Complement in breast milk modifies offspring gut microbiota to promote infant health.

Breastfeeding offers demonstrable benefits to newborns and infants by providing nourishment and immune protection and by shaping the gut commensal microbiota. Although it has been appreciated for decades that breast milk contains complement components, the physiological relevance of complement in breast milk remains undefined. Here, we demonstrate that weanling mice fostered by complement-deficient dams rapidly succumb when exposed to murine pathogen Citrobacter rodentium (CR), whereas pups fostered on complement-containing milk from wild-type dams can tolerate CR challenge. The complement components in breast milk were shown to directly lyse specific members of gram-positive gut commensal microbiota via a C1-dependent, antibody-independent mechanism, resulting in the deposition of the membrane attack complex and subsequent bacterial lysis. By selectively eliminating members of the commensal gut community, complement components from breast milk shape neonate and infant gut microbial composition to be protective against environmental pathogens such as CR.

Infection trains the host for microbiota-enhanced resistance to pathogens.

The microbiota shields the host against infections in a process known as colonization resistance. How infections themselves shape this fundamental process remains largely unknown. Here, we show that gut microbiota from previously infected hosts display enhanced resistance to infection. This long-term functional remodeling is associated with altered bile acid metabolism leading to the expansion of taxa that utilize the sulfonic acid taurine. Notably, supplying exogenous taurine alone is sufficient to induce this alteration in microbiota function and enhance resistance. Mechanistically, taurine potentiates the microbiota's production of sulfide, an inhibitor of cellular respiration, which is key to host invasion by numerous pathogens. As such, pharmaceutical sequestration of sulfide perturbs the microbiota's composition and promotes pathogen invasion. Together, this work reveals a process by which the host, triggered by infection, can deploy taurine as a nutrient to nourish and train the microbiota, promoting its resistance to subsequent infection.

Endocannabinoids Inhibit the Induction of Virulence in Enteric Pathogens.

Endocannabinoids are host-derived lipid hormones that fundamentally impact gastrointestinal (GI) biology. The use of cannabis and other exocannabinoids as anecdotal treatments for various GI disorders inspired the search for mechanisms by which these compounds mediate their effects, which led to the discovery of the mammalian endocannabinoid system. Dysregulated endocannabinoid signaling was linked to inflammation and the gut microbiota. However, the effects of endocannabinoids on host susceptibility to infection has not been explored. Here, we show that mice with elevated levels of the endocannabinoid 2-arachidonoyl glycerol (2-AG) are protected from enteric infection by Enterobacteriaceae pathogens. 2-AG directly modulates pathogen function by inhibiting virulence programs essential for successful infection. Furthermore, 2-AG antagonizes the bacterial receptor QseC, a histidine kinase encoded within the core Enterobacteriaceae genome that promotes the activation of pathogen-associated type three secretion systems. Taken together, our findings establish that endocannabinoids are directly sensed by bacteria and can modulate bacterial function.

Cooperative Metabolic Adaptations in the Host Can Favor Asymptomatic Infection and Select for Attenuated Virulence in an Enteric Pathogen.

Pathogen virulence exists on a continuum. The strategies that drive symptomatic or asymptomatic infections remain largely unknown. We took advantage of the concept of lethal dose 50 (LD50) to ask which component of individual non-genetic variation between hosts defines whether they survive or succumb to infection. Using the enteric pathogen Citrobacter, we found no difference in pathogen burdens between healthy and symptomatic populations. Iron metabolism-related genes were induced in asymptomatic hosts compared to symptomatic or naive mice. Dietary iron conferred complete protection without influencing pathogen burdens, even at 1000× the lethal dose of Citrobacter. Dietary iron induced insulin resistance, increasing glucose levels in the intestine that were necessary and sufficient to suppress pathogen virulence. A short course of dietary iron drove the selection of attenuated Citrobacter strains that can transmit and asymptomatically colonize naive hosts, demonstrating that environmental factors and cooperative metabolic strategies can drive conversion of pathogens toward commensalism.

Endoplasmic reticulum stress in the intestinal epithelium initiates purine metabolite synthesis and promotes Th17 cell differentiation in the gut.

