Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.

Clostridioides (formerly Clostridium) difficile is the most common cause of hospital-acquired infection, and advanced age is a risk factor for C. difficile infection. Disruption of the intestinal microbiota and immune responses contribute to host susceptibility and severity of C. difficile infection. However, the specific impact of aging on immune responses during C. difficile infection remains to be well described. This study explores the effect of age on cellular and cytokine immune responses during C. difficile infection. Young mice (2 to 3 months old) and aged mice (22 to 28 months old) were rendered susceptible to C. difficile infection with the antibiotic cefoperazone and then infected with C. difficile strains with varied disease-causing potentials. We observe that the host age and the infecting C. difficile strain influenced the severity of disease associated with infection. Tissue-specific CD45+ immune cell responses occurred at the time of peak disease severity in the ceca and colons of all mice infected with a high-virulence strain of C. difficile; however, significant deficits in intestinal neutrophils and eosinophils were detected in aged mice, with a corresponding decrease in circulating CXCL1, an important neutrophil recruiter and activator. Interestingly, this lack of intestinal granulocyte response in aged mice during severe C. difficile infection was accompanied by a simultaneous increase in circulating white blood cells, granulocytes, and interleukin 17A (IL-17A). These findings demonstrate that age-related alterations in neutrophils and eosinophils and systemic cytokine and chemokine responses are associated with severe C. difficile infection and support a key role for intestinal eosinophils in mitigating C. difficile-mediated disease severity.

Cutaneous lupus erythematosus (CLE) is a chronic inflammatory skin disease characterized by a diverse cadre of clinical presentations. CLE commonly occurs in patients with systemic lupus erythematosus (SLE), and CLE can also develop in the absence of systemic disease. Although CLE is a complex and heterogeneous disease, several studies have identified common signaling pathways, including those of type I interferons (IFNs), that play a key role in driving cutaneous inflammation across all CLE subsets. However, discriminating factors that drive different phenotypes of skin lesions remain to be determined. Thus, we sought to understand the skin-associated cellular and transcriptional differences in CLE subsets and how the different types of cutaneous inflammation relate to the presence of systemic lupus disease. In this study, we utilized two distinct cohorts comprising a total of 150 CLE lesional biopsies to compare discoid lupus erythematosus (DLE), subacute cutaneous lupus erythematosus (SCLE), and acute cutaneous lupus erythematosus (ACLE) in patients with and without associated SLE. Using an unbiased approach, we demonstrated a CLE subtype-dependent gradient of B cell enrichment in the skin, with DLE lesions harboring a more dominant skin B cell transcriptional signature and enrichment of B cells on immunostaining compared to ACLE and SCLE. Additionally, we observed a significant increase in B cell signatures in the lesional skin from patients with isolated CLE compared with similar lesions from patients with systemic lupus. This trend was driven primarily by differences in the DLE subgroup. Our work thus shows that skin-associated B cell responses distinguish CLE subtypes in patients with and without associated SLE, suggesting that B cell function in skin may be an important link between cutaneous lupus and systemic disease activity.

Innate immunity contributes to the pathogenesis of inflammatory bowel disease (IBD). However, the mechanisms of IBD mediated by innate immunity are incompletely understood and there are limited models of spontaneous innate immune colitis to address this question. Here we describe a new robust model of colitis occurring in the absence of adaptive immunity. RAG1-deficient mice expressing TNFAIP3 in intestinal epithelial cells (TRAG mice) spontaneously developed 100% penetrant, early-onset colitis that was limited to the colon and dependent on intestinal microbes but was not transmissible to co-housed littermates. TRAG colitis was associated with increased mucosal numbers of innate lymphoid cells (ILCs) and depletion of ILC prevented colitis in TRAG mice. ILC depletion also therapeutically reversed established colitis in TRAG mice. The colitis in TRAG mice was not prevented by interbreeding to mice lacking group 3 ILC nor by depletion of TNF. Treatment with the JAK inhibitor ruxolitinib ameliorated colitis in TRAG mice. This new model of colitis, with its predictable onset and colon-specific inflammation, will have direct utility in developing a more complete understanding of innate immune mechanisms that can contribute to colitis and in pre-clinical studies for effects of therapeutic agents on innate immune-mediated IBD.

Clostridioides difficile infection (CDI) can result in severe disease and death, with no accurate models that allow for early prediction of adverse outcomes. To address this need, we sought to develop serum-based biomarker models to predict CDI outcomes. We prospectively collected sera ≤48 h after diagnosis of CDI in two cohorts. Biomarkers were measured with a custom multiplex bead array assay. Patients were classified using IDSA severity criteria and the development of disease-related complications (DRCs), which were defined as ICU admission, colectomy, and/or death attributed to CDI. Unadjusted and adjusted models were built using logistic and elastic net modeling. The best model for severity included procalcitonin (PCT) and hepatocyte growth factor (HGF) with an area (AUC) under the receiver operating characteristic (ROC) curve of 0.74 (95% confidence interval, 0.67 to 0.81). The best model for 30-day mortality included interleukin-8 (IL-8), PCT, CXCL-5, IP-10, and IL-2Rα with an AUC of 0.89 (0.84 to 0.95). The best model for DRCs included IL-8, procalcitonin, HGF, and IL-2Rα with an AUC of 0.84 (0.73 to 0.94). To validate our models, we employed experimental infection of mice with C. difficile. Antibiotic-treated mice were challenged with C. difficile and a similar panel of serum biomarkers was measured. Applying each model to the mouse cohort of severe and nonsevere CDI revealed AUCs of 0.59 (0.44 to 0.74), 0.96 (0.90 to 1.0), and 0.89 (0.81 to 0.97). In both human and murine CDI, models based on serum biomarkers predicted adverse CDI outcomes. Our results support the use of serum-based biomarker panels to inform Clostridioides difficile infection treatment. IMPORTANCE Each year in the United States, Clostridioides difficile causes nearly 500,000 gastrointestinal infections that range from mild diarrhea to severe colitis and death. The ability to identify patients at increased risk for severe disease or mortality at the time of diagnosis of C. difficile infection (CDI) would allow clinicians to effectively allocate disease modifying therapies. In this study, we developed models consisting of only a small number of serum biomarkers that are capable of predicting both 30-day all-cause mortality and adverse outcomes of patients at time of CDI diagnosis. We were able to validate these models through experimental mouse infection. This provides evidence that the biomarkers reflect the underlying pathophysiology and that our mouse model of CDI reflects the pathogenesis of human infection. Predictive models can not only assist clinicians in identifying patients at risk for severe CDI but also be utilized for targeted enrollment in clinical trials aimed at reduction of adverse outcomes from severe CDI.