Update: Induction of Inflammatory Bowel Disease in Immunodeficient Mice by Injection of Naïve CD4+ T cells (T Cell Transfer Model of Colitis).

The intestinal inflammation induced by injection of naïve CD4+ T cells into lymphocyte-deficient hosts (more commonly known as the T cell transfer model of colitis) shares many features of idiopathic inflammatory bowel disease (IBD) in humans, such as epithelial cell hyperplasia, crypt abscess formation, and dense lamina propria lymphocyte infiltration. As such, it provides a useful tool for studying mucosal immune regulation as it relates to the pathogenesis and treatment of IBD in humans. In the IBD model described here, colitis is induced in Rag (recombination-activating gene)-deficient mice by reconstitution of these mice with naïve CD4+CD45RBhi T cells through adoptive T cell transfer. Although different recipient hosts of cell transfer can be used, Rag-deficient mice are the best characterized and support studies that are both flexible and reproduceable. As described in the Basic Protocol, in most studies the transferred cells consist of naïve CD4+ T cells (CD45RBhi T cells) derived by fluorescence-activated cell sorting from total CD4+ T cells previously purified using immunomagnetic negative selection beads. In a Support Protocol, methods to characterize colonic disease progression are described, including the monitoring of weight loss and diarrhea and the histological assessment of colon pathology. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Induction of IBD in Rag-deficient mice by the transfer of naïve CD4+CD45RBhi T cells Support Protocol: Monitoring development of colitis.

Development and Validation of a Two-Step Predictive Risk Stratification Model for Coronavirus Disease 2019 In-hospital Mortality: A Multicenter Retrospective Cohort Study.

Objectives: An accurate prognostic score to predict mortality for adults with COVID-19 infection is needed to understand who would benefit most from hospitalizations and more intensive support and care. We aimed to develop and validate a two-step score system for patient triage, and to identify patients at a relatively low level of mortality risk using easy-to-collect individual information. Design: Multicenter retrospective observational cohort study. Setting: Four health centers from Virginia Commonwealth University, Georgetown University, the University of Florida, and the University of California, Los Angeles. Patients: Coronavirus Disease 2019-confirmed and hospitalized adult patients. Measurements and Main Results: We included 1,673 participants from Virginia Commonwealth University (VCU) as the derivation cohort. Risk factors for in-hospital death were identified using a multivariable logistic model with variable selection procedures after repeated missing data imputation. A two-step risk score was developed to identify patients at lower, moderate, and higher mortality risk. The first step selected increasing age, more than one pre-existing comorbidities, heart rate >100 beats/min, respiratory rate ≥30 breaths/min, and SpO2 <93% into the predictive model. Besides age and SpO2, the second step used blood urea nitrogen, absolute neutrophil count, C-reactive protein, platelet count, and neutrophil-to-lymphocyte ratio as predictors. C-statistics reflected very good discrimination with internal validation at VCU (0.83, 95% CI 0.79-0.88) and external validation at the other three health systems (range, 0.79-0.85). A one-step model was also derived for comparison. Overall, the two-step risk score had better performance than the one-step score. Conclusions: The two-step scoring system used widely available, point-of-care data for triage of COVID-19 patients and is a potentially time- and cost-saving tool in practice.

Identification of gut microbial species linked with disease variability in a widely used mouse model of colitis.

Experimental mouse models are central to basic biomedical research; however, variability exists across genetically identical mice and mouse facilities making comparisons difficult. Whether specific indigenous gut bacteria drive immunophenotypic variability in mouse models of human disease remains poorly understood. We performed a large-scale experiment using 579 genetically identical laboratory mice from a single animal facility, designed to identify the causes of disease variability in the widely used dextran sulphate sodium mouse model of inflammatory bowel disease. Commonly used treatment endpoint measures-weight loss and intestinal pathology-showed limited correlation and varied across mouse lineages. Analysis of the gut microbiome, coupled with machine learning and targeted anaerobic culturing, identified and isolated two previously undescribed species, Duncaniella muricolitica and Alistipes okayasuensis, and demonstrated that they exert dominant effects in the dextran sulphate sodium model leading to variable treatment endpoint measures. We show that the identified gut microbial species are common, but not ubiquitous, in mouse facilities around the world, and suggest that researchers monitor for these species to provide experimental design opportunities for improved mouse models of human intestinal diseases.

