volume regulation, and intracellular pH homeostasis, resulted in dysbiosis with reduction of butyrate-producing bacteria and decrease of Muc2 production in the intestine in mice. Introducing fecal microbiota transplantation (FMT) and VSL#3 in NHE8 knockout (NHE8KO) mice failed to rebalance the microbiota in these mice. Furthermore, administration of FMT, VSL#3, and sodium butyrate was unable to restore mucin production in the absence of NHE8 in the intestine.

Transcriptional Reprogramming and Resistance to Colonic Mucosal Injury in Poly(ADP-ribose) Polymerase 1 (PARP1)-deficient Mice.

Poly(ADP-ribose) polymerases (PARPs) synthesize and bind branched polymers of ADP-ribose to acceptor proteins using NAD as a substrate and participate in the control of gene transcription and DNA repair. PARP1, the most abundant isoform, regulates the expression of proinflammatory mediator cytokines, chemokines, and adhesion molecules, and inhibition of PARP1 enzymatic activity reduced or ameliorated autoimmune diseases in several experimental models, including colitis. However, the mechanism(s) underlying the protective effects of PARP1 inhibition in colitis and the cell types in which Parp1 deletion has the most significant impact are unknown. The objective of the current study was to determine the impact of Parp1 deletion on the innate immune response to mucosal injury and on the gut microbiome composition. Parp1 deficiency was evaluated in DSS-induced colitis in WT, Parp1(-/-), Rag2(-/-), and Rag2(-/-)×Parp1(-/-) double knock-out mice. Genome-wide analysis of the colonic transcriptome and fecal 16S amplicon profiling was performed. Compared with WT, we demonstrated that Parp1(-/-) were protected from dextran-sulfate sodium-induced colitis and that this protection was associated with a dramatic transcriptional reprogramming in the colon. PARP1 deficiency was also associated with a modulation of the colonic microbiota (increases relative abundance of Clostridia clusters IV and XIVa) and a concomitant increase in the frequency of mucosal CD4(+)CD25(+) Foxp3(+) regulatory T cells. The protective effects conferred by Parp1 deletion were lost in Rag2(-/-) × Parp1(-/-) mice, highlighting the role of the adaptive immune system for full protection.

Characterization of the mycobacterial acyl-CoA carboxylase holo complexes reveals their functional expansion into amino acid catabolism.

Biotin-mediated carboxylation of short-chain fatty acid coenzyme A esters is a key step in lipid biosynthesis that is carried out by multienzyme complexes to extend fatty acids by one methylene group. Pathogenic mycobacteria have an unusually high redundancy of carboxyltransferase genes and biotin carboxylase genes, creating multiple combinations of protein/protein complexes of unknown overall composition and functional readout. By combining pull-down assays with mass spectrometry, we identified nine binary protein/protein interactions and four validated holo acyl-coenzyme A carboxylase complexes. We investigated one of these--the AccD1-AccA1 complex from Mycobacterium tuberculosis with hitherto unknown physiological function. Using genetics, metabolomics and biochemistry we found that this complex is involved in branched amino-acid catabolism with methylcrotonyl coenzyme A as the substrate. We then determined its overall architecture by electron microscopy and found it to be a four-layered dodecameric arrangement that matches the overall dimensions of a distantly related methylcrotonyl coenzyme A holo complex. Our data argue in favor of distinct structural requirements for biotin-mediated γ-carboxylation of α-ß unsaturated acid esters and will advance the categorization of acyl-coenzyme A carboxylase complexes. Knowledge about the underlying structural/functional relationships will be crucial to make the target category amenable for future biomedical applications.

Mycobacterium tuberculosis phosphoenolpyruvate carboxykinase is regulated by redox mechanisms and interaction with thioredoxin.

