Pathobionts in Inflammatory Bowel Disease: Origins, Underlying Mechanisms, and Implications for Clinical Care.

The gut microbiota plays a significant role in the pathogenesis of both forms of inflammatory bowel disease (IBD), namely, Crohn's disease (CD) and ulcerative colitis (UC). Although evidence suggests dysbiosis and loss of beneficial microbial species can exacerbate IBD, many new studies have identified microbes with pathogenic qualities, termed "pathobionts," within the intestines of patients with IBD. The concept of pathobionts initiating or driving the chronicity of IBD has largely focused on the putative aggravating role that adherent invasive Escherichia coli may play in CD. However, recent studies have identified additional bacterial and fungal pathobionts in patients with CD and UC. This review will highlight the characteristics of these pathobionts and their implications for IBD treatment. Beyond exploring the origins of pathobionts, we discuss those associated with specific clinical features and the potential mechanisms involved, such as creeping fat (Clostridium innocuum) and impaired wound healing (Debaryomyces hansenii) in patients with CD as well as the increased fecal proteolytic activity (Bacteroides vulgatus) seen as a biomarker for UC severity. Finally, we examine the potential impact of pathobionts on current IBD therapies, and several new approaches to target pathobionts currently in the early stages of development. Despite recognizing that pathobionts likely contribute to the pathogenesis of IBD, more work is needed to define their modes of action. Determining whether causal relationships exist between pathobionts and specific disease characteristics could pave the way for improved care for patients, particularly for those not responding to current IBD therapies.

Vasoactive intestinal peptide promotes host defense against enteric pathogens by modulating the recruitment of group 3 innate lymphoid cells.

Group 3 innate lymphoid cells (ILC3s) control the formation of intestinal lymphoid tissues and play key roles in intestinal defense. They express neuropeptide vasoactive intestinal peptide (VIP) receptor 2 (VPAC2), through which VIP modulates their function, but whether VIP exerts other effects on ILC3 remains unclear. We show that VIP promotes ILC3 recruitment to the intestine through VPAC1 independent of the microbiota or adaptive immunity. VIP is also required for postnatal formation of lymphoid tissues as well as the maintenance of local populations of retinoic acid (RA)-producing dendritic cells, with RA up-regulating gut-homing receptor CCR9 expression by ILC3s. Correspondingly, mice deficient in VIP or VPAC1 suffer a paucity of intestinal ILC3s along with impaired production of the cytokine IL-22, rendering them highly susceptible to the enteric pathogen Citrobacter rodentium This heightened susceptibility to C. rodentium infection was ameliorated by RA supplementation, adoptive transfer of ILC3s, or by recombinant IL-22. Thus, VIP regulates the recruitment of intestinal ILC3s and formation of postnatal intestinal lymphoid tissues, offering protection against enteric pathogens.

Commensal segmented filamentous bacteria-derived retinoic acid primes host defense to intestinal infection.

Interactions between the microbiota and mammalian host are essential for defense against infection, but the microbial-derived cues that mediate this relationship remain unclear. Here, we find that intestinal epithelial cell (IEC)-associated commensal bacteria, segmented filamentous bacteria (SFB), promote early protection against the pathogen Citrobacter rodentium, independent of CD4+ T cells. SFB induced histone modifications in IECs at sites enriched for retinoic acid receptor motifs, suggesting that SFB may enhance defense through retinoic acid (RA). Consistent with this, inhibiting RA signaling suppressed SFB-induced protection. Intestinal RA levels were elevated in SFB mice, despite the inhibition of mammalian RA production, indicating that SFB directly modulate RA. Interestingly, RA was produced by intestinal bacteria, and the loss of bacterial-intrinsic aldehyde dehydrogenase activity decreased the RA levels and increased infection. These data reveal RA as an unexpected microbiota-derived metabolite that primes innate defense and suggests that pre- and probiotic approaches to elevate RA could prevent or combat infections.

Creating a More Perfect Union: Modeling Intestinal Bacteria-Epithelial Interactions Using Organoids.