CD4+ T-cell-derived IL-10 promotes CNS inflammation in mice by sustaining effector T cell survival.

Interleukin (IL)-10 is considered a prototypical anti-inflammatory cytokine, significantly contributing to the maintenance and reestablishment of immune homeostasis. Accordingly, it has been shown in the intestine that IL-10 produced by Tregs can act on effector T cells, thereby limiting inflammation. Herein, we investigate whether this role also applies to IL-10 produced by T cells during central nervous system (CNS) inflammation. During neuroinflammation, both CNS-resident and -infiltrating cells produce IL-10; yet, as IL-10 has a pleotropic function, the exact contribution of the different cellular sources is not fully understood. We find that T-cell-derived IL-10, but not other relevant IL-10 sources, can promote inflammation in experimental autoimmune encephalomyelitis. Furthermore, in the CNS, T-cell-derived IL-10 acts on effector T cells, promoting their survival and thereby enhancing inflammation and CNS autoimmunity. Our data indicate a pro-inflammatory role of T-cell-derived IL-10 in the CNS.

Development and Validation of the COVID-NoLab and COVID-SimpleLab Risk Scores for Prognosis in 6 US Health Systems.

PURPOSE: Develop and validate simple risk scores based on initial clinical data and no or minimal laboratory testing to predict mortality in hospitalized adults with COVID-19. METHODS: We gathered clinical and initial laboratory variables on consecutive inpatients with COVID-19 who had either died or been discharged alive at 6 US health centers. Logistic regression was used to develop a predictive model using no laboratory values (COVID-NoLab) and one adding tests available in many outpatient settings (COVID-SimpleLab). The models were converted to point scores and their accuracy evaluated in an internal validation group. RESULTS: We identified 1340 adult inpatients with complete data for nonlaboratory parameters and 741 with complete data for white blood cell (WBC) count, differential, c-reactive protein (CRP), and serum creatinine. The COVID-NoLab risk score includes age, respiratory rate, and oxygen saturation and identified risk groups with 0.8%, 11.4%, and 40.4% mortality in the validation group (AUROCC = 0.803). The COVID-SimpleLab score includes age, respiratory rate, oxygen saturation, WBC, CRP, serum creatinine, and comorbid asthma and identified risk groups with 1.0%, 9.1%, and 29.3% mortality in the validation group (AUROCC = 0.833). CONCLUSIONS: Because they use simple, readily available predictors, developed risk scores have potential applicability in the outpatient setting but require prospective validation before use.

Learner Perceptions of Electronic End-of-shift Evaluations on An Emergency Medicine Clerkship.

OBJECTIVES: As students on an emergency medicine (EM) rotation work with different faculty on a daily basis, EM clerkships often incorporate an end-of-shift evaluation to capture sufficient student performance data. Electronic shift evaluations have been shown to increase faculty completion compliance. This study aimed to examine learner perceptions of their individualized feedback during an EM clerkship following the adoption of an electronic evaluation tool. METHODS: This retrospective study examined end-of-rotation surveys that students complete at the conclusion of their EM rotation. Survey respondents used a standard Likert scale (1-5). This study examined responses to the question: "The feedback I received on this rotation was adequate." The study period included the 3 academic years prior to and subsequent to the adoption of an electronic evaluation system (replacing paper end-of-shift evaluations). The primary outcome was the mean Likert score and the secondary outcome was the percentage of students who rated their feedback a "5" or "strongly agree." RESULTS: A total of 491 students responded (83.9% response rate) to the survey during the paper evaluation period, while 427 responded (80.7% response rate) in the electronic period. The mean response improved from 4.02 (paper evaluations) to 4.22 (electronic evaluations; mean difference = 0.20, p < 0.05). The percentage of students who responded with a 5 improved (31% with paper evaluations vs. 41% with electronic evaluations, p < 0.05). CONCLUSIONS: The adoption of an electronic end-of-shift evaluation system was associated with improved learner perception of their feedback as compared to paper evaluations. Electronic evaluations are a useful tool to gather just-in-time data on learner performance.

