Malnutrition increases intestinal permeability and the risk of sepsis, yet mechanisms underlying malnutrition-induced gut barrier dysfunction are poorly defined. Here, we aimed to determine how the gut microbiome and microbiota-derived metabolites influence intestinal barrier function in the malnourished host. We induced malnutrition in specific pathogen-free (SPF) and germ-free (GF) mice using a low-protein, low-fat diet. Colonic permeability was quantified in Ussing chambers and invasive bacteria were cultured from liver and spleen. Targeted metabolomics identified microbial metabolites depleted in malnutrition. Candidate metabolites were screened in human-derived colonoid monolayers and administered to malnourished mice to determine whether gut barrier dysfunction can be rescued. Malnutrition thinned the colonic mucus layer, increased gut barrier permeability, and facilitated bacterial translocation in male, but not female, SPF mice. Malnourished GF mice exhibited normal barrier function. In the malnourished intestine, a subset of microbial short-chain fatty acids, the branched-chain fatty acids (BCFAs), was depleted in SPF mice of both sexes. Treating human-derived colonoid monolayers with BCFAs, especially isovalerate, increased transepithelial electrical resistance and altered the expression of genes associated with epithelial junction complexes. In malnourished male SPF mice, either enemas with isovalerate or gavages with its branched-chain amino acid fermentation substrate, leucine, restored the localization of the integral membrane protein claudin-8 within the colonic crypt and reduced barrier permeability. Together, these findings identify BCFAs, including isovalerate, as microbiota-derived regulators of intestinal junction complexes and barrier integrity. We propose the branched-chain amino acid leucine as a microbiota-directed precision nutrition therapy that could target intestinal barrier dysfunction in malnutrition.

Intestinal IgA, produced by local intestinal B cells, is thought to play a major role in protection against intestinal infections. Rotavirus, a well-characterized intestinal virus, induces a rapid viral-specific intestinal IgA response that occurs in the absence of T cells. Previous work has indicated that dendritic cells facilitate the early IgA response to rotavirus. To determine whether the early Peyer's patch B cell activation associated with rotavirus infection in mice requires dendritic cells, we depleted dendritic cells and assessed B cell activation. Depletion of CD11c+ cells in vivo prior to infection resulted in a complete abrogation of Peyer's patch B cell activation. With the use of in vitro cell-based assays, CD11c+, but not T or CD11b+ cells, was shown to be essential for rotavirus-induced activation of B cells. Investigation of several pathways of B cell activation revealed that dendritic cell expression of MyD88 and signaling through the type I interferon receptor were critical for the ability of the virus to induce B cell activation. These findings indicate that CD11c+ dendritic cells can modulate B cell responses to viruses through toll-like receptor and type I interferon signaling pathways.IMPORTANCEDendritic cells are key mediators of immune responses in the intestine. They can capture and process rotavirus antigens and present these antigens to B cells, which produce critical IgA antibody that is essential for clearance of rotavirus infection and protection from reinfection. In the work presented here, we demonstrate that dendritic cell expression of MyD88, a key component of pattern recognition pathways, and not classical IgA pathway molecules such as BAFF and APRIL, is critical for the ability of the dendritic cell to induce the activation of B cells. Our findings emphasize the important role that dendritic cells play in initiating and regulating immune responses including T cell-independent B cell activation. A consideration of the role of dendritic cells in B cell activation and antibody production is an important feature in the development of therapeutic and preventive modalities to combat intestinal viral infections.

Patients with common variable immunodeficiency (CVID) who develop noninfectious complications (NIC) have worse clinical outcomes than those with infections only (INF). While gut microbiome aberrations have been linked to NIC, reductionist animal models that accurately recapitulate CVID are lacking. Our aim in this study was to uncover potential microbiome roles in the development of NIC in CVID. We performed whole-genome shotgun sequencing on fecal samples from CVID patients with NIC, INF, and their household controls. We also performed fecal microbiota transplants from CVID patients to germ-free mice. We found potentially pathogenic microbes Streptococcus parasanguinis and Erysipelatoclostridium ramosum were enriched in gut microbiomes of CVID patients with NIC. In contrast, Fusicatenibacter saccharivorans and Anaerostipes hadrus, known to suppress inflammation and promote healthy metabolism, were enriched in gut microbiomes of INF CVID patients. Fecal microbiota transplant from NIC, INF, and their household controls into germ-free mice revealed gut dysbiosis patterns only in recipients from CVID patients with NIC, but not in those from INF CVID or household controls recipients. Our findings provide a proof of concept that fecal microbiota transplant from CVID patients with NIC to germ-free mice recapitulates microbiome alterations observed in the donors.

