Tofacitinib uptake by patient-derived intestinal organoids predicts individual clinical responsiveness.

Background & Aims: Despite increasing therapeutic options in the treatment of ulcerative colitis (UC), achieving disease remission remains a major clinical challenge. Nonresponse to therapy is common and clinicians have little guidance in selecting the optimal therapy for an individual patient. This study examined whether patient-derived materials could predict individual clinical responsiveness to the Janus kinase (JAK) inhibitor, tofacitinib, prior to treatment initiation. Method: In 48 patients with UC initiating tofacitinib, we longitudinally collected clinical covariates, stool, and colonic biopsies to analyze the microbiota, transcriptome, and exome variations associated with clinical responsiveness at week 24. We established patient-derived organoids (n = 23) to determine how their viability upon stimulation with proinflammatory cytokines in the presence of tofacitinib related to drug responsiveness in patients. We performed additional biochemical analyses of organoids and primary tissues to identify the mechanism underlying differential tofacitinib sensitivity. Results: The composition of the gut microbiota, rectal transcriptome, inflammatory biomarkers, and exome variations were indistinguishable among UC patients prior to tofacitinib treatment. However, a subset of patient-derived organoids displayed reduced sensitivity to tofacitinib as determined by the ability of the drug to inhibit STAT1 phosphorylation and loss of viability upon cytokine stimulation. Remarkably, sensitivity of organoids to tofacitinib predicted individual clinical patient responsiveness. Reduced responsiveness to tofacitinib was associated with decreased levels of the cationic transporter MATE1, which mediates tofacitinib uptake. Conclusions: Patient-derived intestinal organoids predict and identify mechanisms of individual tofacitinib responsiveness in UC. Specifically, MATE1 expression predicted clinical response to tofacitinib.

Functional characterization of helminth-associated Clostridiales reveals covariates of Treg differentiation.

BACKGROUND: Parasitic helminths influence the composition of the gut microbiome. However, the microbiomes of individuals living in helminth-endemic regions are understudied. The Orang Asli, an indigenous population in Malaysia with high burdens of the helminth Trichuris trichiura, display microbiotas enriched in Clostridiales, an order of spore-forming obligate anaerobes with immunogenic properties. We previously isolated novel Clostridiales that were enriched in these individuals and found that a subset promoted the Trichuris life cycle. In this study, we aimed to further characterize the functional properties of these bacteria. RESULTS: Clostridiales isolates were profiled for their ability to perform 57 enzymatic reactions and produce short-chain fatty acids (SCFAs) and hydrogen sulfide, revealing that these bacteria were capable of a range of activities associated with metabolism and host response. Consistent with this finding, monocolonization of mice with individual isolates identified bacteria that were potent inducers of regulatory T-cell (Treg) differentiation in the colon. Comparisons between variables revealed by these studies identified enzymatic properties correlated with Treg induction and Trichuris egg hatching. CONCLUSION: We identified Clostridiales species that are sufficient to induce high levels of Tregs. We also identified a set of metabolic activities linked with Treg differentiation and Trichuris egg hatching mediated by these newly isolated bacteria. Altogether, this study provides functional insights into the microbiotas of individuals residing in a helminth-endemic region. Video Abstract.

The hyphal-specific toxin candidalysin promotes fungal gut commensalism.

The fungus Candida albicans frequently colonizes the human gastrointestinal tract, from which it can disseminate to cause systemic disease. This polymorphic species can transition between growing as single-celled yeast and as multicellular hyphae to adapt to its environment. The current dogma of C. albicans commensalism is that the yeast form is optimal for gut colonization, whereas hyphal cells are detrimental to colonization but critical for virulence1-3. Here, we reveal that this paradigm does not apply to multi-kingdom communities in which a complex interplay between fungal morphology and bacteria dictates C. albicans fitness. Thus, whereas yeast-locked cells outcompete wild-type cells when gut bacteria are absent or depleted by antibiotics, hyphae-competent wild-type cells outcompete yeast-locked cells in hosts with replete bacterial populations. This increased fitness of wild-type cells involves the production of hyphal-specific factors including the toxin candidalysin4,5, which promotes the establishment of colonization. At later time points, adaptive immunity is engaged, and intestinal immunoglobulin A preferentially selects against hyphal cells1,6. Hyphal morphotypes are thus under both positive and negative selective pressures in the gut. Our study further shows that candidalysin has a direct inhibitory effect on bacterial species, including limiting their metabolic output. We therefore propose that C. albicans has evolved hyphal-specific factors, including candidalysin, to better compete with bacterial species in the intestinal niche.