Disrupted Iron Metabolism and Mortality during Co-infection with Malaria and an Intestinal Gram-Negative Extracellular Pathogen.

Individuals with malaria exhibit increased morbidity and mortality when infected with Gram-negative (Gr-) bacteria. To explore this experimentally, we performed co-infection of mice with Plasmodium chabaudi and Citrobacter rodentium, an extracellular Gr- bacterial pathogen that infects the large intestine. While single infections are controlled effectively, co-infection results in enhanced virulence that is characterized by prolonged systemic bacterial persistence and high mortality. Mortality in co-infected mice is associated with disrupted iron metabolism, elevated levels of plasma heme, and increased mitochondrial reactive oxygen species (ROS) production by phagocytes. In addition, iron acquisition by the bacterium plays a key role in pathogenesis because co-infection with a mutant C. rodentium strain lacking a critical iron acquisition pathway does not cause mortality. These results indicate that disrupted iron metabolism may drive mortality during co-infection with C. rodentium and P. chabaudi by both altering host immune responses and facilitating bacterial persistence.

Why do intestinal epithelial cells express MHC class II?

Intestinal epithelial cells (IECs) constitute the border between the vast antigen load present in the intestinal lumen and the mucosal immune compartment. Their ability to express antigen processing and presentation machinery evokes the question whether IECs function as non-conventional antigen-presenting cells. Major histocompatibility complex (MHC) class II expression by non-haematopoietic cells, such as IECs, is tightly regulated by the class II transactivator (CIITA) and is classically induced by IFN-γ. As MHC class II expression by IECs is upregulated under inflammatory conditions, it has been proposed to activate effector CD4+ T (Teff) cells. However, other studies have reported contradictory results and instead suggested a suppressive role of antigen presentation by IECs, through regulatory T (Treg)-cell activation. Recent studies investigating the role of MHC class II + exosomes released by IECs also reported conflicting findings of either immune enhancing or immunosuppressive activities. Moreover, in addition to modulating inflammatory responses, recent findings suggest that MHC class II expression by intestinal stem cells may elicit crosstalk that promotes epithelial renewal. A more complete understanding of the different consequences of IEC MHC class II antigen presentation will guide future efforts to modulate this pathway to selectively invoke protective immunity while maintaining tolerance to beneficial antigens.

Multi-center implementation of automated age-adjusted D-dimer results reduces unnecessary PE imaging.

BACKGROUND: Several previous studies have investigated the clinical utility of age-adjusted D-dimer cutoffs for diagnosing pulmonary embolism (PE). OBJECTIVES: We performed a pre/post implementation study, using data from a mid-Atlantic healthcare system comprising 6 hospitals and 400,000 ED visits to determine whether implementing age adjusted D-dimer cutoffs reduced the number of imaging tests performed. METHODS: Retrospective study of all patients who had a D-dimer performed during ED visits between September 2015 to September 2018. On March 21, 2017, the D-dimer upper limit of normal system-wide was increased for patients over 50 to: Age (years) x 0.01µg/mL. D-dimer results were displayed as normal or high based on automated age adjustment. EHR Chart review was performed 1.5 years prior to implementation of age-adjusted D-dimer cutoffs, as well as 1.5 years after to evaluate mortality and test accuracy characteristics such as false negative rates. Comparisons were made using chi-square testing. RESULTS: 22,302 D-dimers were performed pre-implementation of which 10,837 (48.6%) were positive resulting in 7218 (32.3%) imaging studies. After implementation of age-adjusted d-dimer, 25,082 were performed of which 10,851 (43.2%) were positive resulting in 7017 (28.0%) imaging studies. (pre: 48.6%, post: 43.2%; p < 0.01). A significantly lower proportion of patients had a positive d-dimer (pre: 48.6%, post: 43.2%; p < 0.01) and underwent imaging post-implementation (pre: 32.3%, post: 28.0%; p < 0.05) a relative risk reduction of 13.3. This absolute risk reduction of 4.4% is associated with 1104 less scans in the post-implementation group while still increasing test accuracy from 53.7% to 59.2% (p < 0.05). CONCLUSION: Implementation of an automated age-adjusted D-dimer positive reference value reduced CT and V/Q imaging in this population by 4.4% while increasing test accuracy in a regional, heterogeneous six-hospital system.