Tuberculosis remains a major health concern worldwide. Eradication of its causative agent, the bacterial pathogen Mycobacterium tuberculosis, is particularly challenging due to a vast reservoir of latent carriers of the disease. Despite the misleading terminology of a so-called dormant state associated with latent infections, the bacteria have to maintain basic metabolic activities. Hypoxic conditions have been widely used as an in vitro system to study this dormancy. Such studies identified a rearrangement of central carbon metabolism to exploit fermentative processes caused by the lack of oxygen. Phosphoenolpyruvate carboxykinase (Pck; EC 4.1.1.32) is the enzyme at the center of these metabolic rearrangements. Although Pck is associated with gluconeogenesis under standard growth conditions, the enzyme can catalyze the reverse reaction, supporting anaplerosis of the tricarboxylic acid cycle, under conditions leading to slowed or stopped bacterial replication. To study the mechanisms that regulate the switch between two Pck functions, we systematically investigated factors influencing the gluconeogenic and anaplerotic reaction kinetics. We demonstrate that a reducing environment, as found under hypoxia-triggered non-replicating conditions, accelerates the reaction in the anaplerotic direction. Furthermore, we identified proteins that interact with Pck. The interaction between Pck and the reduced form of mycobacterial thioredoxin, gene expression of which is increased under hypoxic conditions, also increased the Pck anaplerotic activity. We thus propose that a reducing environment and the protein-protein interaction with thioredoxin in particular enable the Pck anaplerotic function under fermentative growth conditions.

Post-translational loss of renal TRPV5 calcium channel expression, Ca(2+) wasting, and bone loss in experimental colitis.

BACKGROUND & AIMS: Dysregulated Ca(2+) homeostasis likely contributes to the etiology of inflammatory bowel disease-associated loss of bone mineral density. Experimental colitis leads to decreased expression of Klotho, a protein that supports renal Ca(2+) reabsorption by stabilizing the transient receptor potential vanilloid 5 (TRPV5) channel on the apical membrane of distal tubule epithelial cells. METHODS: Colitis was induced in mice via administration of 2,4,6-trinitrobenzenesulfonic acid (TNBS) or transfer of CD4(+)interleukin-10(-/-) and CD4(+), CD45RB(hi) T cells. We investigated changes in bone metabolism, renal processing of Ca(2+), and expression of TRPV5. RESULTS: Mice with colitis had normal serum levels of Ca(2+) and parathormone. Computed tomography analysis showed a decreased density of cortical and trabecular bone, and there was biochemical evidence for reduced bone formation and increased bone resorption. Increased fractional urinary excretion of Ca(2+) was accompanied by reduced levels of TRPV5 protein in distal convoluted tubules, with a concomitant increase in TRPV5 sialylation. In mouse renal intermedullary collecting duct epithelial (mIMCD3) cells transduced with TRPV5 adenovirus, the inflammatory cytokines tumor necrosis factor, interferon-γ, and interleukin-1ß reduced levels of TRPV5 on the cell surface, leading to its degradation. Cytomix induced interaction between TRPV5 and UBR4 (Ubiquitin recoginition 4), an E3 ubiquitin ligase; knockdown of UBR4 with small interfering RNAs prevented cytomix-induced degradation of TRPV5. The effects of cytokines on TRPV5 were not observed in cells stably transfected with membrane-bound Klotho; TRPV5 expression was preserved when colitis was induced with TNBS in transgenic mice that overexpressed Klotho or in mice with T-cell transfer colitis injected with soluble recombinant Klotho. CONCLUSIONS: After induction of colitis in mice via TNBS administration or T-cell transfer, tumor necrosis factor and interferon-γ reduced the expression and activity of Klotho, which otherwise would protect TRPV5 from hypersialylation and cytokine-induced TRPV5 endocytosis, UBR4-dependent ubiquitination, degradation, and urinary wasting of Ca(2+).

Exposure to chlorinated drinking water alters the murine fecal microbiota.