Intestinal IL-17-producing T helper (Th17) cells are dependent on adherent microbes in the gut for their development. However, how microbial adherence to intestinal epithelial cells (IECs) promotes Th17 cell differentiation remains enigmatic. Here, we found that Th17 cell-inducing gut bacteria generated an unfolded protein response (UPR) in IECs. Furthermore, subtilase cytotoxin expression or genetic removal of X-box binding protein 1 (Xbp1) in IECs caused a UPR and increased Th17 cells, even in antibiotic-treated or germ-free conditions. Mechanistically, UPR activation in IECs enhanced their production of both reactive oxygen species (ROS) and purine metabolites. Treating mice with N-acetyl-cysteine or allopurinol to reduce ROS production and xanthine, respectively, decreased Th17 cells that were associated with an elevated UPR. Th17-related genes also correlated with ER stress and the UPR in humans with inflammatory bowel disease. Overall, we identify a mechanism of intestinal Th17 cell differentiation that emerges from an IEC-associated UPR.

A nonredundant role for T cell-derived interleukin 22 in antibacterial defense of colonic crypts.

Interleukin (IL)-22 is central to immune defense at barrier sites. We examined the contributions of innate lymphoid cell (ILC) and T cell-derived IL-22 during Citrobacter rodentium (C.r) infection using mice that both report Il22 expression and allow lineage-specific deletion. ILC-derived IL-22 activated STAT3 in C.r-colonized surface intestinal epithelial cells (IECs) but only temporally restrained bacterial growth. T cell-derived IL-22 induced a more robust and extensive activation of STAT3 in IECs, including IECs lining colonic crypts, and T cell-specific deficiency of IL-22 led to pathogen invasion of the crypts and increased mortality. This reflected a requirement for T cell-derived IL-22 for the expression of a host-protective transcriptomic program that included AMPs, neutrophil-recruiting chemokines, and mucin-related molecules, and it restricted IFNγ-induced proinflammatory genes. Our findings demonstrate spatiotemporal differences in the production and action of IL-22 by ILCs and T cells during infection and reveal an indispensable role for IL-22-producing T cells in the protection of the intestinal crypts.

A Dietary Fiber-Deprived Gut Microbiota Degrades the Colonic Mucus Barrier and Enhances Pathogen Susceptibility.

Despite the accepted health benefits of consuming dietary fiber, little is known about the mechanisms by which fiber deprivation impacts the gut microbiota and alters disease risk. Using a gnotobiotic mouse model, in which animals were colonized with a synthetic human gut microbiota composed of fully sequenced commensal bacteria, we elucidated the functional interactions between dietary fiber, the gut microbiota, and the colonic mucus barrier, which serves as a primary defense against enteric pathogens. We show that during chronic or intermittent dietary fiber deficiency, the gut microbiota resorts to host-secreted mucus glycoproteins as a nutrient source, leading to erosion of the colonic mucus barrier. Dietary fiber deprivation, together with a fiber-deprived, mucus-eroding microbiota, promotes greater epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. Our work reveals intricate pathways linking diet, the gut microbiome, and intestinal barrier dysfunction, which could be exploited to improve health using dietary therapeutics.

Glucuronic acid confers colonization advantage to enteric pathogens.

Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.

Diet-driven differential response of Akkermansia muciniphila modulates pathogen susceptibility.

The erosion of the colonic mucus layer by a dietary fiber-deprived gut microbiota results in heightened susceptibility to an attaching and effacing pathogen, Citrobacter rodentium. Nevertheless, the questions of whether and how specific mucolytic bacteria aid in the increased pathogen susceptibility remain unexplored. Here, we leverage a functionally characterized, 14-member synthetic human microbiota in gnotobiotic mice to deduce which bacteria and functions are responsible for the pathogen susceptibility. Using strain dropouts of mucolytic bacteria from the community, we show that Akkermansia muciniphila renders the host more vulnerable to the mucosal pathogen during fiber deprivation. However, the presence of A. muciniphila reduces pathogen load on a fiber-sufficient diet, highlighting the context-dependent beneficial effects of this mucin specialist. The enhanced pathogen susceptibility is not owing to altered host immune or pathogen responses, but is driven by a combination of increased mucus penetrability and altered activities of A. muciniphila and other community members. Our study provides novel insights into the mechanisms of how discrete functional responses of the same mucolytic bacterium either resist or enhance enteric pathogen susceptibility.

