Inflamed Gut Research Articles

Intestinal Epithelium-Derived Luminally Released Extracellular Vesicles in Sepsis Exhibit the Ability to Suppress TNF-a and IL-17A Expression in Mucosal Inflammation.

Sepsis is a systemic inflammatory disorder induced by a dysregulated immune response to infection resulting in dysfunction of multiple critical organs, including the intestines. Previous studies have reported contrasting results regarding the abilities of exosomes circulating in the blood of sepsis mice and patients to either promote or suppress inflammation. Little is known about how the gut epithelial cell-derived exosomes released in the intestinal luminal space during sepsis affect mucosal inflammation. To study this question, we isolated extracellular vesicles (EVs) from intestinal lavage of septic mice. The EVs expressed typical exosomal (CD63 and CD9) and epithelial (EpCAM) markers, which were further increased by sepsis. Moreover, septic-EV injection into inflamed gut induced a significant reduction in the messaging of pro-inflammatory cytokines TNF-α and IL-17A. MicroRNA (miRNA) profiling and reverse transcription and quantitative polymerase chain reaction (RT-qPCR) revealed a sepsis-induced exosomal increase in multiple miRNAs, which putatively target TNF-α and IL-17A. These results imply that intestinal epithelial cell (IEC)-derived luminal EVs carry miRNAs that mitigate pro-inflammatory responses. Taken together, our study proposes a novel mechanism by which IEC EVs released during sepsis transfer regulatory miRNAs to cells, possibly contributing to the amelioration of gut inflammation.

<p>All are Equal, Some are More Equal: Targeting IL 12 and 23 in IBD – A Clinical Perspective</p>

Chronic inflammatory diseases like inflammatory bowel diseases (IBD) or psoriasis represents a worldwide health burden. Researchers provided great achievements in understanding the origin of these diseases leading to improved therapeutic options. The discovery of cytokines like tumor necrosis factor-α or transforming growth factor-β are examples for these efforts. Interleukin 12 (IL 12) and interleukin 23 (IL 23) represent different important cytokines in this regard. They both belong to the interleukin 12 family and are related by sharing the subunit p40. Ustekinumab is an antibody that blocks p40 and thereby interleukins 12 and 23. Trials showed promising results in treating IBD patients with this drug. Consequently, new questions arose about the distinct features of IL 12 and 23. This review focuses on these interleukins regarding their functions in the healthy and inflamed gut and provides an overview about the results from in vitro and in vivo studies as well as clinical trials.

Thymosin alpha 1 exerts beneficial extrapulmonary effects in cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disorder caused by mutations in the gene encoding for the ion channel Cystic Fibrosis Transmembrane conductance Regulator (CFTR). Long considered a lung disease for the devastating impact on the respiratory function, the recent diagnostic and therapeutic advances have shed the light on the extra-pulmonary manifestations of CF, including gastrointestinal, hepatobiliary and pancreatic symptoms. We have previously demonstrated that thymosin alpha1 (Tα1), a naturally occurring immunomodulatory peptide, displays multi-sided beneficial effects in CF that concur in ameliorating the lung inflammatory pathology. In the present study, by resorting to murine models of gut inflammation with clinical relevance for CF patients, we demonstrate that Tα1 can also have beneficial effects in extrapulmonary pathology. Specifically, Tα1 restored barrier integrity and immune homeostasis in the inflamed gut of CF mice as well as in mice with the metabolic syndrome, a disorder that may arise in CF patients with high caloric intake despite pancreatic sufficiency. The protective effects of Tα1 also extended to pancreas and liver, further emphasizing the beneficial effects of Tα1 in extra-pulmonary complications of CF. By performing wide-ranging multi-organ anti-inflammatory effects, Tα1 could potentially integrate current therapeutic approaches to tackle the complex symptomatology of CF disease.

The Cu(II) Reductase RclA Protects Escherichia coli against the Combination of Hypochlorous Acid and Intracellular Copper.