An abundant body of scientific studies and regulatory guidelines substantiates antimicrobial efficacy of freshwater chlorination ensuring drinking water safety in large populations worldwide. In contrast to the purposeful use of chlorination ensuring antimicrobial safety of drinking water, only a limited body of research has addressed the molecular impact of chlorinated drinking water exposure on the gut microbiota. Here, for the first time, we have examined the differential effects of drinking water regimens stratified by chlorination agent [inorganic (HOCl) versus chloramine (TCIC)] on the C57BL/6J murine fecal microbiota. To this end, we exposed C57BL/6J mice to chlorinated drinking water regimens followed by fecal bacterial microbiota analysis at the end of the three-week feeding period employing 16S rRNA sequencing. α-diversity was strongly reduced when comparing chlorinated versus control drinking water groups and community dissimilarities (ß-diversity) were significant between groups even when comparing HOCl and TCIC. We detected significant differences in fecal bacterial composition as a function of drinking water chlorination observable at the phylum and genus levels. Differential abundance analysis of select amplicon sequence variants (ASVs) revealed changes as a function of chlorination exposure [up: Lactobacillus ASV1; Akkermansia muciniphila ASV7; Clostridium ss1 ASV10; down: Ileibacterium valens ASV5; Desulfovibrio ASV11; Lachnospiraceae UCG-006 ASV15]. Given the established complexity of murine and human gastrointestinal microbiota and their role in health and disease, the translational relevance of the chlorination-induced changes documented by us for the first time in the fecal murine microbiota remains to be explored.

Transplant of microbiota from Crohn's disease patients to germ-free mice results in colitis.

Although the role of the intestinal microbiota in the pathogenesis of inflammatory bowel disease (IBD) is beyond debate, attempts to verify the causative role of IBD-associated dysbiosis have been limited to reports of promoting the disease in genetically susceptible mice or in chemically induced colitis. We aimed to further test the host response to fecal microbiome transplantation (FMT) from Crohn's disease patients on mucosal homeostasis in ex-germ-free (xGF) mice. We characterized and transferred fecal microbiota from healthy patients and patients with defined Crohn's ileocolitis (CD_L3) to germ-free mice and analyzed the resulting microbial and mucosal homeostasis by 16S profiling, shotgun metagenomics, histology, immunofluorescence (IF) and RNAseq analysis. We observed a markedly reduced engraftment of CD_L3 microbiome compared to healthy control microbiota. FMT from CD_L3 patients did not lead to ileitis but resulted in colitis with features consistent with CD: a discontinued pattern of colitis, more proximal colonic localization, enlarged isolated lymphoid follicles and/or tertiary lymphoid organ neogenesis, and a transcriptomic pattern consistent with epithelial reprograming and promotion of the Paneth cell-like signature in the proximal colon and immune dysregulation characteristic of CD. The observed inflammatory response was associated with persistently increased abundance of Ruminococcus gnavus, Erysipelatoclostridium ramosum, Faecalimonas umbilicate, Blautia hominis, Clostridium butyricum, and C. paraputrificum and unexpected growth of toxigenic C. difficile, which was below the detection level in the community used for inoculation. Our study provides the first evidence that the transfer of a dysbiotic community from CD patients can lead to spontaneous inflammatory changes in the colon of xGF mice and identifies a signature microbial community capable of promoting colonization of pathogenic and conditionally pathogenic bacteria.

Reduced colonic microbial diversity is associated with colitis in NHE3-deficient mice.

Chronic inflammation and enteric infections are frequently associated with epithelial Na(+)/H(+) exchange (NHE) inhibition. Alterations in electrolyte transport and in mucosal pH associated with inflammation may represent a key mechanism leading to changes in the intestinal microbial composition. NHE3 expression is essential for the maintenance of the epithelial barrier function. NHE3(-/-) mice develop spontaneous distal chronic colitis and are highly susceptible to dextran sulfate (DSS)-induced mucosal injury. Spontaneous colitis is reduced with broad-spectrum antibiotics treatment, thus highlighting the importance of the microbiota composition in NHE3 deficiency-mediated colitis. We herein characterized the colonic microbiome of wild-type (WT) and NHE3(-/-) mice housed in a conventional environment using 454 pyrosequencing. We demonstrated a significant decrease in the phylogenetic diversity of the luminal and mucosal microbiota of conventional NHE3(-/-) mice compared with WT. Rederivation of NHE3(-/-) mice from conventional to a barrier facility eliminated the signs of colitis and decreased DSS susceptibility. Reintroduction of the conventional microflora into WT and NHE3(-/-) mice from the barrier facility resulted in the restoration of the symptoms initially described in the conventional environment. Interestingly, qPCR analysis of the microbiota composition in mice kept in the barrier facility compared with reconventionalized mice showed a significant reduction of Clostridia classes IV and XIVa. Therefore, the gut microbiome plays a prominent role in the pathogenesis of colitis in NHE3(-/-) mice, and, reciprocally, NHE3 also plays a critical role in shaping the gut microbiota. NHE3 deficiency may be a critical contributor to dysbiosis observed in patients with inflammatory bowel disease.