Understanding immune-microbiota interactions in the intestine.

The past two decades have seen an explosion in research that aims to understand how the dynamic interplay with the gut microbiota impacts host health and disease, establishing a role for the gut microbiota in a plethora of pathologies. Understanding how health-promoting microbiota are established and how beneficial host-microbiota interactions are maintained is of immense biomedical importance. Despite the enormous progress that has been made, our knowledge of the specific microbiota members that mediate these effects and the mechanisms underlying these interactions is rudimentary. The dearth of information regarding the nature of advantageous host-microbiota interactions, and the factors that cause these relationships to go awry, has hampered our ability to realize the therapeutic potential of the microbiota. Here we discuss key issues that limit current knowledge and describe a path forwards to improving our understanding of the contributions of the microbiota to host health.

Emergency Department Computed Tomography Use for Non-traumatic Abdominal Pain: Minimal Variability.

INTRODUCTION: Variability in the use of computed tomography (CT) between providers in the emergency department (ED) suggests that CT is ordered on a provider rather than a patient level. We aimed to evaluate the variability of CT ordering practices for non-traumatic abdominal pain (NTAP) across physicians in the ED using patient-visit and physician-level factors. METHODS: We conducted a retrospective study among 6,409 ED visits for NTAP from January 1 to December 31, 2012, at a large, urban, academic, tertiary-care hospital. We used a two-level hierarchical logistic regression model to estimate inter-physician variation. Intraclass correlation coefficient (ICC) was calculated. RESULTS: The hierarchical logistic regression analyses showed that patient-visit factors including younger age, arrival mode by ambulance, prior CT, >79 ED arrivals in the previous four hours, and ultrasound had statistically significant negative associations with physician CT ordering, while surgical team admission and white blood count (WBC) >12.5 K/millimeter cubed (mm3) had statistically significant positive associations with physician CT ordering. With physician-level factors, only physicians with >21 years experience after medical school graduation showed statistical significance negatively associated with physician CT ordering. Our data demonstrated increased CT ordering from the mean in only one out of 43 providers (2.3%), which indicated limited variation across physicians to order CT. After adjusting for patient-visit and physician-level factors, the calculated ICC was 1.46%. CONCLUSION: We found minimal physician variability in CT ordering practices for NTAP. Patient-visit factors such as age, arrival mode, admission team, prior CT, ED arrivals in previous four hours, ultrasound, and WBC count were found to largely influence CT ordering practices.

Defens-IN! Human α-Defensin 5 Acts as an Unwitting Double Agent to Promote Shigella Infection.

Shigella pathogenesis has confounded researchers for years because of its narrow host selectivity and extraordinary infectious capability. In this issue of Immunity, Xu et al. (2018) identify a cunning mechanism whereby Shigella hijacks human α-defensin 5 to enhance its adhesion and subsequent invasion.

Epithelial autophagy controls chronic colitis by reducing TNF-induced apoptosis.

Genome-wide association studies (GWAS) linking polymorphisms in ATG16L1 with susceptibility to inflammatory bowel disease (IBD) have prompted mucosal immunologists to investigate the functional roles of macroautophagy/autophagy in different cell types in the gut. Here we present a recent study that addressed 2 key questions: in which cell type is autophagy deficiency most detrimental during chronic colitis and what is the functional role of autophagy in those cells? We report that autophagy in intestinal epithelial cells (IECs) acts to limit intestinal inflammation by protecting them from TNF-induced apoptosis and we discuss the potential implications for IBD treatment.