Enterobacteria, including Escherichia coli, bloom to high levels in the gut during inflammation and strongly contribute to the pathology of inflammatory bowel diseases. To survive in the inflamed gut, E. coli must tolerate high levels of antimicrobial compounds produced by the immune system, including toxic metals like copper and reactive chlorine oxidants such as hypochlorous acid (HOCl). Here, we show that extracellular copper is a potent detoxifier of HOCl and that the widely conserved bacterial HOCl resistance enzyme RclA, which catalyzes the reduction of copper(II) to copper(I), specifically protects E. coli against damage caused by the combination of HOCl and intracellular copper. E. coli lacking RclA was highly sensitive to HOCl when grown in the presence of copper and was defective in colonizing an animal host. Our results indicate that there is unexpected complexity in the interactions between antimicrobial toxins produced by innate immune cells and that bacterial copper status is a key determinant of HOCl resistance and suggest an important and previously unsuspected role for copper redox reactions during inflammation.IMPORTANCE During infection and inflammation, the innate immune system uses antimicrobial compounds to control bacterial populations. These include toxic metals, like copper, and reactive oxidants, including hypochlorous acid (HOCl). We have now found that RclA, a copper(II) reductase strongly induced by HOCl in proinflammatory Escherichia coli and found in many bacteria inhabiting epithelial surfaces, is required for bacteria to resist killing by the combination of intracellular copper and HOCl and plays an important role in colonization of an animal host. This finding indicates that copper redox chemistry plays a critical and previously underappreciated role in bacterial interactions with the innate immune system.

Biological Therapy in Inflammatory Bowel Disease Patients Partly Restores Intestinal Innate Lymphoid Cell Subtype Equilibrium.

Patients with Crohn disease (CD) and ulcerative colitis (UC) suffer from chronic relapsing intestinal inflammation. While many studies focused on adaptive immunity, less is known about the role of innate immune cells in these diseases. Innate lymphoid cells (ILCs) are recently identified cells with a high cytokine-producing capacity at mucosal barriers. The aim was to study the impact of biological treatment on ILC in CD and UC. Patients initiating anti–tumor necrosis factor (TNF), ustekinumab, or vedolizumab treatment were prospectively followed up and peripheral and intestinal ILCs were determined. In the inflamed gut tissue of patients with inflammatory bowel disease, we found an increase of ILC1 and in immature NKp44− ILC3, whereas there was a decrease of mature NKp44+ ILC3 when compared to healthy controls (HCs). Similar but less pronounced changes in ILC1 were observed in blood, whereas circulating NKp44− ILC3 were decreased. Fifteen percent of CD patients had NKp44+ ILC3 in blood and these cells were not detected in blood of HCs or UC patients. Therapy with three different biologicals (ustekinumab targeting the IL-12/23 cytokines, anti-TNF and vedolizumab) partly restored intestinal ILC subset equilibrium with a decrease of ILC1 (except for ustekinumab) and an increase of NKp44+ ILC3. Anti-TNF also mobilized more NKp44+ ILC3 in circulation. As ILC1 are proinflammatory cells and as NKp44+ ILC3 contribute to homeostasis of intestinal mucosa, the observed effects of biologicals on ILCs might contribute to their clinical efficacy.

Translational research into the effects of cigarette smoke on inflammatory mediators and epithelial TRPV1 in Crohn's disease.

Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increased IL-8 transcription in ileum of controls (p < 0,001; n = 18-20/group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p <0,001; n = 20/group). In the colon, TRPV1 mRNA levels were decreased (p = 0,046) in smoking healthy controls (n = 20/group). Likewise, healthy mice chronically exposed to cigarette smoke (n = 10/group) showed elevated ileal Cxcl2 (p = 0,0075) and colonic Kc mRNA levels (p = 0,0186), whereas TRPV1 mRNA and protein levels were elevated in the ileum (p = 0,0315). Although cigarette smoke exposure prior to trinitrobenzene sulphonic acid administration did not alter disease activity, increased pro-inflammatory cytokine production was observed in the distal colon (Kc: p = 0,0273; Cxcl2: p = 0,104; Il1-β: p = 0,0796), in parallel with the increase of Trpv1 mRNA (p < 0,001). We infer that CS affects pro-inflammatory cytokine expression in healthy and inflamed gut, and that the simultaneous modulation of TRPV1 may point to a potential involvement of TRPV1 in cigarette smoke-induced production of inflammatory mediators.

Growth Hormone Reprograms Macrophages toward an Anti-Inflammatory and Reparative Profile in an MAFB-Dependent Manner.