S1PR1 inhibition induces proapoptotic signaling in T cells and limits humoral responses within lymph nodes.

Effective immunity requires a large, diverse naive T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we addressed how S1P enables T cell survival and the implications for patients treated with S1PR1 antagonists. We found that S1PR1 limited apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization were required to prevent this proapoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naive T cells limited B cell responses. Our findings highlighted an effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggested both limitations and additional uses of this important class of drugs.

Cytokine signature in convalescent SARS-CoV-2 patients with inflammatory bowel disease receiving vedolizumab.

While differential antibody responses SARS-CoV-2 in patients with inflammatory bowel disease (IBD) receiving infliximab and vedolizumab are well-characterized, the immune pathways underlying these differences remain unknown. Prior to COVID-19 vaccine development, we screened 235 patients with IBD receiving biological therapy for antibodies to SARS-CoV-2 and measured serum cytokines. In seropositive patients, we prospectively collected clinical data. We found a cytokine signature in patients receiving vedolizumab who are seropositive compared with seronegative for SARS-CoV-2 antibodies that may be linked to repeated SARS-CoV-2 infections. However, there were no differences between seropositive and seronegative patients receiving infliximab. In this single-center cohort of patients with IBD with anti-SARS-CoV-2 antibodies at the onset of the COVID-19 pandemic, and therefore without influence of vaccination, there is a cytokine signature in patients receiving vedolizumab but not infliximab. These findings lay the groundwork for further studies on immune consequences of viral infection in patients with IBD, which is postulated to evolve from aberrant host-microbe responses.

Mitigation of Osteoclast-Mediated Arthritic Bone Remodeling By Short Chain Fatty Acids.

OBJECTIVE: The objective for this study was to evaluate the effects of short chain fatty acids (SCFAs) on arthritic bone remodeling. METHODS: We treated a recently described preclinical murine model of psoriatic arthritis (PsA), R26STAT3Cstopfl/fl CD4Cre mice, with SCFA-supplemented water. We also performed in vitro osteoclast differentiation assays in the presence of serum-level SCFAs to evaluate the direct impact of these microbial metabolites on maturation and function of osteoclasts. We further characterized the molecular mechanism of SCFAs by transcriptional analysis. RESULTS: The osteoporosis condition in R26STAT3Cstopfl/fl CD4Cre animals is attributed primarily to robust osteoclast differentiation driven by an expansion of osteoclast progenitor cells (OCPs), accompanied by impaired osteoblast development. We show that SCFA supplementation can rescue the osteoporosis phenotype in this model of PsA. Our in vitro experiments revealed an inhibitory effect of the SCFAs on osteoclast differentiation, even at very low serum concentrations. This suppression of osteoclast differentiation enabled SCFAs to impede osteoporosis development in R26STAT3Cstopfl/fl CD4Cre mice. Further interrogation revealed that bone marrow-derived OCPs from diseased mice expressed a higher level of SCFA receptors than those of control mice and that the progenitor cells in the bone marrow of SCFA-treated mice presented a modified transcriptomic landscape, suggesting a direct impact of SCFAs on bone marrow progenitors in the context of osteoporosis. CONCLUSION: We demonstrated how gut microbiota-derived SCFAs can regulate distal pathology (ie, osteoporosis) and identified a potential therapeutic option for restoring bone density in rheumatic disease, further highlighting the critical role of the gut-bone axis in these disorders.

Beyond antiviral: Interferon induced by bacteria maintains tolerance in the gut.

Type I interferons are best known for their antiviral role. Here, Ayala et al. (https://doi.org/10.1084/jem.20230063) reveal that commensal bacteria elicit tonic type I interferons to prime dendritic cells and induce regulatory T cells that maintain a tolerogenic intestinal milieu.

Temporal Tracking of Plasma Cells in vivo Using J-chain CreERT2 Reporter System.