Intestinal organoids have become indispensable tools for many gastrointestinal researchers, advancing their studies of nontransformed intestinal epithelial cells, and their roles in an array of diseases, including inflammatory bowel disease and colon cancer. In many cases. these diseases, as well as many enteric infections, reflect pathogenic interactions between bacteria and the gut epithelium. The complexity of studying this microbe-epithelial interface in vivo has led to significant focus on modeling this cross-talk using organoid models. Considering how quickly the organoid field is advancing, it can be difficult to keep up to date with the latest techniques, as well as their respective strengths and weaknesses. This review addresses the advantages of using organoids derived from adult stem cells and the recently identified differences that biopsy location and patient age can have on organoids and their interactions with microbes. Several approaches to introducing bacteria in a relevant (apical) manner (ie, microinjecting 3-dimensional spheroids, polarity-reversed organoids, and 2-dimensional monolayers) also are addressed, as are the key readouts that can be obtained using these models. Lastly, the potential for new approaches, such as air-liquid interface, to facilitate studying bacterial interactions with important but understudied epithelial subsets such as goblet cells and their products, is evaluated.

Autotaxin stimulates LPA2 receptor in macrophages and exacerbates dextran sulfate sodium-induced acute colitis.

Autotaxin (ATX) is a secreted enzyme that hydrolyzes lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA) and choline. ATX has been implicated in multiple chronic inflammatory diseases, but little is known about its role in the development of inflammatory bowel disease (IBD). Here, we investigated how ATX contributed to intestinal inflammation during colitis. We found that ATX expression levels were upregulated in the intestines of ulcerative colitis (UC) patients in acute state as well as in the intestines of dextran sulfate sodium (DSS)-induced colitis mice, which is likely due to increased infiltration of inflammatory cells including macrophages. Intriguingly, the inhibition of ATX activity led to reduced production of inflammatory cytokines, as well as attenuated colitis. These findings suggest that ATX may display strong pro-inflammatory properties. Supporting this, treatment with recombinant mouse ATX (rmATX) increased the production of inflammatory cytokines and enzymes in mouse macrophage cell line RAW264.7 and bone marrow-derived macrophages (BMDM), whereas silencing ATX by siRNA reduced LPS-stimulated production of pro-inflammatory factors. Notably, we found that the levels of LPA2 (an LPA receptor) were dramatically upregulated in rmATX-treated RAW264.7 cells and DSS-treated mice. Gene silencing of lpa2 in RAW264.7 cells by siRNA led to reduced production of inflammatory cytokines. Moreover, adenovirus-mediated delivery of lpa2 short hairpin RNA into DSS-treated mice ameliorated colitis. Collectively, our research suggests that ATX may exacerbate DSS-induced colitis by activating LPA2 receptor in macrophages and represent a promising target for the treatment of IBD. KEY MESSAGES: Increased ATX expression and secretion in colitic colons are likely due to increased infiltration of inflammatory cells including macrophages. Recombinant ATX promotes, but ATX silencing inhibits, the production of inflammatory cytokines in LPS-stimulated RAW264.7 cells and BMDM. •LPA2 mediates the pro-inflammatory effects of ATX on macrophages. Inhibition of ATX and downregulation of LPA2 ameliorate DSS-induced colitis.

Direct Clinical Evidence Recommending the Use of Proteinase K or Dithiothreitol to Pretreat Sputum for Detection of SARS-CoV-2.

One of the primary tools for diagnosing COVID-19 is the nucleic acid-based real-time reverse transcriptase-polymerase chain reaction (RT-PCR) test performed on respiratory specimens. The detection rate of SARS-CoV-2 in lower respiratory specimens (such as sputum) is higher than that for upper respiratory specimens (such as nasal and pharyngeal swabs). However, sputum specimens are usually quite viscous, requiring a homogenization process prior to nucleic acid (NA) extraction for RT-PCR. Sputum specimens from COVID-19 and non-COVID-19 patients were treated with four commonly used reagents-saline, N-acetyl-L-cysteine (NALC), proteinase K (PK), and dithiothreitol (DTT), prior to NA extraction. These reagents were then compared for their performance in diagnosing COVID-19 in real clinical practice. The detection rate of SARS-CoV-2 in PK- or DTT-treated sputum was comparable, and higher than that in sputum treated with NALC or saline. While there was a 4.8% (1/21) false negative rate for the PK- and DTT-treated sputum, neither treatment showed any false positive cases among patients with non-COVID diseases. Moreover, sputum pretreated with saline, NALC, PK or DTT showed higher detection rates of SARS-CoV-2 as compared to pharyngeal swabs. Taken together, we provide direct evidence recommending the use of PK or DTT to pretreat sputum samples to facilitate SARS-CoV-2 detection by clinical laboratories. Moreover, our methods should help to standardize the procedure of processing sputum specimens and improve the ability to detect SARS-CoV-2 in these samples.