Growth hormone (GH), a pleiotropic hormone secreted by the pituitary gland, regulates immune and inflammatory responses. In this study, we show that GH regulates the phenotypic and functional plasticity of macrophages both in vitro and in vivo. Specifically, GH treatment of GM-CSF-primed monocyte-derived macrophages promotes a significant enrichment of anti-inflammatory genes and dampens the proinflammatory cytokine profile through PI3K-mediated downregulation of activin A and upregulation of MAFB, a critical transcription factor for anti-inflammatory polarization of human macrophages. These in vitro data correlate with improved remission of inflammation and mucosal repair during recovery in the acute dextran sodium sulfate-induced colitis model in GH-overexpressing mice. In this model, in addition to the GH-mediated effects on other immune cells, we observed that macrophages from inflamed gut acquire an anti-inflammatory/reparative profile. Overall, these data indicate that GH reprograms inflammatory macrophages to an anti-inflammatory phenotype and improves resolution during pathologic inflammatory responses.

Faecal cytokine profiling as a marker of intestinal inflammation in acutely decompensated cirrhosis.

Background & AimsGut dysbiosis and inflammation perpetuate loss of gut barrier integrity (GBI) and pathological bacterial translocation (BT) in cirrhosis, contributing to infection risk. Little is known about gut inflammation in cirrhosis and how this differs in acute decompensation (AD). We developed a novel approach to characterise intestinal immunopathology by quantifying faecal cytokines (FCs) and GBI markers.MethodsFaeces and plasma were obtained from patients with stable cirrhosis (SC; n = 16), AD (n = 47), and healthy controls (HCs; n = 31). A panel of 15 cytokines and GBI markers, including intestinal fatty-acid-binding protein-2 (FABP2), d-lactate, and faecal calprotectin (FCAL), were quantified by electrochemiluminescence/ELISA. Correlations between analytes and clinical metadata with univariate and multivariate analyses were performed.ResultsFaecal (F) IL-1β, interferon gamma, tumour necrosis factor alpha, IL-21, IL-17A/F, and IL-22 were significantly elevated in AD vs. SC (q <0.01). F-IL-23 was significantly elevated in AD vs. HC (p = 0.0007). FABP2/d-lactate were significantly increased in faeces in AD vs. SC and AD vs. HC (p <0.0001) and in plasma (p = 0.0004; p = 0.011). F-FABP2 correlated most strongly with disease severity (Spearman's rho: Child-Pugh 0.466; p <0.0001; model for end-stage liver disease 0.488; p <0.0001). FCAL correlated with plasma IL-21, IL-1β, and IL-17F only and none of the faecal analytes. F-cytokines and F-GBI markers were more accurate than plasma in discriminating AD from SC.ConclusionsFC profiling represents an innovative approach to investigating the localised intestinal cytokine micro-environment in cirrhosis. These data reveal that AD is associated with a highly inflamed and permeable gut barrier. FC profiles are very different from the classical innate-like features of systemic inflammation. There is non-specific upregulation of TH1/TH17 effector cytokines and those known to mediate intestinal barrier damage. This prevents mucosal healing in AD and further propagates BT and systemic inflammation.Lay summaryThe gut barrier is crucial in cirrhosis in preventing infection-causing bacteria that normally live in the gut from accessing the liver and other organs via the bloodstream. Herein, we characterised gut inflammation by measuring different markers in stool samples from patients at different stages of cirrhosis and comparing this to healthy people. These markers, when compared with equivalent markers usually measured in blood, were found to be very different in pattern and absolute levels, suggesting that there is significant gut inflammation in cirrhosis related to different immune system pathways to that seen outside of the gut. This provides new insights into gut-specific immune disturbances that predispose to complications of cirrhosis, and emphasises that a better understanding of the gut-liver axis is necessary to develop better targeted therapies.

High microbiota reactivity of adult human intestinal IgA requires somatic mutations.

