Inflamed Gut Research Articles

Dual action tofacitinib-loaded PLGA nanoparticles alleviate colitis in an IBD mouse model.

Inflammatory bowel disease (IBD) affects over 7 million people worldwide and significant side effects are associated with current therapies such as tofacitinib citrate (TFC), which is linked to increased risks of malignancy and congestive heart issues. To mitigate these systemic adverse effects, localised drug delivery via nano-sized carriers to inflamed gut tissues represents a promising approach. Herein, we aimed to optimise the synthesis of nanoparticles (NPs) using a low molecular weight grade of Poly(lactic-co-glycolic acid) (PLGA) 50:50 loaded with TFC. This approach leverages the dual anti-inflammatory action of TFC and the local production of anti-inflammatory short-chain fatty acids from the degradation of PLGA by colonic gut microbiota. NPs were produced by nanoprecipitation and characterised for their drug release profile in vitro. The efficacy of the enhanced PLGA-TFC NPs was then tested in a C57BL/6 DSS colitis mouse model. The release profile of TFC from the enhanced PLGA NPs showed a 40% burst release within the first hour, followed by up to 80% drug release in the colonic environment. Notably, the degradation of PLGA by colonic gut microbiota did not significantly influence TFC release. In the mouse model, neither PLGA NPs alone nor TFC alone showed significant effects on weight loss compared to the TFC-loaded PLGA NPs, emphasising the enhanced efficacy potential of the combined formulation. Altogether, these results suggest a promising role of NP delivery systems in enhancing TFC efficacy, marking a significant step towards reducing dosage and associated side effects in IBD treatment. This study underscores the potential of PLGA-TFC NPs in providing targeted and effective therapy for IBD.

Bromodomain-Containing 4 Is a Positive Regulator of Interleukin-34 Production in the Gut.

Experimental evidence suggests that, in the inflamed gut of inflammatory bowel disease (IBD) patients, interleukin-34 (IL-34) triggers detrimental signaling pathways. Factors/mechanisms regulating IL-34 production in IBD remain poorly characterized. Bromodomain-containing 4 (BRD4), a transcriptional and epigenetic regulator, is over-expressed in IBD, and studies in cancer cells suggest that BRD4 might positively control IL-34 expression. This study aimed to assess whether, in IBD, BRD4 regulates IL-34 expression. In IBD, there was an up-regulation of both IL-34 and BRD4 compared to the controls, and the two proteins co-localized in both lamina propria mononuclear cells (LPMCs) and epithelial cells. Flow cytometry analysis of CD45+ LPMCs confirmed that the percentages of IL-34- and BRD4-co-expressing cells were significantly higher in IBD than in the controls and showed that more than 80% of the IL-34-positive CD45-LPMCs expressed BRD4. IL-34 and BRD4 were mainly expressed by T cells and macrophages. IL-34 expression was reduced in IBD LPMCs transfected with BRD4 antisense oligonucleotide and in the colons of mice with dextran sulfate sodium-induced colitis treated with JQ1, a pharmacological inhibitor of BRD4. These data indicate that BRD4 is a positive regulator of IL-34 in IBD, further supporting the pathogenic role of BRD4 in IBD-associated mucosal inflammation.

Cultured fecal microbial community and its impact as fecal microbiota transplantation treatment in mice gut inflammation

