A TMPRSS2 inhibitor acts as a pan-SARS-CoV-2 prophylactic and therapeutic.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced owing to emerging variants of concern1,2. Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against variants of concern3,4. Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs) such as TMPRSS2; these proteases cleave the viral spike protein to expose the fusion peptide for cell entry, and thus have an essential role in the virus lifecycle5,6. Here we identify and characterize a small-molecule compound, N-0385, which exhibits low nanomolar potency and a selectivity index of higher than 106 in inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids7. In Calu-3 cells it inhibits the entry of the SARS-CoV-2 variants of concern B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma) and B.1.617.2 (Delta). Notably, in the K18-human ACE2 transgenic mouse model of severe COVID-19, we found that N-0385 affords a high level of prophylactic and therapeutic benefit after multiple administrations or even after a single administration. Together, our findings show that TTSP-mediated proteolytic maturation of the spike protein is critical for SARS-CoV-2 infection in vivo, and suggest that N-0385 provides an effective early treatment option against COVID-19 and emerging SARS-CoV-2 variants of concern.

Diet and deprivation in pregnancy: a rat model to investigate the effects of the maternal diet on the growth of the dam and its offspring.

The offspring of women in the poorest socio-economic groups in Western societies have an increased risk of developing non-communicable disease in adult life. Deprivation is closely related to the consumption of a diet with an excess of energy (sugar and fat), salt and a shortage of key vitamins. To test the hypothesis that this diet adversely affects the development and long-term health of the offspring, we have formulated two rodent diets, one with a nutrient profile corresponding to the diet of pregnant women in the poorest socio-economic group (DEP) and a second that incorporated current UK recommendations for the diet in pregnancy (REC). Female rats were fed the experimental diets for the duration of gestation and lactation and the offspring compared with those from a reference group fed the AIN-93G diet. The growth trajectory of DEP and REC offspring was reduced compared with the AIN-93G. The REC offspring diet had a transient increase in adipose reserves at weaning, but by 30 weeks of age the body composition of all three groups was similar. The maternal diet had no effect on the homoeostatic model assessment index or the insulin tolerance of the offspring. Changes in hepatic gene expression in the adult REC offspring were consistent with an increased hepatic utilisation of fatty acids and a reduction in de novo lipogenesis. These results show that despite changes in growth and adiposity maternal metabolic adaptation minimises the adverse consequences of the imbalanced maternal diet on the metabolism of the offspring.

Flagellin-specific human CAR Tregs for immune regulation in IBD.

Regulatory T cell (Treg) therapy is a promising strategy to treat inflammatory bowel disease (IBD). Data from animal models has shown that Tregs specific for intestinal antigens are more potent than polyclonal Tregs at inhibiting colitis. Flagellins, the major structural proteins of bacterial flagella, are immunogenic antigens frequently targeted in IBD subjects, leading to the hypothesis that flagellin-specific Tregs could be an effective cell therapy for IBD. We developed a novel chimeric antigen receptor (CAR) specific for flagellin derived from Escherichia coli H18 (FliC). We used this CAR to confer FliC-specificity to human Tregs and investigated their therapeutic potential. FliC-CAR Tregs were activated by recombinant FliC protein but not a control flagellin protein, demonstrating CAR specificity and functionality. In a humanized mouse model, expression of the FliC-CAR drove preferential migration to the colon and expression of the activation marker PD1. In the presence of recombinant FliC protein in vitro, FliC-CAR Tregs were significantly more suppressive than control Tregs and promoted the establishment of colon-derived epithelial cell monolayers. These results demonstrate the potential of FliC-CAR Tregs to treat IBD and more broadly show the therapeutic potential of CARs targeting microbial-derived antigens.

Adaptive immune response to Clostridium difficile infection: A perspective for prevention and therapy.

Clostridium difficile infection (CDI) is one of the most important nosocomial illnesses and a major cause of morbidity and mortality. While initial treatment of CDI is usually successful, unprovoked relapses remain an important and frustrating problem. This review examines the literature describing the natural immune response to CDI, and to what extent it can explain the propensity for relapses. In particular, we discuss studies on antibody and, to a lesser extent, B cell and T cell responses in CDI. Despite years of study, there remains incomplete understanding of the natural antibody response to the major pathogenic toxins, TcdA and TcdB, and other bacterial antigens, in CDI. Recent literature suggests that a specific subset of neutralizing antibodies that target the putative carbohydrate-binding domains of TcdB and possibly TcdA have the greatest protective ability. This is further supported by recent successful clinical trials of a humanized monoclonal antibody to the major toxin TcdB. A better understanding of how and why the most protective adaptive immune response develops may lead to improved vaccine and therapeutic targets for recurrent CDI.