Salmonella enterica Infection of Murine and Human Enteroid-Derived Monolayers Elicits Differential Activation of Epithelium-Intrinsic Inflammasomes.

Recent studies have determined that inflammasome signaling plays an important role in driving intestinal epithelial cell (IEC) responses to bacterial infections, such as Salmonella enterica serovar Typhimurium. There are two primary inflammasome pathways, canonical (involving caspase-1) and noncanonical (involving caspase-4 and -5 in humans and caspase-11 in mice). Prior studies identified the canonical inflammasome as the major pathway leading to interleukin-18 (IL-18) release and restriction of S Typhimurium replication in the mouse cecum. In contrast, the human C2Bbe1 colorectal carcinoma cell line expresses little caspase-1 but instead utilizes caspase-4 to respond to S Typhimurium infection. Intestinal enteroid culture has enabled long-term propagation of untransformed IECs from multiple species, including mouse and human. Capitalizing on this technology, we used a genetic approach to directly compare the relative importance of different inflammatory caspases in untransformed mouse and human IECs and transformed human IECs upon S Typhimurium infection in vitro We show that caspase-1 is important for restricting intracellular S Typhimurium replication and initiating IL-18 secretion in mouse IECs but is dispensable in human IECs. In contrast, restriction of intracellular S Typhimurium and production of IL-18 are dependent on caspase-4 in both transformed and untransformed human IECs. Notably, cytosolic replication in untransformed cells from both species was less pronounced than in transformed human cells, suggesting that transformation may impact additional pathways that restrict S Typhimurium replication. Taken together, these data highlight the differences between mouse and human IECs and the utility of studying transformed and untransformed cells in parallel.

Intestinal restriction of Salmonella Typhimurium requires caspase-1 and caspase-11 epithelial intrinsic inflammasomes.

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.

Poor Correlation of Oral Swabs with Esophageal Eosinophil Counts.

Eosinophilic esophagitis (EoE) is a chronic condition that requires repeated endoscopies/biopsies to track the disease and treatment response. This invasive procedure involves risk to the patient and has significant costs. We studied whether the detection of specific proteins (cytokines and eosinophil degranulation products) from oral swabs could serve as a minimally invasive test for EoE. Swabs of the oral cavity (buccal and oropharyngeal) were obtained prior to endoscopy/biopsies in patients with EoE, possible EoE, and non-EoE patients in addition to obtaining additional esophageal biopsy tissue. ELISAs measuring the levels of cytokines IL-5, IL-8, IL-13, and eosinophil degranulation products including major basic protein (MBP), eosinophil derived neurotoxin (EDN), and eosinophil peroxidase (EPO) were performed on the samples. Comparisons were made to peak esophageal eosinophil counts. Tolerability of the swabs was evaluated. 43 patients, 4-17 years old, participated in the study. Swabs were well tolerated and all showed measurable protein. 26 patients had EoE [14 active (> 15 eosinophils/high power field), 12 non-active], 17 patients did not have EoE. Results obtained from oral swabs showed poor correlation with those from esophageal tissue. Only measurement of eosinophil degranulation products EDN and EPO from esophageal tissues showed strong correlations with eosinophil counts. In this study, the levels of cytokines and eosinophil degranulation products detected from oral swabs did not correlate with esophageal eosinophilia, and their detection would be insufficient to displace endoscopy/biopsies.

Enteroids Derived From Inflammatory Bowel Disease Patients Display Dysregulated Endoplasmic Reticulum Stress Pathways, Leading to Differential Inflammatory Responses and Dendritic Cell Maturation.

