Microbiota-mediated inflammation and antimicrobial defense in the intestine.

The diverse microbial populations constituting the intestinal microbiota promote immune development and differentiation, but because of their complex metabolic requirements and the consequent difficulty culturing them, they remained, until recently, largely uncharacterized and mysterious. In the last decade, deep nucleic acid sequencing platforms, new computational and bioinformatics tools, and full-genome characterization of several hundred commensal bacterial species facilitated studies of the microbiota and revealed that differences in microbiota composition can be associated with inflammatory, metabolic, and infectious diseases, that each human is colonized by a distinct bacterial flora, and that the microbiota can be manipulated to reduce and even cure some diseases. Different bacterial species induce distinct immune cell populations that can play pro- and anti-inflammatory roles, and thus the composition of the microbiota determines, in part, the level of resistance to infection and susceptibility to inflammatory diseases. This review summarizes recent work characterizing commensal microbes that contribute to the antimicrobial defense/inflammation axis.

The fire within: microbes inflame tumors.

The immune system and the microbiota mutually interact to maintain homeostasis in the intestine. However, components of the microbiota can alter this balance and promote chronic inflammation, promoting intestinal tumor development. We review recent advances in understanding the complex interactions between the microbiota and the innate and adaptive immune systems and discuss their potential to lead us in new directions for understanding cancer biology and treatment.

Epithelial glycosylation in gut homeostasis and inflammation.

Intestinal epithelial cells apically express glycans, especially α1,2-fucosyl linkages, which work as a biological interface for the host-microbe interaction. Emerging studies have shown that epithelial α1,2-fucosylation is regulated by microbes and by group 3 innate lymphoid cells (ILC3s). Dysregulation of the gene (FUT2) encoding fucosyltransferase 2, an enzyme governing epithelial α1,2-fucosylation, is associated with various human disorders, including infection and chronic inflammatory diseases. This suggests a critical role for an interaction between microbes, epithelial cells and ILC3s mediated via glycan residues. In this Review, using α1,2-fucose and Fut2 gene expression as an example, we describe how epithelial glycosylation is controlled by immune cells and luminal microbes. We also address the pathophysiological contribution of epithelial α1,2-fucosylation to pathogenic and commensal microbes as well as the potential of α1,2-fucose and its regulatory pathway as previously unexploited targets in the development of new therapeutic approaches for human diseases.

Severe Immune-Related Enteritis after In Utero Exposure to Pembrolizumab.

Immune checkpoint blockade has become standard treatment for many types of cancer. Such therapy is indicated most often in patients with advanced or metastatic disease but has been increasingly used as adjuvant therapy in those with early-stage disease. Adverse events include immune-related organ inflammation resembling autoimmune diseases. We describe a case of severe immune-related gastroenterocolitis in a 4-month-old infant who presented with intractable diarrhea and failure to thrive after in utero exposure to pembrolizumab. Known causes of the symptoms were ruled out, and the diagnosis of pembrolizumab-induced immune-related gastroenterocolitis was supported by the results of histopathological assays, immunophenotyping, and analysis of the level of antibodies against programmed cell death protein 1 (PD-1). The infant's condition was successfully treated with prednisolone and infliximab.

Differentiation and Protective Capacity of Virus-Specific CD8+ T Cells Suggest Murine Norovirus Persistence in an Immune-Privileged Enteric Niche.

Noroviruses can establish chronic infections with active viral shedding in healthy humans but whether persistence is associated with adaptive immune dysfunction is unknown. We used genetically engineered strains of mouse norovirus (MNV) to investigate CD8+ T cell differentiation during chronic infection. We found that chronic infection drove MNV-specific tissue-resident memory (Trm) CD8+ T cells to a differentiation state resembling inflationary effector responses against latent cytomegalovirus with only limited evidence of exhaustion. These MNV-specific Trm cells remained highly functional yet appeared ignorant of ongoing viral replication. Pre-existing MNV-specific Trm cells provided partial protection against chronic infection but largely ceased to detect virus within 72 hours of challenge, demonstrating rapid sequestration of viral replication away from T cells. Our studies revealed a strategy of immune evasion by MNV via the induction of a CD8+ T cell program normally reserved for latent pathogens and persistence in an immune-privileged enteric niche.