Citrobacter rodentium infection impairs dopamine metabolism and exacerbates the pathology of Parkinson's disease in mice.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder with indistinct etiology and ill-defined pathophysiology. Intestinal inflammation involved in the pathogenesis of PD, but the underlying mechanism is not fully understood. Citrobacter rodentium (C.R) is a gram-negative bacterium that can be used to induce human inflammatory bowel disease in mice. Here, we investigated whether the proinflammatory effects caused by C.R infection initiate PD-like injury and/or exacerbate PD pathology and extensively studied the underlying mechanism. Mice were gavaged once with C.R and monitored for several pathological features at 9 days post infection. The results showed that C.R delivery in mice induced IBD-like symptoms, including significant weight loss, increased fecal water content, an impaired intestinal barrier, intestinal hyperpermeability and inflammation, and intestinal microbiota disturbances. Notably, C.R infection modified dopamine (DA) metabolism in the brains of both male and female mice. Subsequently, a single high dose of MPTP or normal saline was administered at 6 days post infection. At 3 days after MPTP administration, the feces were collected for 16 S rRNA analysis, and PD-like phenotypes and mechanisms were systemically analyzed. Compared with C.R or MPTP injection alone, the injection of C.R and MPTP combined worsened behavioral performance. Moreover, such combination triggered more severe dopaminergic degeneration and glial cell overactivation in the nigrostriatal pathway of mice. Mechanistically, the combination of C.R and MPTP increased the expression of TLR4 and NF-κB p65 in the colon and striatum and upregulated proinflammatory cytokine expression. Therefore, C.R infection-induced intestinal inflammation can impair dopamine metabolism and exacerbate PD pathological processes.

TRPA1 protects mice from pathogenic Citrobacter rodentium infection via maintaining the colonic epithelial barrier function.

Transient receptor potential ankyrin 1 (TRPA1) is expressed in gastrointestinal tract and plays important roles in intestinal motility and visceral hypersensitivity. However, the potential role of TRPA1 in host defense, particularly against intestinal pathogens, is unknown. Here, we show that Trpa1 knockout mice exhibited increased susceptibility to Citrobacter rodentium infection, associated with the increased severity of diarrhea and intestinal permeability associated with the disrupted tight junctions (TJs) in colonic epithelia. We further demonstrated the expression of TRPA1 in murine colonic epithelial cells (CECs) and human epithelial Caco-2 cells both at protein level and transcription level. Using calcium imaging, TRPA1 agonists allyl isothiocyanates (AITC) and hydrogen peroxide were observed to induce a transient Ca2+ response in Caco-2 cells, respectively. Moreover, TRPA1 knockdown in Caco-2 cells resulted in the decreased expression of TJ proteins, ZO-1 and Occludin, and in the increased paracellular permeabilities and the reduced TEER values of Caco-2 monolayers in vitro. Furthermore, inhibition of TRPA1 by HC-030031 in the confluent Caco-2 cells caused the altered distribution and expression of TJ proteins, ZO-1, Occludin, and Claudin-3, and exacerbated the bacterial endotoxin lipopolysaccharide (LPS)-induced damage to these TJ proteins and actin cytoskeleton. By contrast, AITC pretreatment restored the distribution and expression of these TJ proteins in the confluent Caco-2 cells upon LPS challenge. Our results identify an unrecognized protective role of TRPA1 in host defense against an enteric bacterial pathogen by maintaining colonic epithelium barrier function, at least in part, via preserving the distribution and expression of TJ proteins in CECs.

Possible link between colonization of the gastrointestinal tract by Citrobacter rodentium in C57BL/6 mice and microbiota composition.

Colonization resistance, conferred by the host's microbiota through both direct and indirect protective actions, serves to protect the host from enteric infections. Here, we identified the specific members of the gut microbiota that impact gastrointestinal colonization by Citrobacter rodentium, a murine pathogen causing colonic crypt hyperplasia. The gut colonization levels of C. rodentium in C57BL/6 mice varied among breeding facilities, probably due to differences in microbiota composition. A comprehensive analysis of the microbiota revealed that specific members of the microbiota may influence gut colonization by C. rodentium, thus providing a potential link between the two.

Turicibacterales protect mice from severe Citrobacter rodentium infection.

One of the major contributors to child mortality in the world is diarrheal diseases, with an estimated 800,000 deaths per year. Many pathogens are causative agents of these illnesses, including the enteropathogenic or enterohemorrhagic forms of Escherichia coli. These bacteria are characterized by their ability to cause attaching and effacing lesions in the gut mucosa. Although much has been learned about the pathogenicity of these organisms and the immune response against them, the role of the intestinal microbiota during these infections is not well characterized. Infection of mice with E. coli requires pre-treatment with antibiotics in most mouse models, which hinders the study of the microbiota in an undisturbed environment. Using Citrobacter rodentium as a murine model for attaching and effacing bacteria, we show that C57BL/6 mice deficient in granzyme B expression are highly susceptible to severe disease caused by C. rodentium infection. Although a previous publication from our group shows that granzyme B-deficient CD4+ T cells are partially responsible for this phenotype, in this report, we present data demonstrating that the microbiota, in particular members of the order Turicibacterales, have an important role in conferring resistance. Mice deficient in Turicibacter sanguinis have increased susceptibility to severe disease. However, when these mice are co-housed with resistant mice or colonized with T. sanguinis, susceptibility to severe infection is reduced. These results clearly suggest a critical role for this commensal in the protection against enteropathogens.

