Rajat Madan

Background: Clostridioides difficile infection (CDI) is a major cause of antibiotic-associated diarrhea in the United States. Gut dysbiosis and chronic inflammation are key contributors to CDI susceptibility and severity. Metabolic syndrome (MetS)—defined by central obesity, hypertriglyceridemia, low HDL cholesterol, hypertension, and type 2 diabetes mellitus (T2DM)—is increasingly prevalent worldwide and is characterized by chronic immune dysregulation and alterations in gut microbiota. These pathophysiologic features may overlap with mechanisms that predispose individuals to CDI and its complications. Method: Using a large electronic health record database encompassing 102 health care organizations, we examined the association between metabolic conditions (MetS, obesity, and T2DM) and the risk of CDI diagnosis and severe clinical outcomes. Individuals with a diagnosis of each metabolic condition were compared with matched controls. Results: All 3 metabolic conditions were associated with an increased risk of CDI. The strongest association was observed in patients with MetS (odds ratio [OR], 1.94), followed by obesity (OR, 1.14) and T2DM (OR, 1.11). The impact of metabolic disorders on CDI severity varied based on the specific condition. Patients with MetS and obesity were more likely to develop sepsis, leukocytosis, and neutrophilia and to require ICU admission; however, they had lower risk of hypoalbuminemia, recurrent CDI, and all-cause mortality. In contrast, patients with T2DM had greater odds of developing all of the CDI-associated complications. Conclusions: MetS, obesity, and T2DM were all associated with an increased likelihood of CDI diagnosis. However, their effects on CDI severity varied among the 3 conditions examined—patients with T2DM had the greatest risk of adverse outcomes, including sepsis, ICU admission, recurrent CDI, and mortality.

Neutrophils are key first responders in the host response to Clostridioides difficile infection (CDI). Although a high number of tissue and blood neutrophils clearly correlates with adverse outcomes in CDI patients, their functional role remains poorly defined. Using murine (CMT-93) and human (Caco-2) intestinal epithelial cell (IEC) lines co-cultured with neutrophils and a pre-clinical mouse model of CDI, we show that activated neutrophils exacerbate C. difficile-induced IEC injury. To identify neutrophil subtypes and biological pathways that contribute to CDI-associated IEC damage, we utilized single-cell RNAseq (scRNAseq) to generate the first transcriptomic atlas of murine bone marrow, blood, and colonic neutrophils in acute CDI. Our analyses identified a novel neutrophil population in the colonic lamina propria, "cNeu3," which exhibited high expression of genes associated with neutrophil activation and degranulation. Informed by cNeu3 signature genes from the scRNAseq dataset, we validated the presence of these neutrophils in mouse colon using flow cytometry (CD11b^hiCD63^hiIL-1β^hiMIP-1α^hiMALT1^hi cells). We found that their abundance correlated with increased epithelial damage. Tumor Necrosis Factor (TNF) was sufficient to polarize mouse bone marrow neutrophils to a cNeu3 phenotype in vitro. Finally, TNF-primed human neutrophils worsened C. difficile toxin-induced IEC damage, which was improved when neutrophil degranulation was blocked. Collectively, our data provide novel insights into neutrophil-mediated pathology during CDI.

Neutrophils are dominant cells during acute immune response to Clostridioides difficile infection (CDI). A higher number of infiltrating colonic neutrophils is clearly linked to greater tissue damage and severe CDI (3, 4). However, the mechanism(s) by which neutrophils exacerbate tissue damage in CDI remain unknown. We investigated the role of a neutrophil subset marked by Olfactomedin-4 expression (OLFM4 ⁺ neutrophils) during CDI. Single-cell transcriptomics reveal that Olfm4 is increased in blood neutrophils of infected mice, and these cells exhibit gene signatures characterized by high expression of degranulation genes. In C. difficile -infected mice, OLFM4 ⁺ neutrophils aggregate to areas of severe intestinal epithelial cell (IEC) damage, and plasma OLFM4 was significantly increased in both C. difficile -infected mice and patients. In vitro , OLFM4 ⁺ neutrophils and recombinant OLFM4 protein exacerbated C. difficile toxin-induced IEC damage. In sum, our studies provide novel insights into neutrophil-mediated pathology and highlight the role of OLFM4 ⁺ neutrophils in worsening CDI-induced IEC damage.

Clostridioides difficile, a Gram-positive anaerobic bacterium, is the leading cause of hospital-acquired antibiotic-associated diarrhea worldwide. The severity of C. difficile infection (CDI) varies, ranging from mild diarrhea to life-threatening conditions such as pseudomembranous colitis and toxic megacolon. Central to the pathogenesis of the infection are toxins produced by C. difficile, with toxin A (TcdA) and toxin B (TcdB) as the main virulence factors. Additionally, some strains produce a third toxin known as C. difficile transferase (CDT). Toxins damage the colonic epithelium, initiating a cascade of cellular events that lead to inflammation, fluid secretion, and further tissue damage within the colon. Mechanistically, the toxins bind to cell surface receptors, internalize, and then inactivate GTPase proteins, disrupting the organization of the cytoskeleton and affecting various Rho-dependent cellular processes. This results in a loss of epithelial barrier functions and the induction of cell death. The third toxin, CDT, however, functions as a binary actin-ADP-ribosylating toxin, causing actin depolymerization and inducing the formation of microtubule-based protrusions. In this review, we summarize our current understanding of the interaction between C. difficile toxins and host cells, elucidating the functional consequences of their actions. Furthermore, we will outline how this knowledge forms the basis for developing innovative, toxin-based strategies for treating and preventing CDI.