Dysregulated Immunity to Clostridioides difficile in IBD Patients Without a History of Recognized Infection.

BACKGROUND & AIMS: Clostridioides difficile is a toxin-secreting bacteria that is an urgent antimicrobial resistance threat, with approximately 25% of patients developing recurrent infections. Inflammatory bowel disease (IBD) patients are at increased risk of severe, recurrent C. difficile infection. METHODS: To investigate a role for C. difficile infection in IBD pathogenesis, we collected peripheral blood and stool from 20 each of ulcerative colitis patients, Crohn's disease patients, and healthy control subjects. We used a flow cytometric activation induced marker assay to quantify C. difficile toxin-specific CD4+ T cells and 16S ribosomal RNA sequencing to study microbiome diversity. RESULTS: We found IBD patients had significantly increased levels of C. difficile toxin B-specific CD4+ T cells, but not immunoglobulin G or immunoglobulin A, compared with healthy control subjects. Within antigen-specific CD4+ T cells, T helper type 17 cells and cells expressing the gut homing receptor integrin ß7 were reduced compared with healthy control subjects, similar to our previous study of non-IBD patients with recurrent C. difficile infection. Stool microbiome analysis revealed that gut homing, toxin-specific CD4+ T cells negatively associated with microbial diversity and, along with T helper type 17 cells, positively associated with bacteria enriched in healthy control subjects. CONCLUSIONS: These data suggest that IBD patients, potentially due to underlying intestinal dysbiosis, experience undiagnosed C. difficile infections that result in impaired toxin-specific immunity. This may contribute to the development of inflammatory T cell responses toward commensal bacteria and provide a rationale for C. difficile testing in IBD patients.

Crohn's disease and ulcerative colitis patients with no history of Clostridioides difficile infection had dysregulated T cell immunity to C. difficile toxin B. This was significantly different from healthy control subjects but similar to non­inflammatory bowel disease patients with recurrent C. difficile infection.

Effects of methyl-deficient diets on methionine and homocysteine metabolism in the pregnant rat.

Although the importance of methyl metabolism in fetal development is well recognized, there is limited information on the dynamics of methionine flow through maternal and fetal tissues and on how this is related to circulating total homocysteine concentrations. Rates of homocysteine remethylation in maternal and fetal tissues on days 11, 19, and 21 of gestation were measured in pregnant rats fed diets with limiting or surplus amounts of folic acid and choline at two levels of methionine and then infused with L-[1-(13)C,(2)H(3)-methyl]methionine. The rate of homocysteine remethylation was highest in maternal liver and declined as gestation progressed. Diets deficient in folic acid and choline reduced the production of methionine from homocysteine in maternal liver only in the animals fed a methionine-limited diet. Throughout gestation, the pancreas exported homocysteine for methylation within other tissues. Little or no methionine cycle activity was detected in the placenta at days 19 and 21 of gestation, but, during this period, fetal tissues, especially the liver, synthesized methionine from homocysteine. Greater enrichment of homocysteine in maternal plasma than placenta, even in animals fed the most-deficient diets, shows that the placenta did not contribute homocysteine to maternal plasma. Methionine synthesis from homocysteine in fetal tissues was maintained or increased when the dams were fed folate- and choline-deficient methionine-restricted diets. This study shows that methyl-deficient diets decrease the remethylation of homocysteine within maternal tissues but that these rates are protected to some extent within fetal tissues.

An in vitro chronic damage model impairs inflammatory and regenerative responses in human colonoid monolayers.

Acute damage to the intestinal epithelium can be repaired via de-differentiation of mature intestinal epithelial cells (IECs) to a stem-like state, but there is a lack of knowledge on how intestinal stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). We developed a chronic-injury model in human colonoid monolayers by repeated rounds of air-liquid interface and submerged culture. We use this model to understand how chronic intestinal damage affects the ability of IECs to (1) respond to microbial stimulation, using the Toll-like receptor 5 (TLR5) agonist FliC and (2) regenerate and protect the epithelium from further damage. Repeated rounds of damage impair the ability of IECs to regrow and respond to TLR stimulation. We also identify mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer. This methodology results in a human model of recurrent IEC injury like that which occurs in IBD.

A methyl-deficient diet fed to rat dams during the peri-conception period programs glucose homeostasis in adult male but not female offspring.