Distinct Molecular Features of NleG Type 3 Secreted Effectors Allow for Different Roles during Citrobacter rodentium Infection in Mice.

The NleGs are the largest family of type 3 secreted effectors in attaching and effacing (A/E) pathogens, such as enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and Citrobacter rodentium. NleG effectors contain a conserved C-terminal U-box domain acting as a ubiquitin protein ligase and target host proteins via a variable N-terminal portion. The specific roles of these effectors during infection remain uncertain. Here, we demonstrate that the three NleG effectors-NleG1Cr, NleG7Cr, and NleG8Cr-encoded by C. rodentium DBS100 play distinct roles during infection in mice. Using individual nleGCr knockout strains, we show that NleG7Cr contributes to bacterial survival during enteric infection while NleG1Cr promotes the expression of diarrheal symptoms and NleG8Cr contributes to accelerated lethality in susceptible mice. Furthermore, the NleG8Cr effector contains a C-terminal PDZ domain binding motif that enables interaction with the host protein GOPC. Both the PDZ domain binding motif and the ability to engage with host ubiquitination machinery via the intact U-box domain proved to be necessary for NleG8Cr function, contributing to the observed phenotype during infection. We also establish that the PTZ binding motif in the EHEC NleG8 (NleG8Ec) effector, which shares 60% identity with NleG8Cr, is engaged in interactions with human GOPC. The crystal structure of the NleG8Ec C-terminal peptide in complex with the GOPC PDZ domain, determined to 1.85 Å, revealed a conserved interaction mode similar to that observed between GOPC and eukaryotic PDZ domain binding motifs. Despite these common features, nleG8Ec does not complement the ΔnleG8Cr phenotype during infection, revealing functional diversification between these NleG effectors.

TRAF6 signaling in dendritic cells plays protective role against infectious colitis by limiting C. rodentium infection through the induction of Th1 and Th17 responses.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) plays a pivotal role in the induction of inflammatory responses not only in innate immune cells but also in non-immune cells, leading to the activation of adaptive immunity. Signal transduction mediated by TRAF6, along with its upstream molecule MyD88 in intestinal epithelial cells (IECs) is crucial for the maintenance of mucosal homeostasis following inflammatory insult. The IEC-specific TRAF6-deficient (TRAF6ΔIEC) and MyD88-deficient (MyD88ΔIEC) mice exhibit increased susceptibility to DSS-induced colitis, emphasizing the critical role of this pathway. Moreover, MyD88 also plays a protective role in Citrobacter rodentium (C. rodentium) infection-induced colitis. However, its pathological role of TRAF6 in infectious colitis remains unclear. To investigate the site-specific roles of TRAF6 in response to enteric bacterial pathogens, we infected TRAF6ΔIEC and dendritic cell (DC)-specific TRAF6-deficient (TRAF6ΔDC) mice with C. rodentium and found that the pathology of infectious colitis was exacerbated with significantly decreased survival rates in TRAF6ΔDC mice, but not in TRAF6ΔIEC mice, compared to those in control mice. TRAF6ΔDC mice showed increased bacterial burdens, marked disruption of epithelial and mucosal structures with increased infiltration of neutrophils and macrophages, and elevated cytokine levels in the colon at the late stages of infection. The frequencies of IFN-γ producing Th1 cells and IL-17A producing Th17 cells in the colonic lamina propria were significantly reduced in TRAF6ΔDC mice. Finally, we demonstrated that TRAF6-deficient DCs failed to produce IL-12 and IL-23 in response to C. rodentium stimulation, and to induce both Th1 and Th17 cells in vitro. Thus, TRAF6 signaling in DCs, but not in IECs, protects against colitis induced by C. rodentium infection by producing IL-12 and IL-23 that induce Th1 and Th17 responses in the gut.

Simulated Colonic Fluid Replicates the In Vivo Growth Capabilities of Citrobacter rodentium cpxRA Mutants and Uncovers Additive Effects of Cpx-Regulated Genes on Fitness.