Malnutrition decreases intestinal bile acids, resulting in inefficient nutrient absorption and impaired catch-up growth. Mechanisms by which bile acid depletion occurs in malnutrition are unknown. Using a mouse model of early-life malnutrition, we explored bile acid homeostasis, focusing on transcriptional repression of oxysterol 7α-hydroxylase (CYP7B1), a rate-limiting enzyme in the alternative pathway of bile acid biosynthesis, by sterol regulatory element-binding protein-1c (SREBP-1c), a master regulator of lipid metabolism. Mice were maintained on a low-protein, low-fat, or isocaloric control chow until 8 wk of age, when livers were harvested for proteome profiling, western blot, reverse transcription quantitative real-time PCR, and chromatin immunoprecipitation. Cultured hepatocytes and mice were treated with the SREBP-1c inhibitors fatostatin and betulin to determine whether this therapeutic strategy rescues CYP7B1 expression and bile acid synthesis in malnutrition. Malnutrition decreased the bile acid pool size and altered the expression of multiple hepatic cytochrome P450 enzymes, with profound depletion of CYP7B1, in males but not females. Malnutrition activated SREBP-1c and led to its enrichment at a Cyp7b1 gene regulatory region that featured loss of binding by the basal transcriptional activator specificity protein 1 (SP1). Treatment of cultured hepatocytes or malnourished mice with the SREBP-1c inhibitors fatostatin or betulin increased CYP7B1 expression. Both drugs rescued the bile acid pool size in malnourished mice. These results suggest that malnutrition impairs bile acid synthesis via transcriptional repression of Cyp7b1 by SREBP-1c. SREBP-1c inhibitors restore hepatic CYP7B1 expression and bile acid synthesis.NEW & NOTEWORTHY We applied liver proteomics to a unique mouse model of early-life malnutrition to reveal a novel mechanism of suppression of bile acid synthesis. Malnutrition activates the nuclear protein SREBP-1c, which displaces the transcriptional activator SP1 from the promoter of the Cyp7b1 gene. Two different SREBP-1c inhibitors rescue CYP7B1 expression in vitro and rescue the bile acid pool in malnourished mice. This discovery might facilitate novel adjunct therapies to enhance nutritional rehabilitation in malnourished children.

Abstract Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

ABSTRACT Enteric pathogen rotavirus (RV) primarily infects mature enterocytes at the tips of the intestinal villi; however, the role of secretory Paneth and goblet cells in RV pathogenesis remains unappreciated. Atoh1 knockout mice (Atoh1cKO) were used to conditionally delete Paneth, goblet, and enteroendocrine cells in the epithelium to investigate the role of secretory cells in RV infection. Unexpectedly, the number of infected enterocytes and the amount of RV shedding in the stool were greatly decreased following secretory cell deletion. Resistance to RV infection persisted for 7 days after virus inoculation, and Atoh1 knockout mice co-housed with infected wild-type mice were uninfected, based on lack of shedding virus, despite the highly infectious nature of RV. This response was directly proportional to the extent of secretory cell deletion, with infection predominantly occurring in areas containing intact secretory cells. RV infection of Muc2 knockout mice recapitulated the secretory cell deletion phenotype, indicating that goblet cell loss is responsible for attenuated infection. Transcriptome analysis of Atoh1cKO intestine via single-cell RNA sequencing revealed downregulation of MHC II molecules specifically in tip enterocytes, and MHC II -/- mice were likewise resistant to RV infection. These data suggest a previously unknown role for both MUC2 and MHC II expression in susceptibility to RV infection. IMPORTANCE Rotavirus (RV) is a highly contagious pathogen that primarily infects mature intestinal enterocytes. Murine rotavirus readily infects infant and adult mice, enabling evaluation of RV infection and immunity. We report that mice lacking secretory cells are one of the few genetically modified mouse lines not susceptible to murine rotavirus. Further investigation revealed loss of mucin 2 (MUC2) expression or major histocompatibility complex II (MCH II) expression recapitulated this rare resistance to rotavirus infection, suggesting a previously unrecognized link between secretory cell products and major histocompatibility complex II expression. Furthermore, these mouse models provide a platform to investigate rotavirus pathogenesis.