The fecal microbiome is identical to the gut microbial communities and provides an easy access to the gut microbiome. Therefore, fecal microbial transplantation (FMT) strategies have been used to alter dysbiotic gut microbiomes with healthy fecal microbiota, successfully alleviating various metabolic disorders, such as obesity, type 2 diabetes, and inflammatory bowel disease (IBD). However, the success of FMT treatment is donor-dependent and variations in gut microbes cannot be avoided. This problem may be overcome by using a cultured fecal microbiome. In this study, a human fecal microbiome was cultured using five different media; growth in brain heart infusion (BHI) media resulted in the highest microbial community cell count. The microbiome (16S rRNA) data demonstrated that the cultured microbial communities were similar to that of the original fecal sample. Therefore, the BHI-cultured fecal microbiome was selected for cultured FMT (cFMT). Furthermore, a dextran sodium sulfate (DSS)-induced mice-IBD model was used to confirm the impact of cFMT. Results showed that cFMT effectively alleviated IBD-associated symptoms, including improved gut permeability, restoration of the inflamed gut epithelium, decreased expression of pro-inflammatory cytokines (IFN-γ, TNF-α, IL-1, IL-6, IL-12, and IL-17), and increased expression of anti-inflammatory cytokines (IL-4 and IL-10). Thus, study’s findings suggest that cFMT can be a potential alternative to nFMT.Key points• In vitro fecal microbial communities were grown in a batch culture using five different media.• Fecal microbial transplantation was performed on DSS-treated mice using cultured and normal fecal microbes.• Cultured fecal microbes effectively alleviated IBD-associated symptoms.Graphical

Lactate promotes the biofilm-to-invasive-planktonic transition in Salmonella enterica serovar Typhimurium via the de novo purine pathway.

Salmonella enterica serovar Typhimurium (S. Typhimurium) infection triggers an inflammatory response that changes the concentration of metabolites in the gut impacting the luminal environment. Some of these environmental adjustments are conducive to S. Typhimurium growth, such as the increased concentrations of nitrate and tetrathionate or the reduced levels of Clostridia-produced butyrate. We recently demonstrated that S. Typhimurium can form biofilms within the host environment and respond to nitrate as a signaling molecule, enabling it to transition between sessile and planktonic states. To investigate whether S. Typhimurium utilizes additional metabolites to regulate its behavior, our study delved into the impact of inflammatory metabolites on biofilm formation. The results revealed that lactate, the most prevalent metabolite in the inflammatory environment, impedes biofilm development by reducing intracellular c-di-GMP levels, suppressing the expression of curli and cellulose, and increasing the expression of flagellar genes. A transcriptomic analysis determined that the expression of the de novo purine pathway increases during high lactate conditions, and a transposon mutagenesis genetic screen identified that PurA and PurG, in particular, play a significant role in the inhibition of curli expression and biofilm formation. Lactate also increases the transcription of the type III secretion system genes involved in tissue invasion. Finally, we show that the pyruvate-modulated two-component system BtsSR is activated in the presence of high lactate, which suggests that lactate-derived pyruvate activates BtsSR system after being exported from the cytosol. All these findings propose that lactate is an important inflammatory metabolite used by S. Typhimurium to transition from a biofilm to a motile state and fine-tune its virulence.IMPORTANCEWhen colonizing the gut, Salmonella enterica serovar Typhimurium (S. Typhimurium) adopts a dynamic lifestyle that alternates between a virulent planktonic state and a multicellular biofilm state. The coexistence of biofilm formers and planktonic S. Typhimurium in the gut suggests the presence of regulatory mechanisms that control planktonic-to-sessile transition. The signals triggering the transition of S. Typhimurium between these two lifestyles are not fully explored. In this work, we demonstrated that in the presence of lactate, the most dominant host-derived metabolite in the inflamed gut, there is a reduction of c-di-GMP in S. Typhimurium, which subsequently inhibits biofilm formation and induces the expression of its invasion machinery, motility genes, and de novo purine metabolic pathway genes. Furthermore, high levels of lactate activate the BtsSR two-component system. Collectively, this work presents new insights toward the comprehension of host metabolism and gut microenvironment roles in the regulation of S. Typhimurium biology during infection.