Citrobacter rodentium is an attaching and effacing (A/E) pathogen used to model enteropathogenic and enterohemorrhagic Escherichia coli infections in mice. During colonization, C. rodentium must adapt to stresses in the gastrointestinal tract, such as antimicrobial peptides, pH changes, and bile salts. The Cpx envelope stress response (ESR) is a two-component system used by some bacteria to remediate stress by modulating gene expression, and it is necessary for C. rodentium pathogenesis in mice. Here, we utilized simulated colonic fluid (SCF) to mimic the gastrointestinal environment, which we show strongly induces the Cpx ESR and highlights a fitness defect specific to the ΔcpxRA mutant. While investigating genes in the Cpx regulon that may contribute to C. rodentium pathogenesis, we found that the absence of the Cpx ESR resulted in higher expression of the locus of enterocyte effacement (LEE) master regulator, ler, and that the genes yebE, ygiB, bssR, and htpX relied on CpxRA for proper expression. We then determined that CpxRA and select gene mutants were essential for proper growth in SCF when in the presence of extraneous stressors and in competition. Although none of the Cpx-regulated gene mutants exhibited marked virulence phenotypes in vivo, the ΔcpxRA mutant had reduced colonization and attenuated virulence, as previously determined, which replicated the in vitro growth phenotypes specific to SCF. Overall, these results indicate that the ΔcpxRA virulence defect is not due to any single Cpx regulon gene examined. Instead, attenuation may be the result of defective growth in the colonic environment resulting from the collective impact of multiple Cpx-regulated genes.

IRE1α-Driven Inflammation Promotes Clearance of Citrobacter rodentium Infection.

Endoplasmic reticulum (ER) stress is intimately linked with inflammation in response to pathogenic infections. ER stress occurs when cells experience a buildup of misfolded or unfolded protein during times of perturbation, such as infections, which facilitates the unfolded protein response (UPR). The UPR involves multiple host pathways in an attempt to reestablish homeostasis, which oftentimes leads to inflammation and cell death if unresolved. The UPR is activated to help resolve some bacterial infections, and the IRE1α pathway is especially critical in mediating inflammation. To understand the role of the IRE1α pathway of the UPR during enteric bacterial infection, we employed Citrobacter rodentium to study host-pathogen interactions in intestinal epithelial cells and the murine gastrointestinal (GI) tract. C. rodentium is an enteric mouse pathogen that is similar to the human pathogens enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively), for which we have limited small-animal models. Here, we demonstrate that both C. rodentium and EPEC induced the UPR in intestinal epithelial cells. UPR induction during C. rodentium infection correlated with the onset of inflammation in bone marrow-derived macrophages (BMDMs). Our previous work implicated IRE1α and NOD1/2 in ER stress-induced inflammation, which we observed were also required for proinflammatory gene induction during C. rodentium infection. C. rodentium induced IRE1α-dependent inflammation in mice, and inhibiting IRE1α led to a dysregulated inflammatory response and delayed clearance of C. rodentium. This study demonstrates that ER stress aids inflammation and clearance of C. rodentium through a mechanism involving the IRE1α-NOD1/2 axis.

Blocking GARP-mediated activation of TGF-ß1 did not alter innate or adaptive immune responses to bacterial infection or protein immunization in mice.

Transmembrane protein GARP binds latent TGF-ß1 to form GARP:(latent)TGF-ß1 complexes on the surface of several cell types including Tregs, B-cells, and platelets. Upon stimulation, these cells release active TGF-ß1. Blocking TGF-ß1 activation by Tregs with anti-GARP:TGF-ß1 mAbs overcomes resistance to PD1/PD-L1 blockade and induces immune-mediated regressions of murine tumors, indicating that Treg-derived TGF-ß1 inhibits anti-tumor immunity. TGF-ß1 exerts a vast array of effects on immune responses. For example, it favors differentiation of TH17 cells and B-cell switch to IgA production, two important processes for mucosal immunity. Here, we sought to determine whether treatment with anti-GARP:TGF-ß1 mAbs would perturb immune responses to intestinal bacterial infection. We observed no aggravation of intestinal disease, no systemic dissemination, and no alteration of innate or adaptative immune responses upon oral gavage of C. rodentium in highly susceptible Il22r-/- mice treated with anti-GARP:TGF-ß1 mAbs. To examine the effects of GARP:TGF-ß1 blockade on Ig production, we compared B cell- and TH cell- responses to OVA or CTB protein immunization in mice carrying deletions of Garp in Tregs, B cells, or platelets. No alteration of adaptive immune responses to protein immunization was observed in the absence of GARP on any of these cells. Altogether, we show that antibody-mediated blockade of GARP:TGF-ß1 or genetic deletion of Garp in Tregs, B cells or platelets, do not alter innate or adaptive immune responses to intestinal bacterial infection or protein immunization in mice. Anti-GARP:TGF-ß1 mAbs, currently tested for cancer immunotherapy, may thus restore anti-tumor immunity without severely impairing other immune defenses. PRéCIS: Immunotherapy with GARP:TGF-ß1 mAbs may restore anti-tumor immunity without impairing immune or inflammatory responses required to maintain homeostasis or host defense against infection, notably at mucosal barriers.