Plasma cells (PCs) are essential for humoral immunity, as they are responsible for the production of antibodies and contribute to immunological memory. Despite their importance, differentiating between long-lived and short-lived PCs in vivo remains a challenge due to a lack of specific markers to distinguish these populations. Addressing this gap, our study introduces a novel J-chain CreERT2 GFP allele (IgJCreERT2) for precise genetic studies of PCs. This model takes advantage of PC-restricted expression of the J-chain gene, enabling temporal and cell-specific tracking of PCs utilizing a tamoxifen-inducible Cre recombinase. Our in vitro and in vivo validation studies of the inducible Cre allele confirmed the fidelity and utility of this model and demonstrated the model's ability to trace the long-lived PC population in vivo following immunization. The IgJCreERT2 model allowed for detailed analysis of surface marker expression on PCs, revealing insights into PC heterogeneity and characteristics. Our findings not only validate the IgJCreERT2 mouse as a reliable tool for studying PCs but also facilitate the investigation of PC dynamics and longevity, particularly in the context of humoral immunity and vaccine responses. This model represents a significant advancement for the in-depth study of PCs in health and disease, offering a new avenue for the exploration of PC biology and immunological memory.

Spatiotemporal-social association predicts immunological similarity in rewilded mice.

Environmental influences on immune phenotypes are well-documented, but our understanding of which elements of the environment affect immune systems, and how, remains vague. Behaviors, including socializing with others, are central to an individual's interaction with its environment. We therefore tracked behavior of rewilded laboratory mice of three inbred strains in outdoor enclosures and examined contributions of behavior, including associations measured from spatiotemporal co-occurrences, to immune phenotypes. We found extensive variation in individual and social behavior among and within mouse strains upon rewilding. In addition, we found that the more associated two individuals were, the more similar their immune phenotypes were. Spatiotemporal association was particularly predictive of similar memory T and B cell profiles and was more influential than sibling relationships or shared infection status. These results highlight the importance of shared spatiotemporal activity patterns and/or social networks for immune phenotype and suggest potential immunological correlates of social life.

Colonization with extended spectrum beta-lactamase and carbapenemases producing Enterobacteriaceae among hospitalized patients at the global level: A systematic review and meta-analysis.

BACKGROUND: Gut commensal bacteria can mediate resistance against pathogenic bacteria. However, exposure to antibiotics and hospitalization may facilitate the emergence of multidrug resistant bacteria. We aimed to conduct a systematic review and meta-analysis to provide comprehensive evidence about colonization rate of extended spectrum beta-lactamase and carbapenemases producing Enterobacteriaceae. METHOD: We used PubMed, Google Scholar and Web of Science data bases to search studies from January 1, 2016 to August10, 2022 about colonization rate of extended spectrum beta-lactamase and carbapenemase producing Enterobacteriaceae. Data were extracted from eligible studies and analyzed using Stata version 16 software. The quality of included studies was assessed using the Joanna Briggs Institute Critical Appraisal tools, and publication bias was assessed using funnel plot and eggers test. RESULTS: We identified 342 studies from the comprehensive data search and data were extracted from 20 studies. The pooled estimate of extended spectrum beta-lactamase and carbapenemase producing Enterobacteriaceae were 45.6%(95%CI: 34.11-57-10) and 16.19% (95% CI: 5.46-26.91) respectively. The predominant extended spectrum beta-lactamase producers were E. coli,32.99% (95% CI: 23.28-42.69) and K. pneumoniae, 11.43% (95% CI:7.98-14.89). Prolonged hospitalization was linked to carbapenemase producing Enterobacteriaceae colonization with the odds of 14.77 (95% CI: -1.35-30.90) at admission and 45.63 (95% CI: 0.86-92.12) after ≥7 days of admission. CONCLUSION: The pooled estimate of extended spectrum beta-lactamase and carbapenemase producing Enterobacteriaceae were high. This indicates the need for strong mitigation strategies to minimize the spread of multidrug-resistant bacteria at the healthcare facilities.

Sphingosine 1-phosphate receptor 1 inhibition induces a pro-apoptotic signaling cascade in T cells.

Effective immunity requires a large, diverse naïve T cell repertoire circulating among lymphoid organs in search of antigen. Sphingosine 1-phosphate (S1P) and its receptor S1PR1 contribute by both directing T cell migration and supporting T cell survival. Here, we address how S1P enables T cell survival, and the implications for patients treated with S1PR1 antagonists. Contrary to expectations, we found that S1PR1 limits apoptosis by maintaining the appropriate balance of BCL2 family members via restraint of JNK activity. Interestingly, the same residues of S1PR1 that enable receptor internalization are required to prevent this pro-apoptotic cascade. Findings in mice were recapitulated in ulcerative colitis patients treated with the S1PR1 antagonist ozanimod, and the loss of naïve T cells limited B cell responses. Our findings highlight an unexpected effect of S1PR1 antagonists on the ability to mount immune responses within lymph nodes, beyond their effect on lymph node egress, and suggest both limitations and novel uses of this important class of drugs.