Longitudinal Characterization of the Gut Microbiota in the Diabetic ZDSD Rat Model and Therapeutic Potential of Oligofructose.

The complex development of type 2 diabetes (T2D) creates challenges for studying the progression and treatment of the disease in animal models. A newly developed rat model of diabetes, the Zucker Diabetic Sprague Dawley (ZDSD) rat, closely parallels the progression of T2D in humans. Here, we examine the progression of T2D and associated changes in the gut microbiota in male ZDSD rats and test whether the model can be used to examine the efficacy of potential therapeutics such as prebiotics, specifically oligofructose, that target the gut microbiota. Bodyweight, adiposity, and fed/fasting blood glucose and insulin were recorded over the course of the study. Glucose and insulin tolerance tests were performed, and feces collected at 8, 16, and 24 weeks of age for short-chain fatty acids and microbiota analysis using 16s rRNA gene sequencing. At the end of 24 weeks of age, half of the rats were supplemented with 10% oligofructose and tests were repeated. We observed a transition from healthy/nondiabetic to prediabetic and overtly diabetic states, via worsened insulin and glucose tolerance and significant increases in fed/fasted glucose, followed by a significant decrease in circulating insulin. Acetate and propionate levels were significantly increased in the overt diabetic state compared to healthy and prediabetic. Microbiota analysis demonstrated alterations in the gut microbiota with shifts in alpha and beta diversity as well as alterations in specific bacterial genera in healthy compared to prediabetic and diabetic states. Oligofructose treatment improved glucose tolerance and shifted the cecal microbiota of the ZDSD rats during late-stage diabetes. These findings underscore the translational potential of ZDSD rats as a model of T2D and highlight potential gut bacteria that could impact the development of the disease or serve as a biomarker for T2D. Additionally, oligofructose treatment was able to moderately improve glucose homeostasis.

Hyperarid soil microbial community response to simulated rainfall.

The exceptionally long and protracted aridity in the Atacama Desert (AD), Chile, provides an extreme, terrestrial ecosystem that is ideal for studying microbial community dynamics under hyperarid conditions. Our aim was to characterize the temporal response of hyperarid soil AD microbial communities to ex situ simulated rainfall (5% g water/g dry soil for 4 weeks) without nutrient amendment. We conducted replicated microcosm experiments with surface soils from two previously well-characterized AD hyperarid locations near Yungay at 1242 and 1609 masl (YUN1242 and YUN1609) with distinct microbial community compositions and average soil relative humidity levels of 21 and 17%, respectively. The bacterial and archaeal response to soil wetting was evaluated by 16S rRNA gene qPCR, and amplicon sequencing. Initial YUN1242 bacterial and archaeal 16S rRNA gene copy numbers were significantly higher than for YUN1609. Over the next 4 weeks, qPCR results showed significant increases in viable bacterial abundance, whereas archaeal abundance decreased. Both communities were dominated by 10 prokaryotic phyla (Actinobacteriota, Proteobacteria, Chloroflexota, Gemmatimonadota, Firmicutes, Bacteroidota, Planctomycetota, Nitrospirota, Cyanobacteriota, and Crenarchaeota) but there were significant site differences in the relative abundances of Gemmatimonadota and Chloroflexota, and specific actinobacterial orders. The response to simulated rainfall was distinct for the two communities. The actinobacterial taxa in the YUN1242 community showed rapid changes while the same taxa in the YUN1609 community remained relatively stable until day 30. Analysis of inferred function of the YUN1242 microbiome response implied an increase in the relative abundance of known spore-forming taxa with the capacity for mixotrophy at the expense of more oligotrophic taxa, whereas the YUN1609 community retained a stable profile of oligotrophic, facultative chemolithoautotrophic and mixotrophic taxa. These results indicate that bacterial communities in extreme hyperarid soils have the capacity for growth in response to simulated rainfall; however, historic variations in long-term hyperaridity exposure produce communities with distinct putative metabolic capacities.

Corrigendum: Hyperarid soil microbial community response to simulated rainfall.