Homeostasis and inflammation in the intestine.

The gut is home to our largest collection of microbes. The ability of the immune system to coevolve with the microbiota during postnatal life allows the host and microbiota to coexist in a mutually beneficial relationship. Failure to achieve or maintain equilibrium between a host and its microbiota has negative consequences for both intestinal and systemic health. In this Review, we consider the many cellular and molecular methods by which inflammatory responses are regulated to maintain intestinal homeostasis and the disease states that can ensue when this balance is lost.

Fasting increases microbiome-based colonization resistance and reduces host inflammatory responses during an enteric bacterial infection.

Reducing food intake is a common host response to infection, yet it remains unclear whether fasting is detrimental or beneficial to an infected host. Despite the gastrointestinal tract being the primary site of nutrient uptake and a common route for infection, studies have yet to examine how fasting alters the host's response to an enteric infection. To test this, mice were fasted before and during oral infection with the invasive bacterium Salmonella enterica serovar Typhimurium. Fasting dramatically interrupted infection and subsequent gastroenteritis by suppressing Salmonella's SPI-1 virulence program, preventing invasion of the gut epithelium. Virulence suppression depended on the gut microbiota, as Salmonella's invasion of the epithelium proceeded in fasting gnotobiotic mice. Despite Salmonella's restored virulence within the intestines of gnotobiotic mice, fasting downregulated pro-inflammatory signaling, greatly reducing intestinal pathology. Our study highlights how food intake controls the complex relationship between host, pathogen and gut microbiota during an enteric infection.

Advances in understanding of the innate immune response to human norovirus infection using organoid models.

Norovirus is the leading cause of epidemic and endemic acute gastroenteritis worldwide and the most frequent cause of foodborne illness in the United States. There is no specific treatment for norovirus infections and therapeutic interventions are based on alleviating symptoms and limiting viral transmission. The immune response to norovirus is not completely understood and mechanistic studies have been hindered by lack of a robust cell culture system. In recent years, the human intestinal enteroid/human intestinal organoid system (HIE/HIO) has enabled successful human norovirus replication. Cells derived from HIE have also successfully been subjected to genetic manipulation using viral vectors as well as CRISPR/Cas9 technology, thereby allowing studies to identify antiviral signaling pathways important in controlling norovirus infection. RNA sequencing using HIE cells has been used to investigate the transcriptional landscape during norovirus infection and to identify antiviral genes important in infection. Other cell culture platforms such as the microfluidics-based gut-on-chip technology in combination with the HIE/HIO system also have the potential to address fundamental questions on innate immunity to human norovirus. In this review, we highlight the recent advances in understanding the innate immune response to human norovirus infections in the HIE system, including the application of advanced molecular technologies that have become available in recent years such as the CRISPR/Cas9 and RNA sequencing, as well as the potential application of single cell transcriptomics, viral proteomics, and gut-on-a-chip technology to further elucidate innate immunity to norovirus.

Innate Lymphoid Cells and Intestinal Inflammatory Disorders.

Innate lymphoid cells (ILCs) are a population of lymphoid cells that do not express T cell or B cell antigen-specific receptors. They are largely tissue-resident and enriched at mucosal sites to play a protective role against pathogens. ILCs mimic the functions of CD4 T helper (Th) subsets. Type 1 innate lymphoid cells (ILC1s) are defined by the expression of signature cytokine IFN-γ and the master transcription factor T-bet, involving in the type 1 immune response; ILC2s are characterized by the expression of signature cytokine IL-5/IL-13 and the master transcription factor GATA3, participating in the type 2 immune response; ILC3s are RORγt-expressing cells and are capable of producing IL-22 and IL-17 to maintain intestinal homeostasis. The discovery and investigation of ILCs over the past decades extends our knowledge beyond classical adaptive and innate immunology. In this review, we will focus on the roles of ILCs in intestinal inflammation and related disorders.