BACKGROUND AND AIMS: Endoplasmic reticulum [ER] stress in intestinal epithelial cells [IECs] contributes to the pathogenesis of inflammatory bowel disease [IBD]. We hypothesized that ER stress changes innate signalling in human IECs, augmenting toll-like receptor [TLR] responses and inducing pro-inflammatory changes in underlying dendritic cells [DCs]. METHODS: Caco-2 cells and primary human colon-derived enteroid monolayers were exposed to ATP [control stressor] or thapsigargin [Tg] [ER stress inducer], and were stimulated with the TLR5 agonist flagellin. Cytokine release was measured by an enzyme immunoassay. ER stress markers CHOP, GRP78 and XBP1s/u were measured via quantitative PCR and Western blot. Monocyte-derived DCs [moDCs] were cultured with the IEC supernatants and their activation state was measured. Responses from enteroids derived from IBD patients and healthy control participants were compared. RESULTS: ER stress enhanced flagellin-induced IL-8 release from Caco-2 cells and enteroids. Moreover, conditioned media activated DCs to become pro-inflammatory, with increased expression of CD80, CD86, MHCII, IL-6, IL-15 and IL-12p70 and decreased expression of CD103 and IL-10. Flagellin-induced IL-8 production correlated with DC activation, suggesting a common stress pathway. Moreover, there were distinct differences in cytokine expression and basal ER stress between IBD and healthy subject-derived enteroid monolayers, suggesting a dysregulated ER stress pathway in IBD-derived enteroids. CONCLUSIONS: Cellular stress enhances TLR5 responses in IECs, leading to increased DC activation, indicating a previously unknown mechanistic link between epithelial ER stress and immune activation in IBD. Furthermore, dysregulated ER stress may be propagated from the intestinal epithelial stem cell niche in IBD patients.

Suppressive and Gut-Reparative Functions of Human Type 1 T Regulatory Cells.

BACKGROUND & AIMS: T-regulatory (Treg) cells suppress the immune response to maintain homeostasis. There are 2 main subsets of Treg cells: FOXP3 (forkhead box protein 3)-positive Treg cells, which do not produce high levels of effector cytokines, and type 1 Treg (Tr1) cells, which are FOXP3-negative and secrete interleukin (IL) 10. IL10 is an anti-inflammatory cytokine, so Tr1 cells might be used in the treatment of inflammatory bowel diseases. We aimed to develop methods to isolate and expand human Tr1 cells and define their functions. METHODS: We obtained blood and colon biopsy samples from patients with Crohn's disease or ulcerative colitis or healthy individuals (controls). CD4+ T cells were isolated from blood samples and stimulated with anti-CD3 and anti-CD28 beads, and Tr1 cells were purified by using an IL10 cytokine-capture assay and cell sorting. FOXP3-positive Treg cells were sorted as CD4+CD25highCD127low cells from unstimulated cells. Tr1 and FOXP3-positive Treg cells were expanded, and phenotypes and gene expression profiles were compared. T cells in peripheral blood mononuclear cells from healthy donors were stimulated with anti-CD3 and anti-CD28 beads, and the suppressive abilities of Tr1 and FOXP3-positive Treg cells were measured. Human colon organoid cultures were established, cultured with supernatants from Tr1 or FOXP3-positive cells, and analyzed by immunofluorescence and flow cytometry. T84 cells (human colon adenocarcinoma epithelial cells) were incubated with supernatants from Tr1 or FOXP3-positive cells, and transepithelial electrical resistance was measured to determine epithelial cell barrier function. RESULTS: Phenotypes of Tr1 cells isolated from control individuals vs patients with Crohn's disease or ulcerative colitis did not differ significantly after expansion. Tr1 cells and FOXP3-positive Treg cells suppressed proliferation of effector T cells, but only Tr1 cells suppressed secretion of IL1B and tumor necrosis factor from myeloid cells. Tr1 cells, but not FOXP3-positive Treg cells, isolated from healthy individuals and patients with Crohn's disease or ulcerative colitis secreted IL22, which promoted barrier function of human intestinal epithelial cells. Tr1 cell culture supernatants promoted differentiation of mucin-producing goblet cells in intestinal organoid cultures. CONCLUSIONS: Human Tr1 cells suppress proliferation of effector T cells (adaptive immune response) and production of IL1B and TNF by myeloid cells (inmate immune response). They also secrete IL22 to promote barrier function. They might be developed as a cell-based therapy for intestinal inflammatory disorders.