[This corrects the article DOI: 10.3389/fmicb.2023.1202266.].

Differential effects of plant-based flours on metabolic homeostasis and the gut microbiota in high-fat fed rats.

BACKGROUND: The gut microbiome is a salient contributor to the development of obesity, and diet is the greatest modifier of the gut microbiome, which highlights the need to better understand how specific diets alter the gut microbiota to impact metabolic disease. Increased dietary fiber intake shifts the gut microbiome and improves energy and glucose homeostasis. Dietary fibers are found in various plant-based flours which vary in fiber composition. However, the comparative efficacy of specific plant-based flours to improve energy homeostasis and the mechanism by which this occurs is not well characterized. METHODS: In experiment 1, obese rats were fed a high fat diet (HFD) supplemented with four different plant-based flours for 12 weeks. Barley flour (BF), oat bran (OB), wheat bran (WB), and Hi-maize amylose (HMA) were incorporated into the HFD at 5% or 10% total fiber content and were compared to a HFD control. For experiment 2, lean, chow-fed rats were switched to HFD supplemented with 10% WB or BF to determine the preventative efficacy of flour supplementation. RESULTS: In experiment 1, 10% BF and 10% WB reduced body weight and adiposity gain and increased cecal butyrate. Gut microbiota analysis of WB and BF treated rats revealed increases in relative abundance of SCFA-producing bacteria. 10% WB and BF were also efficacious in preventing HFD-induced obesity; 10% WB and BF decreased body weight and adiposity, improved glucose tolerance, and reduced inflammatory markers and lipogenic enzyme expression in liver and adipose tissue. These effects were accompanied by alterations in the gut microbiota including increased relative abundance of Lactobacillus and LachnospiraceaeUCG001, along with increased portal taurodeoxycholic acid (TDCA) in 10% WB and BF rats compared to HFD rats. CONCLUSIONS: Therapeutic and preventative supplementation with 10%, but not 5%, WB or BF improves metabolic homeostasis, which is possibly due to gut microbiome-induced alterations. Specifically, these effects are proposed to be due to increased concentrations of intestinal butyrate and circulating TDCA.

Curcumin inhibits interferon-γ signaling in colonic epithelial cells.

Curcumin (diferulolylmethane) is an anti-inflammatory phenolic compound found effective in preclinical models of inflammatory bowel diseases (IBD) and in ulcerative colitis patients. Pharmacokinetics of curcumin and its poor systemic bioavailability suggest that it targets preferentially intestinal epithelial cells. The intestinal epithelium, an essential component of the gut innate defense mechanisms, is profoundly affected by IFN-γ, which can disrupt the epithelial barrier function, prevent epithelial cell migration and wound healing, and prime epithelial cells to express major histocompatibility complex class II (MHC-II) molecules and to serve as nonprofessional antigen-presenting cells. In this report we demonstrate that curcumin inhibits IFN-γ signaling in human and mouse colonocytes. Curcumin inhibited IFN-γ-induced gene transcription, including CII-TA, MHC-II genes (HLA-DRα, HLA-DPα1, HLA-DRß1), and T cell chemokines (CXCL9, 10, and 11). Acutely, curcumin inhibited Stat1 binding to the GAS cis-element, prevented Stat1 nuclear translocation, and reduced Jak1 phosphorylation and phosphorylation of Stat1 at Tyr(701). Longer exposure to curcumin led to endocytic internalization of IFNγRα followed by lysosomal fusion and degradation. In summary, curcumin acts as an IFN-γ signaling inhibitor in colonocytes with biphasic mechanisms of action, a phenomenon that may partially account for the beneficial effects of curcumin in experimental colitis and in human IBD.

Oligofructose restores postprandial short-chain fatty acid levels during high-fat feeding.