Salmonella Typhimurium Adhesin OmpV Activates Host Immunity To Confer Protection against Systemic and Gastrointestinal Infection in Mice.

Salmonella enterica Typhimurium is a rod-shaped Gram-negative bacterium that mostly enters the human body through contaminated food. It causes a gastrointestinal disorder called salmonellosis in humans and typhoid-like systemic disease in mice. OmpV, an outer membrane protein of S. Typhimurium, helps in adhesion and invasion of bacteria to intestinal epithelial cells and thus plays a vital role in the pathogenesis of S. Typhimurium. In this study, we have shown that intraperitoneal immunization with OmpV is able to induce high IgG production and protection against systemic disease. Further, oral immunization with OmpV-incorporated proteoliposome (OmpV-proteoliposome [PL]) induces production of high IgA antibody levels and protection against gastrointestinal infection. Furthermore, we have shown that OmpV induces Th1 bias in systemic immunization with purified OmpV, but both Th1 and Th2 polarization in oral immunization with OmpV-proteoliposome (PL). Additionally, we have shown that OmpV activates innate immune cells, such as monocytes, macrophages, and intestinal epithelial cells, in a Toll-like receptor 2 (TLR2)-dependent manner. Interestingly, OmpV is recognized by the TLR1/2 heterodimer in monocytes, but by both TLR1/2 and TLR2/6 heterodimers in macrophages and intestinal epithelial cells. Further, downstream signaling involves MyD88, interleukin-1 receptor-associated kinase (IRAK)-1, mitogen-activated protein kinase (MAPK) (both p38 and Jun N-terminal protein kinase (JNK)), and transcription factors NF-κB and AP-1. Due to its ability to efficiently activate both the innate and adaptive immune systems and protective efficacy, OmpV can be a potential vaccine candidate against S. Typhimurium infection. Further, the fact that OmpV can be recognized by both TLR1/2 and TLR2/6 heterodimers increases its potential to act as good adjuvant in other vaccine formulations.

Immunogenicity and Efficacy of Live-Attenuated Salmonella Typhimurium Vaccine Candidate CVD 1926 in a Rhesus Macaque Model of Gastroenteritis.

Salmonella Typhimurium is a common cause of foodborne gastroenteritis and a less frequent but important cause of invasive disease, especially in developing countries. In our previous work, we showed that a live-attenuated S. Typhimurium vaccine (CVD 1921) was safe and immunogenic in rhesus macaques, although shed for an unacceptably long period (10 days) postimmunization. Consequently, we engineered a new strain, CVD 1926, which was shown to be safe and immunogenic in mice, as well as less reactogenic in mice and human cell-derived organoids than CVD 1921. In this study, we assessed the reactogenicity and efficacy of CVD 1926 in rhesus macaques. Animals were given two doses of either CVD 1926 or saline perorally. The vaccine was well-tolerated, with shedding in stool limited to a mean of 5 days. All CVD 1926-immunized animals had both a serological and a T cell response to vaccination. At 4 weeks postimmunization, animals were challenged with wild-type S. Typhimurium I77. Unvaccinated (saline) animals had severe diarrhea, with two animals succumbing to infection. Animals receiving CVD 1926 were largely protected, with only one animal having moderate diarrhea. Vaccine efficacy in this gastroenteritis model was 80%. S. Typhimurium vaccine strain CVD 1926 was safe and effective in rhesus macaques and shed for a shorter period than other previously tested live-attenuated vaccine strains. This strain could be combined with other live-attenuated Salmonella vaccine strains to create a pan-Salmonella vaccine.

STAT2 dependent Type I Interferon response promotes dysbiosis and luminal expansion of the enteric pathogen Salmonella Typhimurium.