Purpose Patients with non-infectious complications have worse clinical outcomes in common variable immunodeficiency (CVID) than those with infections-only. Non-infectious complications are associated with gut microbiome aberrations, but there are no reductionist animal models that emulate CVID. Our aim in this study was to uncover potential microbiome roles in the development of non-infectious complications in CVID. Methods We examined fecal whole genome shotgun sequencing from patients CVID, and non-infectious complications, infections-only, and their household controls. We also performed Fecal Microbiota transplant from CVID patients to Germ-Free Mice. Results We found potentially pathogenic microbes Streptococcus parasanguinis and Erysipelatoclostridium ramosum were enriched in gut microbiomes of CVID patients with non-infectious complications. In contrast, Fusicatenibacter saccharivorans and Anaerostipes hadrus , known to suppress inflammation and promote healthy metabolism, were enriched in gut microbiomes of infections-only CVID patients. Fecal microbiota transplant from non-infectious complications, infections-only, and their household controls into germ-free mice revealed gut dysbiosis patterns in recipients from CVID patients with non-infectious complications, but not infections-only CVID, or household controls recipients. Conclusion Our findings provide a proof of concept that fecal microbiota transplant from CVID patients with non-infectious complications to Germ-Free mice recapitulates microbiome alterations observed in the donors.

BACKGROUND:Early-life events impact maturation of the gut microbiome, enteric nervous system, and gastrointestinal motility. We examined three regions of gastric tissue to determine how maternal separation and gut microbes influence the structure and motor function of specific regions of the neonatal mouse stomach. METHODS:Germ-free and conventionally housed C57BL/6J mouse pups underwent timed maternal separation (TmSep) or nursed uninterrupted (controls) until 14 days of life. We assessed gastric emptying by quantifying the progression of gavaged fluorescein isothiocyanate (FITC)-dextran. With isolated rings of forestomach, corpus, and antrum, we measured tone and contractility by force transduction, gastric wall thickness by light microscopy, and myenteric plexus neurochemistry by whole-mount immunostaining. KEY RESULTS:Regional gastric sampling revealed site-specific differences in contractile patterns and myenteric plexus structure. In neonatal mice, TmSep prolonged gastric emptying. In the forestomach, TmSep increased contractile responses to carbachol, decreased muscularis externa and mucosa thickness, and increased the relative proportion of myenteric plexus nNOS+ neurons. Germ-free conditions did not appreciably alter the structure or function of the neonatal mouse stomach and did not impact the changes caused by TmSep. CONCLUSIONS AND INFERENCES:A regional sampling approach facilitates site-specific investigations of murine gastric motor physiology and histology to identify site-specific alterations that may impact gastrointestinal function. Delayed gastric emptying in TmSep is associated with a thinner muscle wall, exaggerated cholinergic contractile responses, and increased proportions of inhibitory myenteric plexus nNOS+ neurons in the forestomach. Gut microbes do not profoundly affect the development of the neonatal mouse stomach or the gastric pathophysiology that results from TmSep.

The nutrient sensing nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) regulates the host response to short-term fasting by inducing hepatic transcriptional programming of ketogenesis, fatty acid oxidation and transport, and autophagy. This adaptation is ineffective in chronically undernourished individuals, among whom dyslipidemia and hepatic steatosis are common. We recently reported that hepatic PPARα protein is profoundly depleted in male mice undernourished by a low-protein, low-fat diet. Here, we identify PPARα as a deacetylation target of the NAD-dependent deacetylase sirtuin-1 (SIRT1) and link this to the decrease in PPARα protein levels in undernourished liver. Livers from undernourished male mice expressed high levels of SIRT1, with decreased PPARα acetylation and strongly decreased hepatic PPARα protein. In cultured hepatocytes, PPARα protein levels were decreased by transiently transfecting constitutively active SIRT1 or by treating cells with the potent SIRT1 activator resveratrol, while silencing SIRT1 increased PPARα protein levels. SIRT1 expression is correlated with increased PPARα ubiquitination, suggesting that protein loss is due to proteasomal degradation. In accord with these findings, the dramatic loss of hepatic PPARα in undernourished male mice was completely restored by treating mice with the proteasome inhibitor bortezomib. Similarly, treating undernourished mice with the SIRT1 inhibitor selisistat/EX-527 completely restored hepatic PPARα protein. These data suggest that induction of SIRT1 in undernutrition results in hepatic PPARα deacetylation, ubiquitination, and degradation, highlighting a new mechanism that mediates the liver's failed adaptive metabolic responses in chronic undernutrition.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiomewas char-acterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treat-ment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila rein-troduction restored mucusthinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neu-tropenic fever and may guide future microbiome-based preventive strategies.