Methyl deficiencies have been implicated in metabolic programming during the periods of oocyte and embryo development. Semisynthetic methyl-deficient diets (MD) with no folic acid, 0.05% choline, and approximately one-half the recommended content of methionine were fed to female rats for 3 wk prior to mating and for the first 5 d of gestation. During the period of MD feeding, plasma homocysteine concentrations were approximately twice those of rats fed the complete (CON) diet. From d 5, both groups received a complete semipurified AIN diet until birth. On d 8, plasma homocysteine concentrations did not differ between the 2 groups. Thereafter, dams and offspring were fed a nonpurified diet for the remainder of the experiment. At 6 mo of age, the homeostatic model assessment (HOMA) index of the male MD offspring tended to be 32% higher (P = 0.053) and peak insulin during an oral glucose tolerance test (oGTT) was 39% higher (P < 0.05) compared with the male CON offspring. There was no difference in the response to an oGTT in the female offspring at 6 mo of age. The increased HOMA index of male MD offspring persisted to 12 mo of age. The peak glucose concentration during oGTT was 23% higher (P < 0.05) in MD compared with the CON males despite 39% greater (P < 0.05) peak insulin concentrations. This study shows that in rats, a physiologically relevant methyl-deficient diet fed during the period of oocyte maturation and preimplantation development programs gender-specific changes in glucose handling by the offspring.

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice.

The COVID-19 pandemic caused by the SARS-CoV-2 virus remains a global public health crisis. Although widespread vaccination campaigns are underway, their efficacy is reduced against emerging variants of concern (VOCs) 1,2 . Development of host-directed therapeutics and prophylactics could limit such resistance and offer urgently needed protection against VOCs 3,4 . Attractive pharmacological targets to impede viral entry include type-II transmembrane serine proteases (TTSPs), such as TMPRSS2, whose essential role in the virus lifecycle is responsible for the cleavage and priming of the viral spike protein 5-7 . Here, we identify and characterize a small-molecule compound, N-0385, as the most potent inhibitor of TMPRSS2 reported to date. N-0385 exhibited low nanomolar potency and a selectivity index of >10 6 at inhibiting SARS-CoV-2 infection in human lung cells and in donor-derived colonoids 8 . Importantly, N-0385 acted as a broad-spectrum coronavirus inhibitor of two SARS-CoV-2 VOCs, B.1.1.7 and B.1.351. Strikingly, single daily intranasal administration of N-0385 early in infection significantly improved weight loss and clinical outcomes, and yielded 100% survival in the severe K18-human ACE2 transgenic mouse model of SARS-CoV-2 disease. This demonstrates that TTSP-mediated proteolytic maturation of spike is critical for SARS-CoV-2 infection in vivo and suggests that N-0385 provides a novel effective early treatment option against COVID-19 and emerging SARS-CoV-2 VOCs.

DNA methylation, insulin resistance, and blood pressure in offspring determined by maternal periconceptional B vitamin and methionine status.

A complex combination of adult health-related disorders can originate from developmental events that occur in utero. The periconceptional period may also be programmable. We report on the effects of restricting the supply of specific B vitamins (i.e., B(12) and folate) and methionine, within normal physiological ranges, from the periconceptional diet of mature female sheep. We hypothesized this would lead to epigenetic modifications to DNA methylation in the preovulatory oocyte and/or preimplantation embryo, with long-term health implications for offspring. DNA methylation is a key epigenetic contributor to maintenance of gene silencing that relies on a dietary supply of methyl groups. We observed no effects on pregnancy establishment or birth weight, but this modest early dietary intervention led to adult offspring that were both heavier and fatter, elicited altered immune responses to antigenic challenge, were insulin-resistant, and had elevated blood pressure-effects that were most obvious in males. The altered methylation status of 4% of 1,400 CpG islands examined by restriction landmark genome scanning in the fetal liver revealed compelling evidence of a widespread epigenetic mechanism associated with this nutritionally programmed effect. Intriguingly, more than half of the affected loci were specific to males. The data provide the first evidence that clinically relevant reductions in specific dietary inputs to the methionine/folate cycles during the periconceptional period can lead to widespread epigenetic alterations to DNA methylation in offspring, and modify adult health-related phenotypes.

How do immune and mesenchymal cells influence the intestinal epithelial cell compartment in inflammatory bowel disease? Let's crosstalk about it!