Proinflammatory cytokine levels and host genetic makeup are key determinants of Clostridioides difficile infection (CDI) outcomes. We previously reported that blocking the inflammatory cytokine macrophage migration inhibitory factor (MIF) ameliorates CDI. Here, we determined kinetics of MIF production and its association with a common genetic variant in leptin receptor (LEPR) using blood from patients with CDI. We found highest plasma MIF early after C difficile exposure and in individuals who express mutant/derived LEPR. Our data suggest that early-phase CDI provides a possible window of opportunity in which MIF targeting, potentially in combination with LEPR genotype, could have therapeutic utility.

Clostridioides difficile is the most important cause of healthcare-associated diarrhea in the United States. The high incidence and recurrence rates of C. difficile infection (CDI), associated with high morbidity and mortality, pose a public health challenge. Although antibiotics targeting C. difficile bacteria are the first treatment choice, antibiotics also disrupt the indigenous gut flora and, therefore, create an environment that is favorable for recurrent CDI. The challenge of treating CDI is further exacerbated by the rise of antibiotic-resistant strains of C. difficile, placing it among the top five most urgent antibiotic resistance threats in the USA. The evolution of antibiotic resistance in C. difficile involves the acquisition of new resistance mechanisms, which can be shared among various bacterial species and different C. difficile strains within clinical and community settings. This review provides a summary of commonly used diagnostic tests and antibiotic treatment strategies for CDI. In addition, it discusses antibiotic treatment and its resistance mechanisms. This review aims to enhance our current understanding and pinpoint knowledge gaps in antimicrobial resistance mechanisms in C. difficile, with an emphasis on CDI therapies.

Neutrophils are key first responders to Clostridioides difficile infection (CDI). Excessive tissue and blood neutrophils are associated with worse histopathology and adverse outcomes, however their functional role during CDI remains poorly defined. Utilizing intestinal epithelial cell (IEC)-neutrophil co-cultures and a pre-clinical animal model of CDI, we show that neutrophils exacerbate C. difficile -induced IEC injury. We utilized cutting-edge single-cell transcriptomics to illuminate neutrophil subtypes and biological pathways that could exacerbate CDI-associated IEC damage. As such, we have established the first transcriptomics atlas of bone marrow (BM), blood, and colonic neutrophils after CDI. We found that CDI altered the developmental trajectory of BM and blood neutrophils towards populations that exhibit gene signatures associated with pro-inflammatory responses and neutrophil-mediated tissue damage. Similarly, the transcriptomic signature of colonic neutrophils was consistent with hyper-inflammatory and highly differentiated cells that had amplified expression of cytokine-mediated signaling and degranulation priming genes. One of the top 10 variable features in colonic neutrophils was the gene for neutrophil glycoprotein, Olfactomedin 4 (OLFM4). CDI enhanced OLFM4 mRNA and protein expression in neutrophils, and OLFM4 ⁺ cells aggregated to areas of severe IEC damage. Compared to uninfected controls, both humans and mice with CDI had higher concentrations of circulating OLFM4; and in mice, OLFM4 deficiency resulted in faster recovery and better survival after infection. Collectively, these studies provide novel insights into neutrophil-mediated pathology after CDI and highlight the pathogenic role of OLFM4 ⁺ neutrophils in regulating CDI-induced IEC damage. One Sentence Summary: Utilizing single-cell transcriptomics, IEC-epithelial co-cultures, and pre-clinical models of CDI, we have identified a subset of neutrophils that are marked by OLFM4 expression as pathogenic determinants of IEC barrier damage after CDI.

Neutrophils are key first-responders in the innate immune response to C. difficile infection (CDI) and play a central role in disease pathogenesis. Studies have clearly shown that tissue neutrophil numbers need to be tightly regulated for optimal CDI outcomes: while excessive colonic neutrophilia is associated with severe CDI, neutrophil depletion also results in worse outcomes. However, the biological mechanisms that control CDI-induced neutrophilia remain poorly defined. C-X-C chemokine receptor 2 (CXCR2) is a chemotactic receptor that is critical in neutrophil mobilization from bone marrow to blood and tissue sites. We have previously reported that a single nucleotide polymorphism (SNP) in leptin receptor (LEPR), present in up to 50% of people, influenced CDI-induced neutrophil CXCR2 expression and tissue neutrophilia. Homozygosity for mutant LEPR (i.e. RR genotype) was associated with higher CXCR2 expression and more tissue neutrophils. Here, we investigated the biological mechanisms that regulate neutrophil CXCR2 expression after CDI, and the influence of host genetics on this process. Our data reveal that: a) CXCR2 plays a key role in CDI-induced neutrophil extravasation from blood to colonic tissue; b) plasma from C. difficile-infected mice upregulated CXCR2 on bone marrow neutrophils; c) plasma from C. difficile-infected RR mice induced a higher magnitude of CXCR2 upregulation and had more IL-1β; and d) IL-1β neutralization reduced CXCR2 expression on bone marrow and blood neutrophils and their subsequent accrual to colonic tissue. In sum, our data indicate that IL-1β is a key molecular mediator that communicates between gastro-intestinal tract (i.e. site of CDI) and bone marrow (i.e. primary neutrophil reservoir) and regulates the intensity of CDI-induced tissue neutrophilia by modulating CXCR2 expression. Further, our studies highlight the importance of host genetics in affecting these innate immune responses and provide novel insights into the mechanisms by which a common SNP influences CDI-induced neutrophilia.