The gut is home to the body's largest population of plasma cells. In healthy individuals, IgA is the dominating isotype, whereas patients with inflammatory bowel disease also produce high concentrations of IgG. In the gut lumen, secretory IgA binds pathogens and toxins but also the microbiota. However, the antigen specificity of IgA and IgG for the microbiota and underlying mechanisms of antibody binding to bacteria are largely unknown. Here we show that microbiota binding is a defining property of human intestinal antibodies in both healthy and inflamed gut. Some bacterial taxa were commonly targeted by different monoclonal antibodies, whereas others selectively bound single antibodies. Interestingly, individual human monoclonal antibodies from both healthy and inflamed intestines bound phylogenetically unrelated bacterial species. This microbiota cross-species reactivity did not correlate with antibody polyreactivity but was crucially dependent on the accumulation of somatic mutations. Therefore, our data suggest that a system of affinity-matured, microbiota cross-species-reactive IgA is a common aspect of SIgA-microbiota interactions in the gut.

Current, New and Future Therapeutic Targets in Inflammatory Bowel Disease: A Systematic Review

Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are chronic relapsing conditions resulting from immune system activity in a genetically predisposed individual. IBD is based on progressive damage to the inflamed gut tissue. As its pathogenesis remains unknown, recent accumulating data have demonstrated that IBD is a complex and multi-factorial disorder correlated with host luminal factors, which lead to an imbalance between pro- and anti-inflammatory signaling. The growing understanding of the molecular mechanisms responsible for IBD has suggested a wide range of potential therapeutic targets to treat this condition. Some patients do not have a satisfactory response to current therapeutic medications such as antitumor necrosis factor (TNF) agents, or their response decreases over time. As a result, IBD therapeutics have been changed recently, with several new agents being evaluated. The identification of various inflammatory cascades has led to forming the idea to have novel medications developed. Medications targeting Janus kinases (JAK), leukocyte trafficking Interleukin (IL) 12/23, and Sphingosine 1 phosphate (S1P) are among these newly developed medications and highlight the role of microbial-host interaction in inflammation as a safe promising strategy. This systematic review aims to summarize different molecular targeting therapeutics, the most potent candidates for IBD treatment in recent studies.

The Intermucosal Connection between the Mouth and Gut in Commensal Pathobiont-Driven Colitis

The precise mechanism by which oral infection contributes to the pathogenesis of extra-oral diseases remains unclear. Here, we report that periodontal inflammation exacerbates gut inflammation invivo. Periodontitis leads to expansion of oral pathobionts, including Klebsiella and Enterobacter species, in the oral cavity. Amassed oral pathobionts are ingested and translocate to the gut, where they activate the inflammasome in colonic mononuclear phagocytes, triggering inflammation. In parallel, periodontitis results in generation of oral pathobiont-reactive Th17 cells in the oral cavity. Oral pathobiont-reactive Th17 cells are imprinted with gut tropism and migrate to the inflamed gut. When in the gut, Th17 cells of oral origin can be activated by translocated oral pathobionts and cause development of colitis, but they are not activated by gut-resident microbes. Thus, oral inflammation, such as periodontitis, exacerbates gut inflammation by supplying the gut with both colitogenic pathobionts and pathogenic Tcells.

Clostridioides difficile Senses and Hijacks Host Heme for Incorporation into an Oxidative Stress Defense System

Clostridioides difficile infection of the colon leads to severe inflammation and damage to the gastrointestinal epithelium due to the production of potent toxins. This inflammatory tissue damage causes the liberation of high concentrations of host heme at infection sites. Here, we identify the C.difficile heme-sensing membrane protein system (HsmRA) and show that this operon induces a protective response that repurposes heme to counteract antimicrobial oxidative stress responses. HsmR senses vertebrate heme, leading to increased expression of the hsmRA operon and subsequent deployment of HsmA to capture heme and reduce redox damage caused by inflammatory mediators of protection and antibiotic therapy. Strains with inactivated hsmR or hsmA have increased sensitivity to redox-active compounds and reduced colonization persistence in a murine model of relapse C.difficile infection. These results define a mechanism exploited by C.difficile to repurpose toxic heme within the inflamed gut as a shield against antimicrobial compounds.