The lipopolysaccharide structure affects the detoxifying ability of intestinal alkaline phosphatases

Lipopolysaccharide (LPS) is one of the most potent mediators of inflammation. In swine husbandry, weaning is associated with LPS-induced intestinal inflammation, resulting in decreased growth rates due to malabsorption of nutrients by the inflamed gut. A potential strategy to treat LPS-mediated disease is administering intestinal alkaline phosphatase (IAP). The latter can detoxify lipid A, the toxic component of LPS, by removal of phosphate groups. Currently, 183 LPS O-serotypes from E. coli have been described, however, comparative experiments to elucidate functional differences between LPS serotypes are scarce. In addition, these functional differences might affect the efficacy of LPS detoxifying enzymes. Here, we evaluated the ability of four LPS serotypes (O26:B6, O55:B5, O111:B4 and O127:B8) derived from Escherichia coli to trigger the secretion of pro-inflammatory cytokines by porcine PBMCs. We also tested the ability of three commercially available IAPs to detoxify these LPS serotypes. The results show that LPS serotypes differ in their ability to trigger cytokine secretion by immune cells, especially at lower concentrations. Moreover, IAPs displayed a different detoxification efficiency of the tested serotypes. Together, this study sheds light on the impact of LPS structure on the detoxification by IAPs. Further research is however needed to elucidate the LPS serotype-specific effects and their implications for the development of novel treatment options to alleviate LPS-induced gut inflammation in weaned piglets.

Dietary Fiber's Physicochemical Properties and Gut Bacterial Dysbiosis Determine Fiber Metabolism in the Gut.

A fiber-rich diet is considered beneficial for gut health. An inflamed gut with a dysbiotic bacterial community can result in altered fiber metabolism depending on the fiber's physicochemical properties. This study examined the effect of fiber's physicochemical properties on fiber fermentation in the presence of healthy and colitis-associated bacteria. Sixteen fibers with different levels of solubility, complexity, and fermentation rate were used in in vitro fermentation with healthy human gut bacteria. Resistant maltodextrins (RMD), pectin (HMP), inulin (ChIn), and wheat bran (WB) were selected for fermentation using ulcerative colitis (UC)-associated bacteria to assess bacterial dysbiosis effect. UC-associated gut microbiota showed a significant reduction in α-and β-diversity indices compared to healthy-associated microbiota. The differences in the gut microbiota composition and diversity between the donors resulted in decreased fermentation rates with UC-associated bacteria. Fiber fermentation metabolites, short-chain fatty acids (SCFA) and gas production were significantly lower in the presence of UC-associated bacteria for all four fibers tested. Overall, we conclude that dietary fiber properties and microbial dysbiosis are influential in fiber fermentation and metabolite production in the gut.

Commensal E. coli limits Salmonella gut invasion during inflammation by producing toxin-bound siderophores in a tonB-dependent manner.

The gastrointestinal tract is densely colonized by a polymicrobial community known as the microbiota which serves as primary line of defence against pathogen invasion. The microbiota can limit gut-luminal pathogen growth at different stages of infection. This can be traced to specific commensal strains exhibiting direct or indirect protective functions. Although these mechanisms hold the potential to develop new approaches to combat enteric pathogens, they remain far from being completely described. In this study, we investigated how a mouse commensal Escherichia coli can outcompete Salmonella enterica serovar Typhimurium (S. Tm). Using a salmonellosis mouse model, we found that the commensal E. coli 8178 strain relies on a trojan horse trap strategy to limit S. Tm expansion in the inflamed gut. Combining mutants and reporter tools, we demonstrated that inflammation triggers the expression of the E. coli 8178 antimicrobial microcin H47 toxin which, when fused to salmochelin siderophores, can specifically alter S. Tm growth. This protective function was compromised upon disruption of the E. coli 8178 tonB-dependent catecholate siderophore uptake system, highlighting a previously unappreciated crosstalk between iron intake and microcin H47 activity. By identifying the genetic determinants mediating S. Tm competition, our work not only provides a better mechanistic understanding of the protective function displayed by members of the gut microbiota but also further expands the general contribution of microcins in bacterial antagonistic relationships. Ultimately, such insights can open new avenues for developing microbiota-based approaches to better control intestinal infections.