Intestinal epithelial cells provide a front line of defense by establishing a barrier against food Ags, pathogens, and commensal microorganisms. This defense includes the establishment of a tolerogenic environment in the gastrointestinal (GI) tract. The intestinal epithelium replenishes itself by cell turnover every 4-5 days, and this process is facilitated by various pathways of communication between the intestinal epithelial cells (IECs), the underlying stromal cell network, and professional immune cells, which together help establish a proper intestinal stem cell (ISC) niche in the crypt. However, during a state of inflammation, such as in inflammatory bowel diseases (IBD), these communication pathways can be altered, and this can lead to the development of inflammatory IECs within the crypt that further drive inflammation. Here, we review the current literature looking at crosstalk between immune cells, stromal cells, and IECs: how does the immune system potentially alter the ISC niche, and how do IECs influence intestinal immunity? We discuss the latest research using single cell RNA sequencing and intestinal organoid cultures to help answer these questions. A better understanding of this complex crosstalk can help lead to a better understanding of intestinal biology in general, and more efficient therapeutic approaches to treat IBD.

Regenerative Intestinal Stem Cells Induced by Acute and Chronic Injury: The Saving Grace of the Epithelium?

The intestinal epithelium is replenished every 3-4 days through an orderly process that maintains important secretory and absorptive functions while preserving a continuous mucosal barrier. Intestinal epithelial cells (IECs) derive from a stable population of intestinal stem cells (ISCs) that reside in the basal crypts. When intestinal injury reaches the crypts and damages IECs, a mechanism to replace them is needed. Recent research has highlighted the existence of distinct populations of acute and chronic damage-associated ISCs and their roles in maintaining homeostasis in several intestinal perturbation models. What remains unknown is how the damage-associated regenerative ISC population functions in the setting of chronic inflammation, as opposed to acute injury. What long-term consequences result from persistent inflammation and other cellular insults to the ISC niche? What particular "regenerative" cell types provide the most efficacious restorative properties? Which differentiated IECs maintain the ability to de-differentiate and restore the ISC niche? This review will cover the latest research on damage-associated regenerative ISCs and epigenetic factors that determine ISC fate, as well as provide opinions on future studies that need to be undertaken to understand the repercussions of the emergence of these cells, their contribution to relapses in inflammatory bowel disease, and their potential use in therapeutics for chronic intestinal diseases.

Mycobacterium avium Subspecies paratuberculosis Infects and Replicates within Human Monocyte-Derived Dendritic Cells.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP), a member of the mycobacteriaceae family, causes Johne's disease in ruminants, which resembles Crohn's disease (CD) in humans. MAP was proposed to be one of the causes of human CD, but the evidence remains elusive. Macrophages were reported to be the only cell where MAP proliferates in ruminants and humans and is likely the major producer of TNFα-associated inflammation. However, whether human dendritic cells (DCs), another major antigen-presenting cell (APC), have the ability to harbor MAP and disseminate infection, remains unknown. METHODS: Human monocyte-derived dendritic cells (moDCs) were infected with MAP and phagocytosis and intracellular survival were quantified by immunofluorescence (IF) and colony counts, respectively. MoDC cytokine expression was measured via ELISA and their activation state was measured via flow cytometry. RESULTS: We showed that MAP can infect and replicate in human moDCs as means to evade the immune system for successful infection, through inhibition of the phago-lysosome fusion via the secretion of protein tyrosine phosphatase PtpA. This mechanism initially led to a state of tolerance in moDCs and then subsequently caused a pro-inflammatory response as infection persisted, characterized by the upregulation of IL-6 and TNFα, and downregulation of IL-10. Moreover, we showed that moDCs have the ability to phagocytose up to 18% of MAP, when exposed at a multiplicity of infection of 1:1. CONCLUSION: Infection and subsequent proliferation of MAP within moDCs could provide a unique means for the dissemination of MAP to lymphoid tissue, while altering immune responses to facilitate the persistence of infection of host tissues in CD.

Iron deficiency during pregnancy and lactation modifies the fatty acid composition of the brain of neonatal rats.