The mechanisms by which the gut luminal environment is disturbed by the immune system to foster pathogenic bacterial growth and survival remain incompletely understood. Here, we show that STAT2 dependent type I IFN signaling contributes to the inflammatory environment by disrupting hypoxia enabling the pathogenic S. Typhimurium to outgrow the microbiota. Stat2-/- mice infected with S. Typhimurium exhibited impaired type I IFN induced transcriptional responses in cecal tissue and reduced bacterial burden in the intestinal lumen compared to infected wild-type mice. Although inflammatory pathology was similar between wild-type and Stat2-/- mice, we observed decreased hypoxia in the gut tissue of Stat2-/- mice. Neutrophil numbers were similar in wild-type and Stat2-/- mice, yet Stat2-/- mice showed reduced levels of myeloperoxidase activity. In vitro, the neutrophils from Stat2-/- mice produced lower levels of superoxide anion upon stimulation with the bacterial ligand N-formylmethionyl-leucyl-phenylalanine (fMLP) in the presence of IFNα compared to neutrophils from wild-type mice, indicating that the neutrophils were less functional in Stat2-/- mice. Cytochrome bd-II oxidase-mediated respiration enhances S. Typhimurium fitness in wild-type mice, while in Stat2-/- deficiency, this respiratory pathway did not provide a fitness advantage. Furthermore, luminal expansion of S. Typhimurium in wild-type mice was blunted in Stat2-/- mice. Compared to wild-type mice which exhibited a significant perturbation in Bacteroidetes abundance, Stat2-/- mice exhibited significantly less perturbation and higher levels of Bacteroidetes upon S. Typhimurium infection. Our results highlight STAT2 dependent type I IFN mediated inflammation in the gut as a novel mechanism promoting luminal expansion of S. Typhimurium.

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.

The Impact of Circulating Antibody on Group B Streptococcus Intestinal Colonization and Invasive Disease.

Gastrointestinal (GI) colonization with group B Streptococcus (GBS) is an important precursor to late-onset (LO) disease in infants. The host-pathogen interactions that mediate progression to invasive disease remain unknown due, in part, to a paucity of robust model systems. Passively acquired maternal GBS-specific antibodies protect newborns from early-onset disease, yet their impact on GI colonization and LO disease is unexplored. Using murine models of both perinatal and postnatal GBS acquisition, we assessed the kinetics of GBS GI colonization, progression to invasive disease, and the role of GBS-specific IgG production in exposed offspring and juvenile mice at age 12 and 14 days, respectively. We defined LO disease as >7 days of life in the perinatal model. We studied the impact of maternal immunization using a whole-cell GBS vaccine on the duration of intestinal colonization and progression to invasive disease after postnatal GBS exposure in offspring. Animals exhibit sustained GI colonization following both perinatal and postnatal exposure to GBS, with 21% and 27%, respectively, developing invasive disease. Intestinal colonization with GBS induces an endogenous IgG response within 20 days of exposure. Maternal vaccination with whole-cell GBS induces production of GBS-specific IgG in dams that is vertically transmitted to their offspring but does not decrease the duration of GBS intestinal colonization or reduce LO mortality following postnatal GBS exposure. Both perinatal and postnatal murine models of GBS acquisition closely recapitulate the human disease state, in which GBS colonizes the intestine and causes LO disease. We demonstrate both endogenous production of anti-GBS IgG in juvenile mice and vertical transfer of antibodies to offspring following maternal vaccination. These models serve as a platform to study critical host-pathogen interactions that mediate LO GBS disease.

Systemic BK Virus Infection in a Pediatric Patient With Severe Combined Immunodeficiency.