Salmonella Typhimurium expansion in the inflamed murine gut is dependent on aspartate derived from ROS-mediated microbiota lysis

Inflammation boosts the availability of electron acceptors in the intestinal lumen, creating a favorable niche for pathogenic Enterobacteriaceae. However, the mechanisms linking intestinal inflammation-mediated changes in luminal metabolites and pathogen expansion remain unclear. Here, we show that mucosal inflammation induced by Salmonella enterica serovar Typhimurium (S. Tm) infection increases intestinal levels of the amino acid aspartate. S. Tm used aspartate-ammonia lyase (aspA)-dependent fumarate respiration for growth in the murine gut only during inflammation. AspA-dependent growth advantage was abolished in the gut of germ-free mice and restored in gnotobiotic mice colonized with members of the classes Bacteroidia and Clostridia. Reactive oxygen species (ROS) produced during the host response caused lysis of commensal microbes, resulting in the release of microbiota-derived aspartate that was used by S. Tm, in concert with nitrate-dependent anaerobic respiration, to outcompete commensal Enterobacteriaceae. Our findings demonstrate the role of microbiota-derived amino acids in driving respiration-dependent S. Tm expansion during colitis.

Aryl Hydrocarbon Receptor Signalling in the Control of Gut Inflammation.

Aryl hydrocarbon receptor (AHR), a transcription factor activated by many natural and synthetic ligands, represents an important mediator of the interplay between the environment and the host's immune responses. In a healthy gut, AHR activation promotes tolerogenic signals, which help maintain mucosal homeostasis. AHR expression is defective in the inflamed gut of patients with inflammatory bowel diseases (IBD), where decreased AHR signaling is supposed to contribute to amplifying the gut tissue's destructive immune-inflammatory responses. We here review the evidence supporting the role of AHR in controlling the "physiological" intestinal inflammation and summarize the data about the therapeutic effects of AHR activators, both in preclinical mouse models of colitis and in patients with IBD.

Development of Ac2-26 Mesoporous Microparticle System as a Potential Therapeutic Agent for Inflammatory Bowel Diseases.

Inflammatory bowel diseases (IBDs) disrupt the intestinal epithelium, leading to severe chronic inflammation. Current therapies cause adverse effects and are expensive, invasive, and ineffective for most patients. Annexin A1 (AnxA1) is a pivotal endogenous anti-inflammatory and tissue repair protein in IBD. Nanostructured compounds loading AnxA1 or its active N-terminal mimetic peptides improve IBD symptomatology. To further explore their potential as a therapeutic candidate, the AnxA1 N-terminal mimetic peptide Ac2-26 was incorporated into SBA-15 ordered mesoporous silica and covered with EL30D-55 to deliver it by oral treatment into the inflamed gut. The systems SBA-Ac2-26 developed measurements revealed self-assembled rod-shaped particles, likely on the external surface of SBA-15, and 88% of peptide incorporation. SBA-15 carried the peptide Ac2-26 into cultured Raw 264.7 macrophages and Caco-2 epithelial cells. Moreover, oral administration of Eudragit-SBA-15-Ac2-26 (200 μg; once a day; for 4 days) reduced colitis clinical symptoms, inflammation, and improved epithelium recovery in mice under dextran-sodium sulfate-induced colitis. The absorption of SBA-15 in gut epithelial cells is typically low; however, the permeable inflamed barrier can enable microparticles to cross, being phagocyted by macrophages. These findings suggest that Ac2-26 is successfully delivered and binds to its receptors in both epithelial and immune cells, aligning with the clinical results. Our findings demonstrate a simple and cost-effective approach to delivering Ac2-26 orally into the inflamed gut, highlighting its potential as non-invasive IBD therapy.