Risk of Severe Coronavirus Disease 2019 in Patients With Inflammatory Bowel Disease in the United States: A Multicenter Research Network Study

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Adaptogenic flower buds exert cancer preventive effects by enhancing the SCFA-producers, strengthening the epithelial tight junction complex and immune responses

Microbiome therapy has attracted a keen interest from both research and business sectors. Our lab has been applying this “second genome” platform to assess the functionality of herbal medicines with fulfilling results. In this study, we applied this platform to assess the potential cancer-preventive effects of three selected adaptogenic plants. The flower buds from these plants were used to constitute Preparations SL and FSP according to the receipts of two commonly consumed Chinese medicinal decoctions for gastrointestinal discomfort. Preparation SL contains Sophorae japonica and Lonicerae Japonicae, and Preparation FSP contains Sophorae japonica and Gardenia Jasminoides. SL and FSP extracts significantly (p < 0.001) lowered the polyp burden, as well as the expressions of oncogenic signaling molecules, such as MAPK/ERK, PI3K/AKT, and STAT3 in ApcMin/+ mice. The inflamed gut was alleviated by shifting M1 to M2 macrophage phenotypes and the associated immune cytokines. The other remarkable change was on the extracellular tight junction protein complex, where the occludin, ZO-1, ICAM-1, E-cadherin were significantly (p < 0.05) upregulated while the N-cadherin and β-catenin were downregulated in the treated mice. The above physiological changes in the gut epithelial barrier were companied with the changes in gut microbiome. The 16S Sequencing data revealed a marked decrease in the potential pathogens (especially Helicobacter species and hydrogen sulfide producing-bacteria) and the increase in beneficial bacteria (especially for species from the genera of Akkermansia, Barnesiella, Coprococcus, Lachnoclostridium, and Ruminococcus). The majority of which were the short-chain fatty acids (SCFAs) producers. Meanwhile SCFAs-sensing G protein-coupled receptors (GPCRs), including GPR41, GPR43, and GPR109a were also significantly upregulated. In a recent report, we proved that the bacteria-derived SCFAs plays an essential role to the anti-cancer effects of the mushroom polysaccharides and saponins in ApcMin/+ mice. In this study, we further demonstrated that butyrate treatment could enhance the extracellular tight junction protein complex as effective as the treatments with SL and FSP to the ApcMin/+ mice. Our findings provide strong evidence of the vital role of the SCFA-producers and their metabolites to the cancer-preventive properties of the SL and FSP preparations.

Synthesis of a novel PEGylated colon-specific azo-based 4- aminosalicylic acid prodrug.

Objective(s):4-aminosalicylic acid (4-ASA) is an isomer of mesalazine that has recently been shown to be effective against inflammatory bowel disease (IBD), and more specifically, ulcerative colitis. However, the majority of orally administered 4-ASA is readily and extensively absorbed from the stomach and small intestine, so only a small amount is transported to the colon. A mutual ester and azo prodrug of 4-ASA was synthesized with polyethylene glycol (PEG) and dimethylaniline, respectively , to overcome this issue. Materials and Methods:The 4-ASA prodrug was synthesized via a two-step process and then characterized by 1H-NMR. The stability of the prodrug was evaluated in simulated gastric fluid (pH 1.2). Furthermore, the in vitro release profiles of the drug conjugate was evaluated at pH 1.2, as well as pH 6.8 in the absence or presence of rat cecal content. Results:The prepared prodrug was stable at pH 1.2, indicating that it could be protected from the acidic environment of the stomach. Also, the results of drug release at pH 6.8 showed that the amount of 4-ASA released was 63% within 12 hr in the absence of rat cecal content, while in the presence of rat cecal content, 97% of 4-ASA was released from the prodrug in 6 hr. Conclusion:Overall, the synthesized PEGylated azo-based 4-ASA prodrug could be a potential candidate for targeted drug delivery to the inflamed gut tissue in IBD.

Fermentable Fiber Pectin Improves Intestinal Inflammation by Modulating Gut Microbial Metabolites and Inflammasome Activity