Human BK virus (BKV) infection is known to occur mostly during childhood with the establishment of latent infection with no tissue damage or clinical manifestations. However, conditions causing immunosuppression can lead to increased virus replication and tissue damage. Although the tissues most commonly involved are the kidneys, bladder, ureters and, to some extent, brain tissue, there are some reports that suggest that BKV may cause multisystemic infections. In this case, a 12-month-old child was seen to suffer from multiple gastrointestinal infections. This prompted a search for immunodeficiencies, which revealed the presence of severe combined immunodeficiency. The child was eventually hospitalized and continued showing recurrent bouts of gastroenteritis as well as lower respiratory infection. After multiple antibiotic courses, he developed acute kidney injury, a hemophagocytic syndrome, and eventually respiratory failure, which led to his death a year later. Autopsy findings revealed the presence of a disseminated BKV infection involving the kidneys, ureters, leptomeninges, and pancreas. Analysis of the literature failed to show any previous case of BKV pancreatitis. The present case suggests that BKV can damage more tissues than previously reported and may be responsible for systemic infections in immunosuppressed patients.

Immunosensor Based on Long-Period Fiber Gratings for Detection of Viruses Causing Gastroenteritis.

Since the norovirus is the main cause of acute gastroenteritis all over the world, its fast detection is crucial in medical diagnostics. In this work, a rapid, sensitive, and selective optical fiber biosensor for the detection of norovirus virus-like particles (VLPs) is reported. The sensor is based on highly sensitive long-period fiber gratings (LPFGs) coated with antibodies against the main coat protein of the norovirus. Several modification methods were verified to obtain reliable immobilization of protein receptors on the LPFG surface. We were able to detect 1 ng/mL norovirus VLPs in a 40-min assay in a label-free manner. Thanks to the application of an optical fiber as the sensor, there is a possibility to increase the user's safety by separating the measurement point from the signal processing setup. Moreover, our sensor is small and light, and the proposed assay is straightforward. The designed LPFG-based biosensor could be applied in both fast norovirus detection and in vaccine testing.

Influence of histo blood group antigen expression on susceptibility to enteric viruses and vaccines.

PURPOSE OF REVIEW: Gastroenteritis results in substantial morbidity and mortality worldwide, especially in young children in low-and-middle-income settings. Rotavirus and norovirus are the leading causes of viral gastroenteritis. Although introduction of rotavirus vaccines into childhood immunization programmes has reduced disease burden, vaccine effectiveness remains low in developing countries. Norovirus is replacing rotavirus as the most common cause of diarrhea hospitalization in settings where rotavirus vaccines are highly effective. Genetically determined host factors, such as expression of histo blood group antigens (HBGAs) are hypothesized to play key roles in susceptibility to infections and gastroenteritis caused by these virus, as well as influence vaccine take. RECENT FINDINGS: Epidemiology studies provide strong support for virus genotype-dependent effects of host HBGA expression, specifically secretor status on susceptibility to rotavirus and norovirus. Secretor-positive persons are significantly more susceptible to gastroenteritis caused by rotavirus P[8] genotype, and to infection with the GII.4 genotype of human norovirus. There is increasing data on the role of secretor status on rotavirus vaccine take but results are currently conflicting. For analyses involving young infants, maternal HBGA status is an important factor to be considered in future studies. SUMMARY: Genetically determined HBGA expression influences susceptibility to enteric viruses of public health importance.

Emergence of G12P[6] rotavirus strains among hospitalised children with acute gastroenteritis in Belém, Northern Brazil, following introduction of a rotavirus vaccine.