Oral pathobiont Klebsiella chaperon usher pili provide site-specific adaptation for the inflamed gut mucosa

ABSTRACT The ectopic gut colonization by orally derived pathobionts has been implicated in the pathogenesis of various gastrointestinal diseases, including inflammatory bowel disease (IBD). For example, gut colonization by orally derived Klebsiella spp. has been linked to IBD in mice and humans. However, the mechanisms whereby oral pathobionts colonize extra-oral niches, such as the gut mucosa, remain largely unknown. Here, we performed a high-density transposon (Tn) screening to identify genes required for the adaptation of an oral Klebsiella strain to different mucosal sites – the oral and gut mucosae – at the steady state and during inflammation. We find that K. aerogenes, an oral pathobiont associated with both oral and gut inflammation in mice, harbors a newly identified genomic locus named “locus of colonization in the inflamed gut (LIG)” that encodes genes related to iron acquisition (Sit and Chu) and host adhesion (chaperon usher pili [CUP] system). The LIG locus is highly conserved among K. aerogenes strains, and these genes are also present in several other Klebsiella species. The Tn screening revealed that the LIG locus is required for the adaptation of K. aerogenes in its ectopic niche. In particular, we determined K. aerogenes employs a CUP system (CUP1) present in the LIG locus for colonization in the inflamed gut, but not in the oral mucosa. Thus, oral pathobionts likely exploit distinct adaptation mechanisms in their ectopically colonized intestinal niche compared to their native niche.

Development of an Inflamed High Throughput Stem-cell-based Gut Epithelium Model to Assess the Impact of Annexin A1.

Annexin A1 (ANXA1) plays a role in maintaining intestinal hemostasis, especially following mucosal inflammation. The published data about ANXA1 was derived from experimental animal models where there is an overlapping between epithelial and immune cells. There is no in vitro gut epithelial model that can assess the direct effect of ANXA1 on the gut epithelium. We developed high-throughput stem-cell-based murine epithelial cells and bacterial lipopolysaccharides (LPS) were used to induce inflammation. The impact of ANXA1 and its functional part (Ac2-26) was evaluated in the inflamed model. Intestinal integrity was assessed by the transepithelial electrical resistance (TEER), and FITC-Dextran permeability. Epithelial junction proteins were assessed using confocal microscopy and RT-qPCR. Inflammatory cytokines were evaluated by RT-qPCR and ELISA. LPS challenge mediated a damage in the epithelial cells as shown by a drop in the TEER and an increase in FITC-dextran permeability; reduced the expression of epithelial junctional proteins (Occludin, ZO-1, and Cadherin) and increased the expression of the gut leaky protein, Claudin - 2. ANXA1 and Ac2-26 treatment reduced the previous damaging effects. In addition, ANXA1 and Ac2-26 inhibited the inflammatory responses mediated by the LPS and increased the transcription of the anti-inflammatory cytokine, IL-10. ANXA1 and Ac2-26 directly protect the epithelial integrity by affecting the expression of epithelial junction and inflammatory markers. The inflamed gut model is a reliable tool to study intestinal inflammatory diseases, and to evaluate the efficacy of potential anti-inflammatory drugs and the screening of new drugs that could be candidates for inflammatory bowel disease.

Attenuation of Colitis-Induced Visceral Hypersensitivity and Pain by Selective Silencing of TRPV1-Expressing Fibers in Rat Colon.

Transient receptor potential vanilloid 1 (TRPV1) cation channels, expressed on nociceptors, are well established as key contributors to abdominal pain in inflammatory bowel disease (IBD). Previous attempts at blocking these channels have been riddled with side effects. Here, we propose a novel treatment strategy, utilizing the large pore of TRPV1 channels as a drug delivery system to selectively inhibit visceral nociceptors. We induced colitis in rats using intrarectal dinitrobenzene sulfonic acid. Visceral hypersensitivity, spontaneous pain, and responsiveness of the hind paws to noxious heat stimuli were examined before and after the intrarectal application of membrane-impermeable sodium channel blocker (QX-314) alone or together with TRPV1 channel activators or blockers. Intrarectal co-application of QX-314 with TRPV1 channel activator capsaicin significantly inhibited colitis-induced gut hypersensitivity. Furthermore, in the model of colitis, but not in naïve rats, QX-314 alone was sufficient to reverse gut hypersensitivity. The blockade of TRPV1 channels prevented this effect of QX-314. Finally, applying QX-314 alone to the inflamed gut inhibited colitis-induced ongoing pain. Selective silencing of gut nociceptors by a membrane-impermeable sodium channel blocker entering via exogenously or endogenously activated TRPV1 channels diminishes IBD-induced gut hypersensitivity. The lack of effect on naïve rats suggests a selective analgesic effect in the inflamed gut. Our results suggest that in the colitis model, TRPV1 channels are tonically active. Furthermore, our results emphasize the role of TRPV1-expressing nociceptive fibers in colitis-induced pain. These findings provide proof of concept for using charged activity blockers for the blockade of IBD-associated abdominal pain.