Bacterial contact induces polar plug disintegration to mediate whipworm egg hatching.

The bacterial microbiota promotes the life cycle of the intestine-dwelling whipworm Trichuris by mediating hatching of parasite eggs ingested by the mammalian host. Despite the enormous disease burden associated with Trichuris colonization, the mechanisms underlying this transkingdom interaction have been obscure. Here, we used a multiscale microscopy approach to define the structural events associated with bacteria-mediated hatching of eggs for the murine model parasite Trichuris muris. Through the combination of scanning electron microscopy (SEM) and serial block face SEM (SBFSEM), we visualized the outer surface morphology of the shell and generated 3D structures of the egg and larva during the hatching process. These images revealed that exposure to hatching-inducing bacteria catalyzed asymmetric degradation of the polar plugs prior to exit by the larva. Unrelated bacteria induced similar loss of electron density and dissolution of the structural integrity of the plugs. Egg hatching was most efficient when high densities of bacteria were bound to the poles. Consistent with the ability of taxonomically distant bacteria to induce hatching, additional results suggest chitinase released from larva within the eggs degrade the plugs from the inside instead of enzymes produced by bacteria in the external environment. These findings define at ultrastructure resolution the evolutionary adaptation of a parasite for the microbe-rich environment of the mammalian gut.

Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

Antimicrobial overproduction sustains intestinal inflammation by inhibiting Enterococcus colonization.

Loss of antimicrobial proteins such as REG3 family members compromises the integrity of the intestinal barrier. Here, we demonstrate that overproduction of REG3 proteins can also be detrimental by reducing a protective species in the microbiota. Patients with inflammatory bowel disease (IBD) experiencing flares displayed heightened levels of secreted REG3 proteins that mediated depletion of Enterococcus faecium (Efm) from the gut microbiota. Efm inoculation of mice ameliorated intestinal inflammation through activation of the innate immune receptor NOD2, which was associated with the bacterial DL-endopeptidase SagA that generates NOD2-stimulating muropeptides. NOD2 activation in myeloid cells induced interleukin-1ß (IL-1ß) secretion to increase the proportion of IL-22-producing CD4+ T helper cells and innate lymphoid cells that promote tissue repair. Finally, Efm was unable to protect mice carrying a NOD2 gene variant commonly found in IBD patients. Our findings demonstrate that inflammation self-perpetuates by causing aberrant antimicrobial activity that disrupts symbiotic relationships with gut microbes.

Functional characterization of helminth-associated Clostridiales reveals covariates of Treg differentiation.

Parasitic helminths influence the composition of the gut microbiome. However, the microbiomes of individuals living in helminth-endemic regions are understudied. The Orang Asli, an indigenous population in Malaysia with high burdens of the helminth Trichuris trichiura, displayed microbiotas enriched in Clostridiales, an order of spore-forming obligate anaerobes previously shown to have immunogenic properties. We previously isolated novel Clostridiales that were enriched in these individuals and found that a subset promoted the Trichuris life cycle. Here, we further characterized the functional properties of these bacteria. Enzymatic and metabolomic profiling revealed a range of activities associated with metabolism and host response. Consistent with this finding, monocolonization of mice with individual isolates identified bacteria that were potent inducers of regulatory T cell (Treg) differentiation in the colon. Comparisons between variables revealed by these studies identified enzymatic properties correlated with Treg induction and Trichuris egg hatching. These results provide functional insights into the microbiotas of an understudied population.

Immunomodulation by Enteric Viruses.

Enteric viruses display intricate adaptations to the host mucosal immune system to successfully reproduce in the gastrointestinal tract and cause maladies ranging from gastroenteritis to life-threatening disease upon extraintestinal dissemination. However, many viral infections are asymptomatic, and their presence in the gut is associated with an altered immune landscape that can be beneficial or adverse in certain contexts. Genetic variation in the host and environmental factors including the bacterial microbiota influence how the immune system responds to infections in a remarkably viral strain-specific manner. This immune response, in turn, determines whether a given virus establishes acute versus chronic infection, which may have long-lasting consequences such as susceptibility to inflammatory disease. In this review, we summarize our current understanding of the mechanisms involved in the interaction between enteric viruses and the immune system that underlie the impact of these ubiquitous infectious agents on our health.

Rewilding of laboratory mice enhances granulopoiesis and immunity through intestinal fungal colonization.