Trichinella spiralis infection ameliorates the severity of Citrobacter rodentium-induced experimental colitis in mice.

Trichinellosis is a food-borne zoonotic parasitic disease that causes serious harm to human health and the pig breeding industry. However, there are reports that Trichinella spiralis (T. spiralis) infection can treat autoimmune diseases, including enteritis and experimental autoimmune encephalitis (EAE). However, research on the mechanism of T. spiralis infection in infectious enteritis has not been fully elucidated. Therefore, this experiment used Citrobacter rodentium (C. rodentium) to induce colitis in mouse models and explored its underlying mechanisms. In this experiment, a total of 72 C57BL/6 mice were randomly divided into four groups. Experimental mice in the TS and TS + CR groups were orally inoculated with individual T. spiralis larvae. At 21 days postinfection (dpi) with T. spiralis, experimental animals in the CR and TS + CR groups were inoculated by orogastric gavage with C. rodentium. The control group received PBS only. The results indicated that the weight loss and macroscopic and microscopic colon damage of mice in the TS + CR group were significantly decreased compared with those observed in the CR group. The results of flow cytometry showed that the expression levels of IL-4, IL-10 and CD4+CD25+Foxp3+ Tregs were increased (P < 0.05), while the expression levels of IFN-γ, IL-12 and IL-17 were decreased in the spleens and MLNs of the TS + CR experimental mice compared with the colitis model mice. ELISA results revealed that the TS + CR group not only elicited a strong IgG1 response (P < 0.01) but also a low level of IgG2a response (P < 0.05) relative to the CR group. The above results demonstrated that prior exposure of mice to T. spiralis infection ameliorated the severity of C. rodentium-induced infectious colitis.

Infection-induced signals generated at the plasma membrane epigenetically regulate Wnt signaling in vitro and in vivo.

Wnt signaling regulates immunomodulatory functions during infection and inflammation. Employing NCCIT and HCT116 cells, having high endogenous Wnt signaling, we observed elevated levels of low-density lipoprotein receptor-related protein 5/6 (LRP5/6) and Frizzled class receptor 10 (FZD10) and increases in ß-catenin, doublecortin-like kinase 1 (DCLK1), CD44 molecule (CD44), and aldehyde dehydrogenase 1 family member A1 (ALDH1A1). siRNA-induced knockdown of these receptors antagonized TOPflash reporter activity and spheroid growth in vitro and elevated Wnt-inhibitory factor 1 (WIF1) activity. Elevated mRNA and protein levels of LRP5/6 and FZD10 paralleled expression of WNT2b and WNT4 in colonic crypts at days 6 and 12 post-infection with Citrobacter rodentium (CR) and tended to decline at days 20-34. The CR mutant escV or the tankyrase inhibitor XAV939 attenuated these responses. A three-dimensional organoid assay in colonic crypts isolated from CR-infected mice revealed elevated levels of LRP5/6 and FZD10 and ß-catenin co-localization with enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2). Co-immunoprecipitation in the membrane fraction revealed that axin associates with LRP5/6 in CR-infected crypts, and this association was correlated with increased ß-catenin. Colon tumors from either CR-infected ApcPMin/+ or azoxymethane/dextran sodium sulfate (AOM/DSS)-treated mice had high LRP5/6 or FZD10 levels, and chronic Notch blockade through the γ-secretase inhibitor dibenzazepine down-regulated LRP5/6 and FZD10 expression. In CR-responsive CT-26 cells, siRNA-induced LRP5/6 or FZD10 knockdown antagonized TOPflash reporter activity. Elevated miR-153-3p levels correlated with LRP5/6 and FZD10, and miR-153-3p sequestration via a plasmid-based miR inhibitor system attenuated Wnt signaling. We conclude that infection-induced signals from the plasma membrane epigenetically regulate Wnt signaling.