ATP citrate lyase (ACLY)-dependent immunometabolism in mucosal T cells drives experimental colitis in vivo

ObjectiveMucosal T cells play a major role in inflammatory bowel disease (IBD). However, their immunometabolism during intestinal inflammation is poorly understood. Due to its impact on cellular metabolism and proinflammatory...

P165 Guselkumab binding to CD64+ IL-23–producing myeloid cells enhances potency for neutralizing IL-23 signaling

Abstract Background IL-23 is implicated in the pathogenesis of inflammatory bowel disease (IBD) and myeloid cells that express FcγRI (CD64) have been identified as the primary cellular source of IL-23 in inflamed IBD gut tissue. Guselkumab (GUS) and risankizumab (RZB) are monoclonal antibodies (mAbs) specifically directed against the IL-23p19 subunit. GUS is a fully human IgG1 mAb with a native Fc region while RZB is a humanized IgG1 mAb with a mutated Fc region. Here, we evaluated CD64 and IL-23 expression in IBD patient gut biopsies, binding of GUS and RZB to CD64, and the functional consequences of CD64 binding by IL-23p19 subunit mAbs, in in vitro assays. Methods IL23A and FCGR1A (CD64) expression was analyzed from bulk and single-cell RNAseq datasets. Binding of mAbs to IFNγ-primed human monocytes, as well as binding to IL-23–secreting inflammatory monocytes and capture of endogenously secreted IL-23, were assessed by flow cytometry. Internalization of IL-23, GUS, and RZB within CD64+ macrophages was evaluated using live cell confocal imaging. Potency of GUS and RZB for inhibiting IL-23 signaling was determined in a co-culture of THP-1 (a CD64+ monocyte cell line activated to produce IL-23) and an IL-23 reporter cell line (measuring biologically active IL-23). Results Analysis of RNAseq datasets showed that FCGR1A, IL23A, and IL12B were significantly increased in inflamed versus non-inflamed IBD gut biopsies and that IL23A was predominantly expressed by FCGR1A-expressing myeloid cells. In in vitro assays GUS, but not RZB, showed Fc-mediated binding to CD64 on IFNγ-primed monocytes. CD64-bound GUS simultaneously captured IL-23 secreted from the same cells. GUS, but not RZB, bound to the surface of CD64+ macrophages and mediated internalization of IL-23 to low pH intracellular compartments. GUS and RZB demonstrated similar potency for inhibiting signaling by IL-23 present in THP-1–conditioned media. However, in a co-culture of IL-23–producing THP-1 cells with an IL-23–responsive reporter cell line, GUS demonstrated enhanced potency compared to RZB for inhibition of IL-23 signaling. Conclusion Our transcriptomic analysis supported previous observations of CD64+ myeloid cells as a key source of IL-23 production in inflamed IBD gut tissue. GUS binding to CD64 on IL-23–producing cells likely contributed to the enhanced functional potency of GUS compared to RZB for inhibition of IL-23 signaling in the co-culture assay. These in vitro data support a hypothesis for optimal enrichment of GUS in inflamed tissues where CD64+ IL-23–producing myeloid cells are increased and in proximity to IL-23–responsive lymphoid cells, enabling GUS to more potently neutralize IL-23 by targeting IL-23 at its source of production.