Plasmodium vivax Infection Impairs Regulatory T-Cell Suppressive Function During Acute Malaria.

The balance between pro- and antiinflammatory mechanisms is essential to limit immune-mediated pathology, and CD4+ forkhead box P3 (Foxp3+) regulatory T cells (Treg) play an important role in this process. The expression of inhibitory receptors regulates cytokine production by Plasmodium vivax-specific T cells. Our goal was to assess the induction of programmed death-1 (PD-1) and cytotoxic T-lymphocyte antigen (CTLA-4) on Treg during malaria and to evaluate their function. We found that P. vivax infection triggered an increase in circulating Treg and their expression of CTLA-4 and PD-1. Functional analysis demonstrated that Treg from malaria patients had impaired suppressive ability and PD-1+Treg displayed lower levels of Foxp3 and Helios, but had higher frequencies of T-box transcription factor+ and interferon-gamma+ cells than PD-1-Treg. Thus malaria infection alters the function of circulating Treg by triggering increased expression of PD-1 on Treg that is associated with decreased regulatory function and increased proinflammatory characteristics.

Hydrogen Sulfide Reduces Myeloid-Derived Suppressor Cell-Mediated Inflammatory Response in a Model of Helicobacter hepaticus-Induced Colitis.

Chronic inflammation contributes to tumor initiation in colitis-associated colorectal cancer (CRC). Indeed, inflammatory bowel disease (IBD) patients show an increased risk of developing CRC. Cancer immune evasion is a major issue in CRC and preclinical and clinical evidence has defined a critical role for myeloid-derived suppressor cells (MDSCs) that contribute to tumor growth and progression by suppressing T-cells and modulating innate immune responses. MDSCs comprise a heterogeneous population of immature myeloid cells that can be distinct in two subtypes: CD11b+Ly6G+Ly6Clow with granulocytic phenotype (G-MDSCs) and CD11b+Ly6G-Ly6Chigh with monocytic phenotype (M-MDSCs). Hydrogen sulfide (H2S) is an endogenous gaseous signaling molecule that regulates various physiological and pathophysiological functions. In particular, several studies support its anti-inflammatory activity in experimental colitis and ulcer. However, the role of the H2S pathway in innate immune-mediated IBD has not yet been elucidated. To better define a possible link between MDSCs and H2S pathway in colitis-associated CRC development, we used an innate immune-mediated IBD model induced by infection with the bacterium Helicobacter hepaticus (Hh), closely resembling human IBD. Here, we demonstrated an involvement of MDSCs in colitis development. A significant time-dependent increase of both G-MDSCs and M-MDSCs was observed in the colon and in the spleen of Hh-infected mice. Following, we observed that chronic oral administration of the H2S donor DATS reduced colon inflammation by limiting the recruitment of G-MDSCs in the colon of Hh-infected mice. Thus, we identify the metabolic pathway l-cysteine/H2S as a possible new player in the immunosuppressive mechanism responsible for the MDSCs-promoted colitis-associated cancer development.

Metabolic Adaptations of CD4+ T Cells in Inflammatory Disease.

A controlled and self-limiting inflammatory reaction generally results in removal of the injurious agent and repair of the damaged tissue. However, in chronic inflammation, immune responses become dysregulated and prolonged, leading to tissue destruction. The role of metabolic reprogramming in orchestrating appropriate immune responses has gained increasing attention in recent years. Proliferation and differentiation of the T cell subsets that are needed to address homeostatic imbalance is accompanied by a series of metabolic adaptations, as T cells traveling from nutrient-rich secondary lymphoid tissues to sites of inflammation experience a dramatic shift in microenvironment conditions. How T cells integrate information about the local environment, such as nutrient availability or oxygen levels, and transfer these signals to functional pathways remains to be fully understood. In this review, we discuss how distinct subsets of CD4+ T cells metabolically adapt to the conditions of inflammation and whether these insights may pave the way to new treatments for human inflammatory diseases.

Intestinal Epithelial Cell Autophagy Is Required to Protect against TNF-Induced Apoptosis during Chronic Colitis in Mice.