Abstract Objectives Appreciation of the therapeutic benefits of fermentable dietary fibers (FDFs) in inflammatory bowel disease (IBD) is continuously growing. Herein, we examined the effect, and underlying mechanism(s), of FDF pectin on IBD development by using a preclinical model of intestinal inflammation. Methods Chronic colitis was induced in dietary cellulose or pectin-fed WT mice by administering four weekly injections of interleukin (IL)-10 receptor neutralizing antibody. Mice were euthanized two weeks after the last injection, and colitis development was examined by gross colon appearance, serological, and histological markers. Results The control group, which received insoluble fiber cellulose, developed extensive colonic inflammation as evident via colomegaly, splenomegaly, elevated pro-inflammatory cytokines including IL-1β, and distorted colonic crypts. Relative to cellulose, the level of inflammatory cytokines and histological scores were reduced in the pectin-fed mice. To understand the mechanism(s) by which dietary pectin alleviated intestinal inflammation, we analyzed the gut microbiota composition, its metabolic products, and inflammation regulators in our model. Compared to cellulose, pectin reduced the abundance of Verrucomicrobia and elevated acetate, an immune response modulator, in the cecal content. Inline, pectin-fed mice displayed reduced colonic IL-1β and heightened expression of inflammasome component NLRC4 in colonic epithelial crypts. Moreover, the colonic level of IL-1 receptor antagonist (IL-1Ra, a natural endogenous inhibitor of IL-1β), whose expression is partly regulated via NLRC4, was also elevated in mice fed pectin. The lack of pectin-mediated protective effects in NLRC4-deficient and IL-1Ra-deficient mice indicated that the NLRC4-IL-1Ra axis executes the beneficial effects of pectin during gut inflammation. Considering that IL-1Ra restrains the pro-inflammatory activity of IL-1β, this study suggests that pectin derived metabolites promote NLRC4 signaling, which augments IL-1Ra and thus limits IL-1β mediated colonic inflammation. Conclusions Altogether, our study demonstrates that microbial metabolism, of FDFs, and NLRC4 inflammasome axis mediates the beneficial effects of pectin in the inflamed gut. Funding Sources Career Development Award [ID# 597,229] from Crohn's &amp; Colitis Foundation (CCF).

PPARα‐targeted mitochondrial bioenergetics mediate repair of intestinal barriers at the host‐microbe intersection during SIV infection

Chronic gut inflammatory diseases are associated with disruption of intestinal epithelial barriers and impaired mucosal immunity. Human immunodeficiency virus‐1 (HIV) causes depletion of mucosal CD4+ T cells early in infection and disruption of gut epithelium resulting in chronic inflammation and immunodeficiency. Although anti‐retroviral therapy (ART) is effective in suppressing viral replication, it is incapable of restoring the ‘leaky gut’ which poses an impediment for HIV cure efforts. Strategies are needed for rapid repair of the epithelium to protect intestinal microenvironments and immunity in inflamed gut. Using an in vivo non‐human primate intestinal loop model of HIV/AIDS, we identified the pathogenic mechanism underlying sustained disruption of gut epithelium and explored rapid repair of gut epithelium at the intersection of microbial metabolism. Molecular, immunological, and metabolomic analyses revealed marked loss of peroxisomal proliferator‐activated receptor alpha (PPARα) signaling, predominant impairment of mitochondrial function and epithelial disruption both in vivo and in vitro. To elucidate pathways regulating intestinal epithelial integrity, we introduced probiotic Lactobacillus plantarum into Simian immunodeficiency virus (SIV)‐inflamed intestinal lumen. Rapid recovery of the epithelium occurred within 5 hours of L. plantarum administration and independent of mucosal CD4+ T cell recovery and in the absence of ART. This intestinal barrier repair was driven by L. plantarum‐induced PPARα activation and restoration of mitochondrial structure and fatty acid beta‐oxidation. Our findings elucidate the critical role of PPARα 1) at the intersection between microbial metabolism and epithelial repair in virally‐inflamed gut and 2) as a potential mitochondrial target for restoring gut barriers in other infectious or gut inflammatory diseases. These data highlight an opportunity to investigate the evolutionary synergism between host mitochondria and gut microbes to restore mucosal health in lieu of full immune recovery.Support or Funding InformationThis study was supported by the National Institutes of Health grant R01 AI123105. KRC received a pre‐doctoral training grant F30 AI150462.

A gardian of gut epithelial barrier from inflammation: the elastase inhibitor ELAFIN