P017 An activity-based chemiluminescence assay targeting granzyme A for the detection of Inflammatory Bowel Diseases

Abstract Background Inflammatory Bowel Diseases (IBD) represent a pressing global health concern lacking precise diagnostic tools for accurate patient stratification. Conventional methods, such as colonoscopy are extremely invasive whilst generic inflammatory markers (such as CRP or Calprotectin), are not specific enough and pose limitations in assessing disease activity, urging the need for novel, non-invasive diagnostic approaches which can rapidly inform clinicians on the inflammatory status of a patients bowels. Methods Our study focused on developing a non-invasive chemiluminescence assay targeting active granzyme A (GzmA) in stool supernatants. Employing a multifaceted approach encompassing functional, chemical, and imaging assays on human cells and gut tissue biopsies, we discerned elevated GzmA levels predominantly secreted by CD8+ T cells in inflamed gut tissues. Results The design of GzmA-INH, a specific inhibitor for GzmA, underscored the prevalence of active extracellular GzmA and it’s role in driving inflammation in the guts - revealing promising potential as a novel IBD biomarker. Further, we synthesized GzmA-CL, a highly reactive and selective chemiluminescent reporter for GzmA. Integrating GzmA-CL into a robust antibody-coated 96-well plate assay (Figure 1a), we analyzed 30 IBD biosamples along with healthy controls. Notably, substantially higher GzmA levels were observed in IBD patients, with discernible distinctions between ulcerative colitis (UC) and Crohn’s disease (CD) (Figure 1b). These levels correlated positively with established biomarkers, including faecal calprotectin and immune cell counts. Conclusion Our investigation highlighted active extracellular GzmA as a noteworthy biomarker in gut inflammation. The rapid, selective, and sensitive nature of our chemiluminescence assay presents promising prospects for precise IBD diagnosis and advancements in personalized medicine.

Contribution of bacterial and host factors to pathogen "blooming" in a gnotobiotic mouse model for Salmonella enterica serovar Typhimurium-induced enterocolitis.

Inflammation has a pronounced impact on the intestinal ecosystem by driving an expansion of facultative anaerobic bacteria at the cost of obligate anaerobic microbiota. This pathogen "blooming" is also a hallmark of enteric Salmonella enterica serovar Typhimurium (S. Tm) infection. Here, we analyzed the contribution of bacterial and host factors to S. Tm "blooming" in a gnotobiotic mouse model for S. Tm-induced enterocolitis. Mice colonized with the Oligo-Mouse-Microbiota (OMM12), a minimal bacterial community, develop fulminant colitis by day 4 after oral infection with wild-type S. Tm but not with an avirulent mutant. Inflammation leads to a pronounced reduction in overall intestinal bacterial loads, distinct microbial community shifts, and pathogen blooming (relative abundance >50%). S. Tm mutants attenuated in inducing gut inflammation generally elicit less pronounced microbiota shifts and reduction in total bacterial loads. In contrast, S. Tm mutants in nitrate respiration, salmochelin production, and ethanolamine utilization induced strong inflammation and S. Tm "blooming." Therefore, individual Salmonella-specific inflammation-fitness factors seem to be of minor importance for competition against this minimal microbiota in the inflamed gut. Finally, we show that antibody-mediated neutrophil depletion normalized gut microbiota loads but not intestinal inflammation or microbiota shifts. This suggests that neutrophils equally reduce pathogen and commensal bacterial loads in the inflamed gut.

Gut microbiota carbon and sulfur metabolisms support Salmonella infections.