OBJECTIVE: Obesity is associated with consumption of a Western diet low in dietary fiber, while prebiotics reduce body weight. Fiber induces short-chain fatty acid (SCFA) production, and SCFA administration is beneficial to host metabolic homeostasis. However, the role of endogenous SCFA signaling in the development of obesity is contentious. Therefore, the primary objective of this study is to evaluate the postprandial time course of SCFA production and uptake in healthy (chow-fed), Western diet-fed (high-fat diet [HFD]) obese, and oligofructose-treated HFD-fed (HFD + OFS) rats. METHODS: Male Sprague-Dawley rats were maintained on chow or HFD for 5 weeks, with or without supplementation of 10% OFS for 3 weeks. SCFAs were measured in the ileum, cecum, colon, portal vein, and vena cava at 0, 2, 4, 6, and 8 hours postprandially. RESULTS: Postprandial cecal and portal vein SCFAs were decreased in obese rats compared with lean chow controls, whereas no differences were observed in fasting SCFA concentrations. OFS supplementation increased SCFA levels in the cecum and portal vein during obesity. Butyrate levels were positively associated with portal glucagon-like peptide 1 and adiposity and with Roseburia relative abundance. CONCLUSIONS: The current study demonstrates that obesity is associated with reduced SCFA production, and that OFS supplementation increases SCFA levels. Additionally, postprandial butyrate production appears to be beneficial to host energy homeostasis.

Decreased NHE3 expression in colon cancer is associated with DNA damage, increased inflammation and tumor growth.

Dysregulation of intra- and extracellular pH in cancer contributes to extracellular matrix remodeling, favors cell migration, proliferation, and metastasis. Although the primary attention has been focused on the role of the ubiquitous Na+/H+ exchanger isoform NHE1, the role of NHE3, the predominant apical isoform in colonic surface epithelium in the pathogenesis of colon cancer has not been investigated. Here, we show that NHE3 mRNA expression is significantly reduced in colorectal cancer patients and that low NHE3 expression is associated with poorer survival. Deletion of NHE3 in ApcMin mice evaluated at 15 weeks of age (significant mortality was observed beyond this time) led to lower body weights, increased mucosal inflammation, increased colonic tumor numbers, evidence of enhanced DNA damage in tumor surface epithelium, and to significant alteration in the gut microbiota. In the absence of the inflammatory and microbial pressors, ca. 70% knockdown of NHE3 expression in SK-CO15 cells led to reduced intracellular pH, elevated apical pH, dramatic differences in their transcriptomic profile, increased susceptibility to DNA damage, increased proliferation, decreased apoptosis and reduced adhesion to extracellular matrix proteins. Our findings suggest that loss of NHE3 in the surface epithelium of colonic tumors has profound consequences for cancer progression and behavior.

Interactive Effect of Combined Intermittent and Sustained Hypoxia and High-Fat Diet on the Colonic Mucosal Microbiome and Host Gene Expression in Mice.

Purpose: Gut dysbiosis can cause cardiometabolic disease. Gut dysbiosis can be independently caused by high-fat diet (HFD) and intermittent hypoxia (IH; characterizing obstructive sleep apnea), but the interactive effect of combined intermittent and sustained hypoxia (IH+SH) (characterizing obesity hypoventilation syndrome) and HFD on gut dysbiosis is unclear. We aimed to investigate the interactive effect of a combination of IH and SH and HFD on proximal colonic microbiota and colonic gene expression pattern. Methods: Male mice (n=16) were randomly received four different combinations of diet (normal versus HFD) and oxygen conditions (normoxia versus IH+SH) for 4 weeks. Bacterial DNA and mucosal epithelial cell RNA from proximal colon were collected for analysis of adherent microbiome and host's gene expression analysis. Results: HFD during IH+SH (22.6 ± 5.73; SD) led to greater Firmicutes: Bacteroidetes ratio than HFD during normoxia (5.89 ± 1.19; p=0.029). HFD significantly decreased microbial diversity as compared to normal diet, but the addition of IH+SH to HFD mildly reversed such effects. When compared to HFD during normoxia, HFD with combination of IH+SH resulted in changes to host mucosal gene expression for apical junctional complexes and adhesion molecules. Specifically, when compared to HFD during normoxia, HFD during IH+SH led to upregulation of Claudin 2 and Syk (tight junction dysfunction and increased mucosal permeability), while the barrier promoting claudin 4 was downregulated. Conclusion: HFD during combined IH and SH causes greater gut dysbiosis and potentially adverse changes in colonic epithelial transcriptome than HFD during normoxia. The latter changes are suggestive of impaired gut barrier function.

Tumor necrosis factor and interferon-gamma down-regulate Klotho in mice with colitis.