Microbiota Inhibit Epithelial Pathogen Adherence by Epigenetically Regulating C-Type Lectin Expression.

Numerous bacterial pathogens infect the mammalian host by initially associating with epithelial cells that line the intestinal lumen. Recent work has revealed that commensal bacteria that reside in the intestine promote defense against pathogenic infection, however whether the microbiota direct host pathways that alter pathogen adherence is not well-understood. Here, by comparing germ-free mice, we identify that the microbiota decrease bacterial pathogen adherence and dampen epithelial expression of the cell surface glycoprotein C-type lectin 2e (Clec2e). Functional studies revealed that overexpression of this lectin promotes adherence of intestinal bacterial pathogens to mammalian cells. Interestingly, microbiota-sensitive downregulation of Clec2e corresponds with decreased histone acetylation of the Clec2e gene in intestinal epithelial cells. Histone deacetylation and transcriptional regulation of Clec2e depends on expression and recruitment of the histone deacetylase HDAC3. Thus, commensal bacteria epigenetically instruct epithelial cells to decrease expression of a C-type lectin that promotes pathogen adherence, revealing a novel mechanism for how the microbiota promote innate defense against infection.

Macrophage ß2-Integrins Regulate IL-22 by ILC3s and Protect from Lethal Citrobacter rodentium-Induced Colitis.

ß2-integrins promote neutrophil recruitment to infected tissues and are crucial for host defense. Neutrophil recruitment is defective in leukocyte adhesion deficiency type-1 (LAD1), a condition caused by mutations in the CD18 (ß2-integrin) gene. Using a model of Citrobacter rodentium (CR)-induced colitis, we show that CD18-/- mice display increased intestinal damage and systemic bacterial burden, compared to littermate controls, ultimately succumbing to infection. This phenotype is not attributed to defective neutrophil recruitment, as it is shared by CXCR2-/- mice that survive CR infection. CR-infected CD18-/- mice feature prominent upregulation of IL-17 and downregulation of IL-22. Exogenous IL-22 administration, but not endogenous IL-17 neutralization, protects CD18-/- mice from lethal colitis. ß2-integrin expression on macrophages is mechanistically linked to Rac1/ROS-mediated induction of noncanonical-NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome-dependent IL-1ß production, which promotes ILC3-derived IL-22. Therefore, ß2-integrins are required for protective IL-1ß-dependent IL-22 responses in colitis, and the identified mechanism may underlie the association of human LAD1 with colitis.

IL-22 Preserves Gut Epithelial Integrity and Promotes Disease Remission during Chronic Salmonella Infection.

The cytokine IL-22 is rapidly induced at barrier surfaces where it regulates host-protective antimicrobial immunity and tissue repair but can also enhance disease severity in some chronic inflammatory settings. Using the chronic Salmonella gastroenteritis model, Ab-mediated neutralization of IL-22 impaired intestinal epithelial barrier integrity and, consequently, exaggerated expression of proinflammatory cytokines. As disease normally resolved, neutralization of IL-22 caused luminal narrowing of the cecum-a feature reminiscent of fibrotic strictures seen in Crohn disease patients. Corresponding to the exaggerated immunopathology caused by IL-22 suppression, Salmonella burdens in the gut were reduced. This enhanced inflammation and pathogen clearance was associated with alterations in gut microbiome composition, including the overgrowth of Bacteroides acidifaciens Our findings thus indicate that IL-22 plays a protective role by limiting infection-induced gut immunopathology but can also lead to persistent pathogen colonization.

Immune Stimulation Using a Gut Microbe-Based Immunotherapy Reduces Disease Pathology and Improves Barrier Function in Ulcerative Colitis.