We seek to identify the role of the microbiome in immune dysregulation in Common Variable Immunodeficiency (CVID).CVID patients have two distinct phenotypes: patients who develop infections-only (INF), and patients who develop autoimmune and inflammatory complications (noninfectious complications (NIC)).Compared to INF-CVID, NIC-CVID patients have higher morbidity and mortality rates.Studies showed associations between NIC-CVID and gut microbiome aberrations, including imbalanced microbial composition, leaky gut, and systemic inflammation.To investigate the role of dysbiosis in the immune responses in CVID, we performed fecal microbiota transplant (FMT) from CVID patients to C57Bl/6J Germ-Free (GF) mice.GF-mice received FMT from NIC-CVID, INF-CVID, or a healthy control (CTL).Thirty days later, gut microbial alpha and beta diversity were significantly different between NIC, INF, and CTL-FMT recipients.Furthermore, FMT recipients from all CVID donors (NIC and INF) had significantly higher serum IgG2b and IgG2c, indicating inflammation.We next treated 50% of the mice in each group with normal saline (NS) and 50% with a single dose of intraperitoneal human IgG (h-IgG).Compared to NS, h-IgG resulted in a significant change in gut microbiome beta diversity in NIC-FMT but not in INF or CTL-FMT recipients.In addition, IgG-treated NIC-FMT recipients had a decrease in the relative abundance of Eggerthella, Bifidobacterium, and Ruminococcaceae genera.Dysregulation of the same genera has been described in CVID patients.Thus, we provide a proof of concept that FMT from CVID patients to GFmice recapitulates the microbiome alteration seen in CVID patients.Our data are from a single donor per group; however, confirmatory experiments using additional donor samples are underway.Our work will help provide new insight into immune dysregulation in CVID.It will also help identify the anti-commensal effects of IgG therapy.Our ultimate goal is to develop therapeutics to modulate the gut microbiome and treat and prevent NIC development in CVID.There is a significant difference in alpha and beta gut microbiome diversity between NIC, INF, and CTL FMT recipients: Beta diversity between GF mice 30 days following FMT.Principal-coordinate analysis plots of Unifraction distances of fecal microbiota generated from 16S rRNA gene sequence analysis.Axis labels indicate the percentage of variance explained by the respective principal coordinate axis.PC1, principal coordinate 1; PC2, principal coordinate 2. Feces were freshly collected from GF (C57Bl/6J) mice (n=8/group, all males, ages 8-12 weeks), mice housed at the Gnotobiotics Core, BCM).Blue: NIC.Red: INF.Green: CTL.

Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first reported in December 2019 in Wuhan, China, and then rapidly spread causing an unprecedented pandemic. A robust serological assay is needed to evaluate vaccine candidates and better understand the epidemiology of coronavirus disease (COVID-19). Methods We used the full-length spike (S) protein of SARS-CoV-2 for the development of qualitative and quantitative IgG and IgA anti-S enzyme linked immunosorbent assays (ELISA). A total of 320 sera used for assay development were comprised of pandemic sera from SARS-CoV-2 infected adults (n=51) and pre-pandemic sera (n=269) including sera from endemic human coronavirus infected adults. Reverse cumulative curves and diagnostic test statistics were evaluated to define the optimal serum dilution and OD cutoff value for IgG anti-S and IgA anti-S ELISAs. The IgG and IgA anti-S, and three functional antibodies (ACE-2 receptor blocking antibody, lentipseudovirus-S neutralizing antibody, and SARS-CoV-2 neutralizing antibody) were measured using additional SARS-CoV-2 PCR positive sera (n=76) and surveillance sera (n=25). Lastly, the IgG and IgA anti-S levels were compared in different demographic groups. Results The optimal serum dilution for the qualitative IgG anti-S ELISA was at 1:1024 yielding a 99.6% specificity, 92.2% sensitivity, 92.9% positive predictive value (PPV), and 99.6% negative predictive value (NPV) at a SARS-CoV-2 seroprevalence of 5%. The optimal serum dilution for the qualitative IgA anti-S ELISA was at 1:128 yielding a 98.9% specificity, 76.5% sensitivity, 78.3% PPV, and 98.8% NPV at the same seroprevalence. Significant correlations were demonstrated between the IgG and IgA (r=0.833 for concentrations, r=0.840 for titers) as well as between IgG and three functional antibodies (r=0.811-0.924 for concentrations, r=0.795-0.917 for titers). The IgG and IgA anti-S levels were significantly higher in males than females (p<0.05), and in adults with moderate/severe symptoms than in adults with mild/moderate symptoms (p<0.001). Conclusion We developed a highly specific and sensitive IgG anti-S ELISA assay to SARS-CoV-2 using full length S protein. The IgG anti-S antibody level was strongly associated with IgA and functional antibody levels in adults with SARS-CoV-2 infection. Gender and disease severity, rather than age, play an important role in antibody levels.