The paucity of blood granulocyte populations such as neutrophils in laboratory mice is a notable difference between this model organism and humans, but the cause of this species-specific difference is unclear. We previously demonstrated that laboratory mice released into a seminatural environment, referred to as rewilding, display an increase in blood granulocytes that is associated with expansion of fungi in the gut microbiota. Here, we find that tonic signals from fungal colonization induce sustained granulopoiesis through a mechanism distinct from emergency granulopoiesis, leading to a prolonged expansion of circulating neutrophils that promotes immunity. Fungal colonization after either rewilding or oral inoculation of laboratory mice with Candida albicans induced persistent expansion of myeloid progenitors in the bone marrow. This increase in granulopoiesis conferred greater long-term protection from bloodstream infection by gram-positive bacteria than by the trained immune response evoked by transient exposure to the fungal cell wall component ß-glucan. Consequently, introducing fungi into laboratory mice may restore aspects of leukocyte development and provide a better model for humans and free-living mammals that are constantly exposed to environmental fungi.

Geography Influences Susceptibility to SARS-CoV-2 Serological Response in Patients With Inflammatory Bowel Disease: Multinational Analysis From the ICARUS-IBD Consortium.

BACKGROUND: Beyond systematic reviews and meta-analyses, there have been no direct studies of serological response to COVID-19 in patients with inflammatory bowel disease (IBD) across continents. In particular, there has been limited data from Asia, with no data reported from India. The ICARUS-IBD (International study of COVID-19 Antibody Response Under Sustained immunosuppression in IBD) consortium assessed serological response to SARS-CoV-2 in patients with IBD in North America, Europe, and Asia. METHODS: The ICARUS-IBD study is a multicenter observational cohort study spanning sites in 7 countries. We report seroprevalence data from 2303 patients with IBD before COVID-19 vaccination between May 2020 and November 2021. SARS-CoV-2 anti-spike and anti-nucleocapsid antibodies were analyzed. RESULTS: The highest and lowest SARS-CoV-2 anti-spike seropositivity rates were found in Asia (81.2% in Chandigarh and 57.9% in Delhi, India; and 0% in Hong Kong). By multivariable analysis, country (India: odds ratio [OR], 18.01; 95% confidence interval [CI], 12.03-26.95; P < .0001; United Kingdom: OR, 2.43; 95% CI, 1.58-3.72; P < .0001; United States: OR, 2.21; 95% CI, 1.27-3.85; P = .005), male sex (OR, 1.46; 95% CI, 1.07-1.99; P = .016), and diabetes (OR, 2.37; 95% CI, 1.04-5.46; P = .039) conferred higher seropositivity rates. Biological therapies associated with lower seroprevalence (OR, 0.22; 95% CI, 0.15-0.33; P < .0001). Multiple linear regression showed associations between anti-spike and anti-nucleocapsid titers with medications (P < .0001) but not with country (P = .3841). CONCLUSIONS: While the effects of medications on anti-SARS-CoV-2 antibody titers in patients with IBD were consistent across sites, geographical location conferred the highest risk of susceptibility to serologically detectable SARS-CoV-2 infection. Over half of IBD patients in India were seropositive prior to vaccination. These insights can help to inform shielding advice, therapeutic choices, and vaccine strategies in IBD patients for COVID-19 and future viral challenges.

In this multinational study of SARS-CoV-2 seroprevalence prior to vaccination, including the first data from India, where over half of patients seroconverted, geographical location conferred the highest risk of susceptibility to serologically detectable infection.

A neonatal mouse model characterizes transmissibility of SARS-CoV-2 variants and reveals a role for ORF8.

Small animal models have been a challenge for the study of SARS-CoV-2 transmission, with most investigators using golden hamsters or ferrets. Mice have the advantages of low cost, wide availability, less regulatory and husbandry challenges, and the existence of a versatile reagent and genetic toolbox. However, adult mice do not robustly transmit SARS-CoV-2. Here we establish a model based on neonatal mice that allows for transmission of clinical SARS-CoV-2 isolates. We characterize tropism, respiratory tract replication and transmission of ancestral WA-1 compared to variants Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2), Omicron BA.1 and Omicron BQ.1.1. We identify inter-variant differences in timing and magnitude of infectious particle shedding from index mice, both of which shape transmission to contact mice. Furthermore, we characterize two recombinant SARS-CoV-2 lacking either the ORF6 or ORF8 host antagonists. The removal of ORF8 shifts viral replication towards the lower respiratory tract, resulting in significantly delayed and reduced transmission in our model. Our results demonstrate the potential of our neonatal mouse model to characterize viral and host determinants of SARS-CoV-2 transmission, while revealing a role for an accessory protein in this context.