Background: Current ulcerative colitis (UC) treatments are focused on symptom management primarily via immune suppression. Despite the current arsenal of immunosuppressant treatments, the majority of patients with UC still experience disease progression. Importantly, aggressive long-term inhibition of immune function comes with consequent risk, such as serious infections and malignancy. There is thus a recognized need for new, safe and effective treatment strategies for people living with UC that work upstream of managing the symptoms of the disease. The objective of this study was to evaluate a microbial-based treatment, QBECO, that functions to productively activate rather than suppress mucosal immune function as a novel approach to treat UC. Methods: Two established models of experimental colitis, namely chemically-induced DSS colitis and the spontaneous colitis that develops in Muc2 deficient mice, were used to assess whether QBECO treatment could ameliorate gastrointestinal disease. A small exploratory 16-week QBECO open-label trial was subsequently conducted to test the safety and tolerability of this approach and also to determine whether similar improvements in clinical disease and histopathology could be demonstrated in patients with moderate-to-severe UC. Results: QBECO treatment successfully reduced inflammation and promoted mucosal and histological healing in both experimental models and in UC patients. The preclinical models of colitis showed that QBECO ameliorated mucosal pathology, in part by reducing inflammatory cell infiltration, primarily that induced by neutrophils and inflammatory T cells. The most rapid and noticeable change observed in QBECO treated UC patients was a marked reduction in rectal bleeding. Conclusion: Collectively, this work demonstrates for the first time that strategically activating immune function rather than suppressing it, not only does not worsen colitis induced-damage, but may lead to an objective reduction in UC disease pathology.

The Intestinal Epithelium: Central Coordinator of Mucosal Immunity.

The gastrointestinal (GI) tract represents a unique challenge to the mammalian immune system. It must tolerate the presence of the luminal microbiota and thus not respond to their products, but still protect the intestinal mucosa from potentially harmful dietary antigens and invading pathogens. The intestinal epithelium, composed of a single layer of cells, is crucial for preserving gut homeostasis and acts both as a physical barrier and as a coordinating hub for immune defense and crosstalk between bacteria and immune cells. We highlight here recent findings regarding communication between microbes and intestinal epithelial cells (IECs), as well as the immune mechanisms employed by distinct IEC subsets to promote homeostasis, emphasizing the central and active role that these cells play in host enteric defense.

Pediatric Eosinophilic Esophagitis Is Associated With Increased Lamina Propria Immunoglobulin G4-Positive Plasma Cells.

OBJECTIVE: Eosinophilic esophagitis (EoE) is considered a TH2-mediated food allergy disease that leads to submucosal esophageal fibrosis and strictures. Recent studies focused on adults with EoE identified a strong association with elevated esophageal IgG4 immunostaining. Our study aimed to determine the association of IgG4 with EoE in pediatric patients. METHODS: Using our local EoE research registry, we identified 41 adequate biopsies from EoE patients. We used 10 age- and sex-matched patients with no diagnostic abnormalities at endoscopy or on biopsy. Using a monoclonal antibody to Immunoglobulin G4 (IgG4), we determined the maximum density of IgG4-positive plasma cells (IgG4-PC) per high-power field (hpf). Using a semi-quantitative assessment, we also graded the noncellular staining of the lamina propria and epithelium. RESULTS: Our EoE cohort consisted predominantly of boys with an average age of 5.9 years and 63% had a documented IgE-based food allergy. Median peak eosinophilia was 40 eosinophils/hpf and the median IgG4-PC density was 39/hpf in the active esophagitis patients, compared with a median of 0 IgG4-PC/hpf in the non-EoE patients (P = 0.0001). EoE patients with a food allergy showed a significantly higher IgG4-PC density (44.5/hpf) than those without a food allergy (8/hpf; P = 0.0385). There was no significant association between IgG4-PC density and peak eosinophilia (r = 0.0011). CONCLUSIONS: We demonstrate that active esophagitis in pediatric EoE patients is associated with elevated levels of IgG4-positive plasma cells, which was more significant in EoE patients with a documented food allergy. Our study also adds to the growing literature that EoE may involve more than just an exaggerated TH2 immune response.

Giardia co-infection promotes the secretion of antimicrobial peptides beta-defensin 2 and trefoil factor 3 and attenuates attaching and effacing bacteria-induced intestinal disease.