BACKGROUND:Slow gastrointestinal (GI) transit occurs in moderate-to-severe malnutrition. Mechanisms underlying malnutrition-associated dysmotility remain unknown, partially due to lack of animal models. This study sought to characterize GI dysmotility in mouse models of malnutrition.METHODS:Neonatal mice were malnourished by timed maternal separation. Alternatively, low-protein, low-fat diet was administered to dams, with malnourished neonates tested at two weeks or weaned to the same chow and tested as young adults. We determined total GI transit time by carmine red gavage, colonic motility by rectal bead latency, and both gastric emptying and small bowel motility with fluorescein isothiocyanate-conjugated dextran. We assessed histology with light microscopy, ex vivo contractility and permeability with force-transduction and Ussing chamber studies, and gut microbiota composition by 16S rDNA sequencing.KEY RESULTS:Both models of neonatal malnutrition and young adult malnourished males but not females exhibited moderate growth faltering, stunting, and grossly abnormal stomachs. Progression of fluorescent dye was impaired in both neonatal models of malnutrition, whereas gastric emptying was delayed only in maternally separated pups and malnourished young adult females. Malnourished young adult males but not females had atrophic GI mucosa, exaggerated intestinal contractile responses, and increased gut barrier permeability. These sex-specific abnormalities were associated with altered gut microbial communities.CONCLUSIONS & INFERENCES:Multiple models of early-life malnutrition exhibit delayed upper GI transit. Malnutrition affects young adult males more profoundly than females. These models will facilitate future studies to identify mechanisms underlying malnutrition-induced pathophysiology and sex-specific regulatory effects.

Background. Clostridioides (Clostridium) difficile colonization is common among infants. Serological sequelae of infant C. difficile colonization are poorly understood. Methods. In this prospective cohort study of healthy infants, stools serially collected between ages 1-2 and 9-12 months were tested for non-toxigenic and toxigenic C. difficile (TCD). Cultured isolates underwent whole-genome sequencing. Serum collected at 9-12 months underwent measurement of IgA, IgG, and IgM against TCD toxins A and B and neutralizing antibody (NAb) titers against toxin B. For comparison, antitoxin IgG and NAb were measured in cord blood from 50 mothers unrelated to study infants. Results. Among 32 infants, 16 (50%) were colonized with TCD; 12 were first colonized >1 month before serology measurements. A variety of sequence types were identified, and there was evidence of putative in-home (enrolled siblings) and outpatient clinic transmission. Infants first colonized with TCD >1 month prior had significantly greater serum antitoxin IgA and IgG against toxins A (P=.02 for both) and B (P=.009 and .008, respectively) compared with non-TCD-colonized infants, and greater IgG compared with unrelated cord blood (P=.005). Five of 12 (42%) colonized infants had detectable NAb titers compared with zero non-TCD-colonized infants (P=.02). Breastfeeding was not associated with differences in serological measurements. Conclusions. TCD colonization is associated with a humoral immune response against toxins A and B, with evidence of toxin B neutralization in vitro. The extent and duration of protection against CDI later in life afforded by natural C. difficile immunization events require further investigation.