BACKGROUND & AIMS: Klotho (KL) is an anti-inflammatory protein that protects the endothelium from nitric oxide (NO)-induced dysfunction, reduces the expression of endothelial adhesion molecules, and potentially regulates T-cell functions. KL deficiency leads to premature senescence and impaired Ca2+/Pi homeostasis, which can lead to inflammatory bowel disease (IBD)-associated osteopenia/osteoporosis. We investigated the changes in renal expression of Kl as a consequence of colitis. METHODS: We studied 3 mouse models of IBD: colitis induced by trinitrobenzene sulfonic acid, colitis induced by microflora (in gnotobiotic interleukin-10(-/-)), and colitis induced by adoptive transfer of CD4(+)CD45RB(high) T cells. Effects of the tumor necrosis factor (TNF) and interferon (IFN)-gamma on Kl expression and the activity of its promoter were examined in renal epithelial cells (mpkDCT4 and mIMCD3). RESULTS: Renal expression of Kl messenger RNA (mRNA) and protein was reduced in all 3 models of IBD. Reduced level of KL correlated with the severity of colitis; the effect was reversed by neutralizing antibodies against TNF. In vitro, TNF inhibited Kl expression, an effect potentiated by IFN-gamma. The combination of TNF and IFN-gamma increased expression of inducible nitric oxide synthase (iNOS) and increased NO production. The effect of IFN-gamma was reproduced by exposure to an NO donor and reversed by the iNOS inhibitor. In cells incubated with TNF and/or IFN-gamma, Kl mRNA stability was unaffected, whereas Kl promoter activity was reduced, indicating that these cytokines regulate Kl at the transcriptional level. CONCLUSIONS: The down-regulation of KL that occurs during inflammation might account for the extraintestinal complications such as abnormalities in bone homeostasis that occur in patients with IBD.

Dynamics of dark fermentation microbial communities in the light of lactate and butyrate production.

BACKGROUND: This study focuses on the processes occurring during the acidogenic step of anaerobic digestion, especially resulting from nutritional interactions between dark fermentation (DF) bacteria and lactic acid bacteria (LAB). Previously, we have confirmed that DF microbial communities (MCs) that fed on molasses are able to convert lactate and acetate to butyrate. The aims of the study were to recognize the biodiversity of DF-MCs able and unable to convert lactate and acetate to butyrate and to define the conditions for the transformation. RESULTS: MCs sampled from a DF bioreactor were grown anaerobically in mesophilic conditions on different media containing molasses or sucrose and/or lactate and acetate in five independent static batch experiments. The taxonomic composition (based on 16S_rRNA profiling) of each experimental MC was analysed in reference to its metabolites and pH of the digestive liquids. In the samples where the fermented media contained carbohydrates, the two main tendencies were observed: (i) a low pH (pH ≤ 4), lactate and ethanol as the main fermentation products, MCs dominated with Lactobacillus, Bifidobacterium, Leuconostoc and Fructobacillus was characterized by low biodiversity; (ii) pH in the range 5.0-6.0, butyrate dominated among the fermentation products, the MCs composed mainly of Clostridium (especially Clostridium_sensu_stricto_12), Lactobacillus, Bifidobacterium and Prevotella. The biodiversity increased with the ability to convert acetate and lactate to butyrate. The MC processing exclusively lactate and acetate showed the highest biodiversity and was dominated by Clostridium (especially Clostridium_sensu_stricto_12). LAB were reduced; other genera such as Terrisporobacter, Lachnoclostridium, Paraclostridium or Sutterella were found. Butyrate was the main metabolite and pH was 7. Shotgun metagenomic analysis of the selected butyrate-producing MCs independently on the substrate revealed C.tyrobutyricum as the dominant Clostridium species. Functional analysis confirmed the presence of genes encoding key enzymes of the fermentation routes. CONCLUSIONS: Batch tests revealed the dynamics of metabolic activity and composition of DF-MCs dependent on fermentation conditions. The balance between LAB and the butyrate producers and the pH values were shown to be the most relevant for the process of lactate and acetate conversion to butyrate. To close the knowledge gaps is to find signalling factors responsible for the metabolic shift of the DF-MCs towards lactate fermentation. Video Abstract.