Iron deficiency is common in pregnant and lactating women and is associated with reduced cognitive development of the offspring. Since iron affects lipid metabolism, the availability of fatty acids, particularly the polyunsaturated fatty acids required for early neural development, was investigated in the offspring of female rats fed iron-deficient diets during gestation and lactation. Subsequent to the dams giving birth, one group of iron-deficient dams was recuperated by feeding an iron-replete diet. Dams and neonates were killed on postnatal days 1, 3 and 10, and the fatty acid composition of brain and stomach contents was assessed by gas chromatography. Changes in the fatty acid profile on day 3 became more pronounced on day 10 with a decrease in the proportion of saturated fatty acids and a compensatory increase in monounsaturated fatty acids. Long-chain polyunsaturated fatty acids in the n-6 family were reduced, but there was no change in the n-3 family. The fatty acid profiles of neonatal brain and stomach contents were similar, suggesting that the change in milk composition may be related to the changes in the neonatal brain. When the dams were fed an iron-sufficient diet at birth, the effects of iron deficiency on the fatty acid composition of lipids in both dam's milk and neonates' brains were reduced. This study showed an interaction between maternal iron status and fatty acid composition of the offspring's brain and suggests that these effects can be reduced by iron repletion of the dam's diet at birth.

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.

The effects of feeding rats diets deficient in folic acid and related methyl donors on the blood pressure and glucose tolerance of the offspring.

In humans poor maternal folate status is associated with a decrease in infant birth weight. As low birth weight increases the risk of cardiovascular and metabolic disease in adults, an inadequate supply of folic acid in the mother's diet may increase the susceptibility of the offspring to disease. We have fed laboratory rats diets deficient in folic acid and the related methyl donors methionine and choline to examine the effects on growth, blood pressure and insulin action in the offspring. Poor folate status transiently increased fetal growth but did not produce a long-term change in body weight. There were, however, small changes in the hearts of the female offspring. When folate deficiency was combined with low intakes of methionine and choline, the kidneys of the male offspring were proportionately smaller, probably because of the limited availability of methionine. There was no effect on the blood pressure of either the male or female offspring. The pancreatic insulin content of fetuses from animals fed the folate-deficient diets were higher than those of the controls. Following an oral glucose challenge, there was a weak trend for glucose-stimulated insulin release to be increased in the offspring of dams fed the folate-deficient diet. The changes in insulin concentrations were, however, much smaller than the corresponding changes observed in the offspring of animals fed protein-deficient diets. These results suggest that folate deficiency during gestation causes modest changes to the insulin axis of the fetus.

Maternal diets deficient in folic acid and related methyl donors modify mechanisms associated with lipid metabolism in the fetal liver of the rat.

Previously we have examined the effects of diets deficient in folic acid ( - F) or folate deficient with low methionine and choline ( - F LM LC) on the relative abundance of soluble proteins in the liver of the pregnant rat. In the present study we report the corresponding changes in the fetal liver at day 21 of gestation. The abundance of eighteen proteins increased when dams were fed the - F diet. When dams were fed the - F LM LC diet, thirty-three proteins increased and eight decreased. Many of the differentially abundant proteins in the fetal liver could be classified into the same functional groups as those previously identified in the maternal liver, namely protein synthesis, metabolism, lipid metabolism and proteins associated with the cytoskeleton and endoplasmic reticulum. The pattern was consistent with reduced cell proliferation in the - F LM LC group but not in the - F group. Metabolic enzymes associated with lipid metabolism changed in both the - F and - F LM LC groups. The mRNA for carnitine palmitoyl transferase were up-regulated and CD36 (fatty acid translocase) down-regulated in the - F group, suggesting increased mitochondrial oxidation of fatty acids as an indirect response to altered maternal lipid metabolism. In the - F LM LC group the mRNA for acetyl CoA carboxylase was down-regulated, suggesting reduced fatty acid synthesis. The mRNA for transcriptional regulators including PPARalpha and sterol response element-binding protein-1c were unchanged. These results suggest that an adequate supply of folic acid and the related methyl donors may benefit fetal development directly by improving lipid metabolism in fetal as well as maternal tissues.

Interactions between nutrients in the maternal diet and the implications for the long-term health of the offspring.

Nutritional science has traditionally used the reductionist approach to understand the roles of individual nutrients in growth and development. The macronutrient dense but micronutrient poor diets consumed by many in the Western world may not result in an overt deficiency; however, there may be situations where multiple mild deficiencies combine with excess energy to alter cellular metabolism. These interactions are especially important in pregnancy as changes in early development modify the risk of developing non-communicable diseases later in life. Nutrient interactions affect all stages of fetal development, influencing endocrine programming, organ development and the epigenetic programming of gene expression. The rapidly developing field of stem cell metabolism reveals new links between cellular metabolism and differentiation. This review will consider the interactions between nutrients in the maternal diet and their influence on fetal development, with particular reference to energy metabolism, amino acids and the vitamins in the B group.