Our understanding of polymicrobial gastrointestinal infections and their effects on host biology remains incompletely understood. Giardia duodenalis is an ubiquitous intestinal protozoan parasite infecting animals and humans. Concomitant infections with Giardia and other gastrointestinal pathogens commonly occur. In countries with poor sanitation, Giardia infection has been associated with decreased incidence of diarrheal disease and fever, and reduced serum inflammatory markers release, via mechanisms that remain obscure. This study analyzed Giardia spp. co-infections with attaching and effacing (A/E) pathogens, and assessed whether and how the presence of Giardia modulates host responses to A/E enteropathogens, and alters intestinal disease outcome. In mice infected with the A/E pathogen Citrobacter rodentium, co-infection with Giardia muris significantly attenuated weight loss, macro- and microscopic signs of colitis, bacterial colonization and translocation, while concurrently enhancing the production and secretion of antimicrobial peptides (AMPs) mouse ß-defensin 3 and trefoil factor 3 (TFF3). Co-infection of human intestinal epithelial cells (Caco-2) monolayers with G. duodenalis trophozoites and enteropathogenic Escherichia coli (EPEC) enhanced the production of the AMPs human ß-defensin 2 (HBD-2) and TFF3; this effect was inhibited with treatment of G. duodenalis with cysteine protease inhibitors. Collectively, these results suggest that Giardia infections are capable of reducing enteropathogen-induced colitis while increasing production of host AMPs. Additional studies also demonstrated that Giardia was able to directly inhibit the growth of pathogenic bacteria. These results reveal novel mechanisms whereby Giardia may protect against gastrointestinal disease induced by a co-infecting A/E enteropathogen. Our findings shed new light on how microbial-microbial interactions in the gut may protect a host during concomitant infections.

Milk Fat Globule Membrane Supplementation in Formula Modulates the Neonatal Gut Microbiome and Normalizes Intestinal Development.

Breast milk has many beneficial properties and unusual characteristics including a unique fat component, termed milk fat globule membrane (MFGM). While breast milk yields important developmental benefits, there are situations where it is unavailable resulting in a need for formula feeding. Most formulas do not contain MFGM, but derive their lipids from vegetable sources, which differ greatly in size and composition. Here we tested the effects of MFGM supplementation on intestinal development and the microbiome as well as its potential to protect against Clostridium difficile induced colitis. The pup-in-a-cup model was used to deliver either control or MFGM supplemented formula to rats from 5 to 15 days of age; with mother's milk (MM) reared animals used as controls. While CTL formula yielded significant deficits in intestinal development as compared to MM littermates, addition of MFGM to formula restored intestinal growth, Paneth and goblet cell numbers, and tight junction protein patterns to that of MM pups. Moreover, the gut microbiota of MFGM and MM pups displayed greater similarities than CTL, and proved protective against C. difficile toxin induced inflammation. Our study thus demonstrates that addition of MFGM to formula promotes development of the intestinal epithelium and microbiome and protects against inflammation.

Tricellular Tight Junction Protein Tricellulin Is Targeted by the Enteropathogenic Escherichia coli Effector EspG1, Leading to Epithelial Barrier Disruption.

Enteropathogenic Escherichia coli (EPEC)-induced diarrhea is often associated with disruption of intestinal epithelial tight junctions. Although studies have shown alterations in the expression and localization of bicellular tight junction proteins during EPEC infections, little is known about whether tricellular tight junction proteins (tTJs) are affected. Using Caco-2 cell monolayers, we investigated if EPEC is capable of targeting the tTJ protein tricellulin. Our results demonstrated that at 4 h postinfection, EPEC induced a significant reduction in tricellulin levels, accompanied by a significant loss of transepithelial resistance (TEER) and a corresponding increase in paracellular permeability. Conversely, cells overexpressing tricellulin were highly resistant to EPEC-induced barrier disruption. Confocal microscopy revealed the distribution of tricellulin into the plasma membrane of infected epithelial cells and confirmed the localization of EPEC aggregates in close proximity to tTJs. Moreover, infections with EPEC strains lacking genes encoding specific type III secreted effector proteins demonstrated a crucial role for the effector EspG1 in modulating tricellulin expression. Complementation studies suggest that the EspG-induced depletion of tricellulin is microtubule dependent. Overall, our results show that EPEC-induced epithelial barrier dysfunction is mediated in part by EspG1-induced microtubule-dependent depletion of tricellulin.