Salmonella enterica serovar Typhimurium is a pervasive enteric pathogen and ongoing global threat to public health. Ecological studies in the Salmonella impacted gut remain underrepresented in the literature, discounting microbiome mediated interactions that may inform Salmonella physiology during colonization and infection. To understand the microbial ecology of Salmonella remodeling of the gut microbiome, we performed multi-omics on fecal microbial communities from untreated and Salmonella-infected mice. Reconstructed genomes recruited metatranscriptomic and metabolomic data providing a strain-resolved view of the expressed metabolisms of the microbiome during Salmonella infection. These data informed possible Salmonella interactions with members of the gut microbiome that were previously uncharacterized. Salmonella-induced inflammation significantly reduced the diversity of genomes that recruited transcripts in the gut microbiome, yet increased transcript mapping was observed for seven members, among which Luxibacter and Ligilactobacillus transcript read recruitment was most prevalent. Metatranscriptomic insights from Salmonella and other persistent taxa in the inflamed microbiome further expounded the necessity for oxidative tolerance mechanisms to endure the host inflammatory responses to infection. In the inflamed gut lactate was a key metabolite, with microbiota production and consumption reported amongst members with detected transcript recruitment. We also showed that organic sulfur sources could be converted by gut microbiota to yield inorganic sulfur pools that become oxidized in the inflamed gut, resulting in thiosulfate and tetrathionate that support Salmonella respiration. This research advances physiological microbiome insights beyond prior amplicon-based approaches, with the transcriptionally active organismal and metabolic pathways outlined here offering intriguing intervention targets in the Salmonella-infected intestine.

Formylated Peptide Receptor-1-Mediated Gut Inflammation as a Therapeutic Target in Inflammatory Bowel Disease.

Formylated peptide receptor (FPR)-1 is a G-coupled receptor that senses foreign bacterial and host-derived mitochondrial formylated peptides (FPs), leading to innate immune system activation. We sought to investigate the role of FPR1-mediated inflammation and its potential as a therapeutic target in inflammatory bowel disease (IBD). We characterized FPR1 gene and protein expression in 8 human IBD (~1000 patients) datasets with analysis on disease subtype, mucosal inflammation, and drug response. We performed in vivo dextran-sulfate sodium (DSS) colitis in C57/BL6 FPR1 knockout mice. In ex vivo studies, we studied the role of mitochondrial FPs and pharmacological blockade of FPR1 using cyclosporin H in human peripheral blood neutrophils. Finally, we assess mitochondrial FPs as a potential mechanistic biomarker in the blood and stools of patients with IBD. Detailed in silico analysis in human intestinal biopsies showed that FPR1 is highly expressed in IBD (n = 207 IBD vs 67 non-IBD controls, P < .001), and highly correlated with gut inflammation in ulcerative colitis (UC) and Crohn's disease (CD) (both P < .001). FPR1 receptor is predominantly expressed in leukocytes, and we showed significantly higher FPR1+ve neutrophils in inflamed gut tissue section in IBD (17 CD and 24 UC; both P < .001). Further analysis in 6 independent IBD (data available under Gene Expression Omnibus accession numbers GSE59071, GSE206285, GSE73661, GSE16879, GSE92415, and GSE235970) showed an association with active gut inflammation and treatment resistance to infliximab, ustekinumab, and vedolizumab. FPR1 gene deletion is protective in murine DSS colitis with lower gut neutrophil inflammation. In the human ex vivo neutrophil system, mitochondrial FP, nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) is a potent activator of neutrophils resulting in higher CD62L shedding, CD63 expression, reactive oxygen species production, and chemotactic capacity; these effects are inhibited by cyclosporin H. We screened for mitochondrial ND6 in IBD (n = 54) using ELISA and detected ND6 in stools with median values of 2.2 gg/mL (interquartile range [IQR] 0.0-4.99; range 0-53.3) but not in blood. Stool ND6 levels, however, were not significantly correlated with paired stool calprotectin, C-reactive protein, and clinical IBD activity. Our data suggest that FPR1-mediated neutrophilic inflammation is a tractable target in IBD; however, further work is required to clarify the clinical utility of mitochondrial FPs as a potential mechanistic marker for future stratification.

Altered B-Cell Expansion and Maturation in Draining Mesenteric Lymph Nodes of Inflamed Gut in Crohn’s Disease

Altered B-Cell Expansion and Maturation in Draining Mesenteric Lymph Nodes of Inflamed Gut in Crohn’s Disease