Species A rotavirus still remains a major cause of acute gastroenteritis in infants and young children. Globally, six genotypes (G1P[8], G2P[4], G3P[8], G4P[8], G9P[8] and G12P[8]) account for >90% of circulating strains; however, genotype G12 in combination with P[6] or P[9] has been detected at increasing rates. We sought to broaden our knowledge about the rotavirus strains circulating during the early post-vaccine-introduction period. Stool samples were obtained from children hospitalised for acute gastroenteritis in Belém, Northern Brazil, from May 2008 to May 2011 and examined by reverse transcription polymerase chain reaction and nucleotide sequencing. A total of 122 out of the original 1076 rotavirus strains were judged to be non-typeable in the first analysis and were therefore re-examined. G2P[4] was the most prevalent genotype (58.0%), followed by G1P[8] (16.9%), and G12P[6] (7.5%). G12P[6] strains were identified at similar rates during the first (2.5%) and second (3.9%) years, and the rate jumped to 15.6% in the third year. Analysis of VP7 sequences of the G12P[6] strains showed that they belonged to lineage III. In addition, co-circulating G12P[6] strains displaying long and short RNA patterns were found to belong to the Wa-like and DS-1-like constellation, respectively. Additional unusual circulating strains G12P[9] and G3P[9] were also identified. This hospital-based study showed a high prevalence of G12P[6] strains in the third year of surveillance. Our results highlight the need for continuous longitudinal monitoring of circulating rotavirus strains after introduction of rotavirus vaccines in Brazil and elsewhere.

Linear epitope binding antibodies against GII.3 Norovirus exhibit no histo-blood group antigens (HBGAs) blocking effects.

Noroviruses are leading cause of acute gastroenteritis worldwide. In our previous study, we established an in vitro histo-blood group antigens (HBGAs) binding blockade assay against GII.3 Norovirus virus like particles (VLPs) with trypsin digestion. In this study, we characterized the blocking antibody binding site and epitope type (linear or conformational) by using hyperimmune sera produced against different antigens. VP1 from Jingzhou402 (GII.3, JZ402) strain was expressed by using pGEX-6p-1 expression vector and the insoluble proteins were purified for immunization in rabbit. Previously characterized chimeric VP1-assembled VLPs (GII.4-VP1/GII.3-P2) were used to immunize guinea pig. Peptides reactive with hyperimmune serum against VLPs derived from the VP1 of JZ402 strain were conjugated with BSA and used to immunize rabbits. Hyperimmune sera against above antigens and JZ402 and JZ403 strain-derived VLPs were used to compare their HBGAs blocking effects. Rabbit anti-GST-VP1 and BSA-peptide conjugated hyperimmune sera demonstrated no blocking effects against the binding of GII.3 and GII.4 NoV VLPs to salivary HBGAs. Guinea pig anti-GII.4-VP1/GII.3-P2 hyperimmune serum blocked the binding of trypsin cleaved GII.3 VLPs to salivary HBGAs with no or very weak blocking effects against the binding of GII.4 VLPs to salivary HBGAs. Our data indicated that HBGAs blocking antibodies primarily bound the P2 domain of GII.3 NoV VP1 and their binding epitopes were most probably conformation-dependent.

TLR9 Regulates the NF-κB-NLRP3-IL-1ß Pathway Negatively in Salmonella-Induced NKG2D-Mediated Intestinal Inflammation.

TLRs are key sensors for conserved bacterial molecules and play a critical role in host defense against invading pathogens. Although the roles of TLRs in defense against pathogen infection and in maintaining gut immune homeostasis have been studied, the precise functions of different TLRs in response to pathogen infection in the gut remain elusive. The present study investigated the role of TLR signaling in defense against the Gram-negative bacterial pathogen Salmonella typhimurium The results indicated that TLR9-deficient mice were more susceptible to S. typhimurium infection compared with wild-type and TLR2- or TLR4-deficient mice, as indicated by more severe intestinal damage and the highest bacterial load. TLR9 deficiency in intestinal epithelial cells (IECs) augmented the activation of NF-κB and NLRP3 inflammasomes significantly, resulting in increased secretion of IL-1ß. IL-1ß increased the expression of NKG2D on intestinal intraepithelial lymphocytes and NKG2D ligands on IECs, resulting in higher susceptibility of IECs to cytotoxicity of intestinal intraepithelial lymphocytes and damage to the epithelial barrier. We proposed that TLR9 regulates the NF-κB-NLRP3-IL-1ß pathway negatively in Salmonella-induced NKG2D-mediated intestinal inflammation and plays a critical role in defense against S. typhimurium infection and in the protection of intestinal integrity.