Genome-wide association studies have linked polymorphisms in the autophagy gene ATG16L1 with susceptibility to inflammatory bowel disease (IBD). However, the cell-type-specific effects of autophagy on the regulation of chronic intestinal inflammation have not been investigated. Here, we assessed the effect of myeloid-specific or intestinal epithelial cell (IEC)-specific deletion of Atg16l1 on chronic colitis triggered by the intestinal opportunistic pathogen Helicobacter hepaticus in mice. Although Atg16l1 deficiency in myeloid cells had little effect on disease, mice selectively lacking Atg16l1 in IECs (Atg16l1VC) developed severely exacerbated pathology, accompanied by elevated pro-inflammatory cytokine secretion and increased IEC apoptosis. Using ex vivo IEC organoids, we demonstrate that autophagy intrinsically controls TNF-induced apoptosis and in vivo blockade of TNF attenuated the exacerbated pathology in Atg16l1VC mice. These findings suggest that the IBD susceptibility gene ATG16L1 and the process of autophagy within the epithelium control inflammation-induced apoptosis and barrier integrity to limit chronic intestinal inflammation.

Epithelial Cell Inflammasomes in Intestinal Immunity and Inflammation.

Pattern recognition receptors (PRR), such as NOD-like receptors (NLRs), sense conserved microbial signatures, and host danger signals leading to the coordination of appropriate immune responses. Upon activation, a subset of NLR initiate the assembly of a multimeric protein complex known as the inflammasome, which processes pro-inflammatory cytokines and mediates a specialized form of cell death known as pyroptosis. The identification of inflammasome-associated genes as inflammatory bowel disease susceptibility genes implicates a role for the inflammasome in intestinal inflammation. Despite the fact that the functional importance of inflammasomes within immune cells has been well established, the contribution of inflammasome expression in non-hematopoietic cells remains comparatively understudied. Given that intestinal epithelial cells (IEC) act as a barrier between the host and the intestinal microbiota, inflammasome expression by these cells is likely important for intestinal immune homeostasis. Accumulating evidence suggests that the inflammasome plays a key role in shaping epithelial responses at the host-lumen interface with many inflammasome components highly expressed by IEC. Recent studies have exposed functional roles of IEC inflammasomes in mucosal immune defense, inflammation, and tumorigenesis. In this review, we present the main features of the predominant inflammasomes and their effector mechanisms contributing to intestinal homeostasis and inflammation. We also discuss existing controversies in the field and open questions related to their implications in disease. A comprehensive understanding of the molecular basis of intestinal inflammasome signaling could hold therapeutic potential for clinical translation.

Oncostatin M drives intestinal inflammation and predicts response to tumor necrosis factor-neutralizing therapy in patients with inflammatory bowel disease.

Inflammatory bowel diseases (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), are complex chronic inflammatory conditions of the gastrointestinal tract that are driven by perturbed cytokine pathways. Anti-tumor necrosis factor-α (TNF) antibodies are mainstay therapies for IBD. However, up to 40% of patients are nonresponsive to anti-TNF agents, which makes the identification of alternative therapeutic targets a priority. Here we show that, relative to healthy controls, inflamed intestinal tissues from patients with IBD express high amounts of the cytokine oncostatin M (OSM) and its receptor (OSMR), which correlate closely with histopathological disease severity. The OSMR is expressed in nonhematopoietic, nonepithelial intestinal stromal cells, which respond to OSM by producing various proinflammatory molecules, including interleukin (IL)-6, the leukocyte adhesion factor ICAM1, and chemokines that attract neutrophils, monocytes, and T cells. In an animal model of anti-TNF-resistant intestinal inflammation, genetic deletion or pharmacological blockade of OSM significantly attenuates colitis. Furthermore, according to an analysis of more than 200 patients with IBD, including two cohorts from phase 3 clinical trials of infliximab and golimumab, high pretreatment expression of OSM is strongly associated with failure of anti-TNF therapy. OSM is thus a potential biomarker and therapeutic target for IBD, and has particular relevance for anti-TNF-resistant patients.