Liver dysfunction, including coagulopathy, is a prominent feature of protein-energy malnutrition. To identify mechanisms underlying malnutrition-associated coagulopathy, we administered a low-protein low-fat diet to lactating dams and examined hepatic transcription and plasma coagulation parameters in young adult weanlings. Malnutrition impacted body composition to a greater extent in male versus female mice. Transcriptional profiles suggested opposing effects of nutrient-sensing nuclear receptors, namely induction of peroxisome proliferator-activated receptor α (PPARα) targets and repression of farnesoid-X-receptor (FXR) targets. Coagulopathy with decreased synthesis of fibrinogen-α (FGA) and factor 11 (F11) was observed in malnourished male animals but not female animals. In primary mouse hepatocytes, FXR agonist increased and PPARα agonist decreased Fga and F11 messenger RNA expression. Nuclear receptor DNA response elements were identified in the Fga and F11 gene regulatory regions, and opposing effects of FXR and PPARα were confirmed with luciferase assays. Unexpectedly, hepatic PPARα protein was markedly depleted in malnourished male liver and was not enriched on Fga or F11 response elements. Rather, there was loss of FXR binding at these response elements. Reduced PPARα protein was associated with loss of hepatocyte peroxisomes, which are necessary for bile acid biosynthesis, and with decreased concentrations of bile acids that function as FXR ligands, most notably the FXR agonist chenodeoxycholic acid. Conclusion: Malnutrition impairs growth and liver synthetic function more severely in male mice than in female mice. Malnourished male mice are coagulopathic and exhibit decreased hepatocyte peroxisomes, FXR agonist bile acids, FXR binding on Fga and F11 gene regulatory elements, and coagulation factor synthesis. These effects are absent in female mice, which have low baseline levels of PPARα, suggesting that nutrient-sensing nuclear receptors regulate coagulation factor synthesis in response to host nutritional status in a sex-specific manner.

The intestinal immune system is emerging as an important contributor to obesity-related insulin resistance, but the role of intestinal B cells in this context is unclear. Here, we show that high fat diet (HFD) feeding alters intestinal IgA + immune cells and that IgA is a critical immune regulator of glucose homeostasis. Obese mice have fewer IgA + immune cells and less secretory IgA and IgA-promoting immune mediators. HFD-fed IgA-deficient mice have dysfunctional glucose metabolism, a phenotype that can be recapitulated by adoptive transfer of intestinal-associated pan-B cells. Mechanistically, IgA is a crucial link that controls intestinal and adipose tissue inflammation, intestinal permeability, microbial encroachment and the composition of the intestinal microbiome during HFD. Current glucose-lowering therapies, including metformin, affect intestinal-related IgA + B cell populations in mice, while bariatric surgery regimen alters the level of fecal secretory IgA in humans. These findings identify intestinal IgA + immune cells as mucosal mediators of whole-body glucose regulation in diet-induced metabolic disease.

Immunoglobulin A (IgA) is the major secretory immunoglobulin isotype found at mucosal surfaces, where it regulates microbial commensalism and excludes luminal factors from contacting intestinal epithelial cells (IECs). IgA is induced by both T cell-dependent and -independent (TI) pathways. However, little is known about TI regulation. We report that IEC endoplasmic reticulum (ER) stress induces a polyreactive IgA response, which is protective against enteric inflammation. IEC ER stress causes TI and microbiotaindependent expansion and activation of peritoneal B1b cells, which culminates in increased lamina propria and luminal IgA. Increased numbers of IgA-producing plasma cells were observed in healthy humans with defective autophagy, who are known to exhibit IEC ER stress. Upon ER stress, IECs communicate signals to the peritoneum that induce a barrier-protective TI IgA response.

Background An important limitation of gastrointestinal motility testing is high variability. Conditions that could contribute to variability, including the duration of pretest fasting and time of day, are rarely reported and have not been examined systematically. This study aimed to explore whether these conditions, as well as age, sex, and strain of mice, affect the results of a standard laboratory test of upper gastrointestinal motility. Methods Male and female 8-week-old C57BL/6J mice received a gastric gavage of fluorescein isothiocyanate (FITC)-conjugated dextran. FITC-dextran distribution was measured 30 minutes later. Mean geometric centers (MGCs) were calculated to determine the effects of short versus prolonged fasting and morning versus afternoon testing. The influence of age was assessed in 2- to 10-week-old animals, and the influence of strain was determined in C57BL/6J, BALB/c, and CD-1 mice. Key results Motility was sexually dimorphic. MGC progressed 19% further in 8-week-old C57BL/6J males versus females when tested in the morning after a short fast. Similar patterns were observed in morning or afternoon testing after overnight fasting. In males, motility was unaffected by time of day; however, MGC progressed 31% further in females tested in the afternoon versus morning after a short fast. Sex differences also were present in CD-1 but not BALB/c mice. Testing in neonates revealed strikingly low variability and no sex differences. Conclusions & inferences Fasting duration, time of day, age, sex, and strain of mice all influence upper gastrointestinal motility testing. Sex differences are not present in neonatal pups, but develop soon after weaning.