In the gastrointestinal tract (GIT), proteolytic activity is tightly regulated to prevent inappropriate and potentially harmful proteolysis. In IBD this regulation is broken and our recent work has demonstrated that elastolytic activity is significantly increased in colonic epithelium from IBD patients. We have shown that the expression of the only elastase released by epithelial cells, ELA2 is up‐regulated; and the selective elastase inhibitor ELAFIN is severely down regulated in colonic epithelium from IBD patients. We showed that oral treatments with ELAFIN‐expressing L. Lactis bacteria restored proteolytic balance in the inflamed gut and was protective against acute and chronic colitis. ELAFIN is a low‐molecular weight molecule (10‐kDa), which specifically inhibits elastases. At N‐terminal terminus, a cationic helix showed anti‐microbial activities and could interact with phospholipids. Moreover ELAFIN is also able to inhibit NFkB and AP‐1 activation but this domain is not localized on protein. Therefore, several mechanisms of action are thus possible to explain ELAFIN’s protective property.ObjectivesIf ELAFIN‐recombinant L. lactis has to be considered as a possible treatment for IBD in human, there is an absolute need to define the mechanisms by which ELAFIN delivery protects against intestinal inflammation.MethodsWe generated and expressed in LAB ELAFIN’s mutants allowing investigation the biological properties of ELAFIN individually. These different forms of ELAFIN‐recombinant LAB were co‐culture with thapsigargin (10mg/ml) and ELAFIN (107 CFU/well). After 6H, cells were additionally exposed to gentamicin (100 mg/ml) to limit bacterial development. The barrier function was measured using FITC‐dextran passage through the epithelial layer. The elastolytic activity and CXCL8 release were quantified from the culture media. Expression of inflammatory mediators was quantified by qPCR.ResultsAddition of thapsigargin for 24H on Caco2 cells induced ER‐stress and disruption of elastolytic balance. ELAFIN expression was decreased with up‐regulation of ELA2, leading to increase of elastolytic activity in medium, disclosed in IBD patients. Concomitantly, barrier function was reduced, and expression of CXCL8, CXCL10 and hBD2 was increased. Co‐culture with ELAFIN expressing‐LAB normalized all these parameters confirming anti‐inflammatory properties of ELAFIN. Neutralization of a‐helix (mutant K44D/K50G) did not modify ELAFIN’s properties. However, disruption of protease inhibitory function (mutant A62G/M63G) led to unability of ELAFIN mutant to modify permeability and inflammatory phenotype induced by exposure of intestinal epithelial cells to thapsigagin.ConclusionThis suggests that anti‐inflammatory activity of ELAFIN is based on the control of elastase activity released by epithelial cells.

Stromal Cells in the Pathogenesis of Inflammatory Bowel Disease.

Up till now, research on inflammatory bowel disease [IBD] has mainly been focused on the immune cells present in the gastrointestinal tract. However, recent insights indicate that stromal cells also play an important and significant role in IBD pathogenesis. Stromal cells in the intestines regulate both intestinal epithelial and immune cell homeostasis. Different subsets of stromal cells have been found to play a role in other inflammatory diseases [e.g. rheumatoid arthritis], and these various stromal subsets now appear to carry out also specific functions in the inflamed gut in IBD. Novel potential therapies for IBD utilize, as well as target, these pathogenic stromal cells. Injection of mesenchymal stromal cells [MSCs] into fistula tracts of Crohn’s disease patients is already approved and used in clinical settings. In this review we discuss the current knowledge of the role of stromal cells in IBD pathogenesis. We further outline recent attempts to modify the stromal compartment in IBD with agents that target or replace the pathogenic stroma.

The Landscape of Adaptive Evolution of a Gut Commensal Bacteria in Aging Mice.

Aging is a complex process, with many associated time-dependent phenotypes. The gut microbiota have long been postulated as an important factor in shaping healthy aging [1, 2]. During aging, changes in the microbiota composition occur, with taxa that are rare in adults becoming dominant in the elderly [3, 4]. Increased inflammation associated with aging is also known to modulate and be modulated by the microbiota [5]. Ecological interactions are known to affect the evolution of bacteria both invitro [6] and invivo [7], but the extent to which these and the host age-dependent inflammatory environment can alter the pattern of evolutionary change of a gut commensal lineage is still unknown [8]. Here, we provide the first genomic analysis of such evolution in cohorts of old mice, under controlled host genetics and lifestyle conditions. We find that Escherichia coli evolution when colonizing the gut of old mice significantly differs from its evolution in young mice. Evolution toward metabolic adaptation is slower in old than young mice, and mutational targets concerning stress-related functions were found specifically in the inflamed gut of old mice. Taking the genetic basis of E.coli short-term evolution as a reflection of the environment it experiences, the sequencing data indicate that aging imposes a more stressful environment to this important colonizer of the mammalian gut.