Clostridioides difficile Toxins: Host Cell Interactions and Their Role in Disease Pathogenesis.

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.

Effect of Leptin Receptor Q223R Polymorphism on Clostridioides difficile Infection-Induced Macrophage Migration Inhibitory Factor Production.

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 Infection: Diagnosis and Treatment Challenges.

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.

Adipocyte inflammation and pathogenesis of viral pneumonias: an overlooked contribution.

Epidemiological evidence establishes obesity as an independent risk factor for increased susceptibility and severity to viral respiratory pneumonias associated with H1N1 influenza and SARS-CoV-2 pandemics. Given the global obesity prevalence, a better understanding of the mechanisms behind obese susceptibility to infection is imperative. Altered immune cell metabolism and function are often perceived as a key causative factor of dysregulated inflammation. However, the contribution of adipocytes, the dominantly altered cell type in obesity with broad inflammatory properties, to infectious disease pathogenesis remains largely ignored. Thus, skewing of adipocyte-intrinsic cellular metabolism may lead to the development of pathogenic inflammatory adipocytes, which shape the overall immune responses by contributing to either premature immunosenescence, delayed hyperinflammation, or cytokine storm in infections. In this review, we discuss the underappreciated contribution of adipocyte cellular metabolism and adipocyte-produced mediators on immune system modulation and how such interplay may modify disease susceptibility and pathogenesis of influenza and SARS-CoV-2 infections in obese individuals.

Leptin Receptor q223r Polymorphism Influences Clostridioides difficile Infection-Induced Neutrophil CXCR2 Expression in an Interleukin-1ß Dependent Manner.

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-1ß; and d) IL-1ß neutralization reduced CXCR2 expression on bone marrow and blood neutrophils and their subsequent accrual to colonic tissue. In sum, our data indicate that IL-1ß 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.

Obeticholic acid ameliorates severity of Clostridioides difficile infection in high fat diet-induced obese mice.

Severe Clostridiodes difficile infection (CDI) is life-threatening and responds poorly to treatment. Obesity is associated with development of severe CDI. Therefore, to define the mechanisms that exacerbate disease severity, we examined CDI pathogenesis in high-fat diet (HFD)-fed obese mice. Compared to control mice, HFD-fed mice failed to clear C. difficile bacteria which resulted in protracted diarrhea, weight loss and colonic damage. After infection, HFD-induced obese mice had an intestinal bile acid (BA) pool that was dominated by primary BAs which are known promoters of C. difficile spore germination, and lacked secondary BAs that inhibit C. difficile growth. Concurrently, synthesis of primary BAs from liver was significantly increased in C. difficile-infected HFD-fed mice. A key pathway that regulates hepatic BA synthesis is via feedback inhibition from intestinal Farnesoid X receptors (FXRs). Our data reveal that the proportion of FXR agonist BAs to FXR antagonist BAs in the intestinal lumen was significantly reduced in HFD-fed mice after CDI. Treatment of HFD-fed mice with an FXR agonist Obeticholic acid, resulted in decreased primary BA synthesis, fewer C. difficile bacteria and better CDI outcomes. Thus, OCA treatment holds promise as a therapy for severe CDI.

Neutralization of macrophage migration inhibitory factor improves host survival after Clostridium difficile infection.

Clostridium difficile is an important cause of nosocomial diarrhea in the western world. Toxins (A, B, and binary toxins) generated by C. difficile bacteria damage intestinal epithelial cells. Hallmarks of host response to C. difficile infection (CDI) include upregulation of inflammatory mediators and tissue infiltration by immune cells. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that is known to enhance the host immune response to infectious pathogens. Additionally, MIF can adversely impact host survival to numerous infections. The role of MIF in the pathogenesis of CDI remains poorly understood. Here, we show that patients with CDI had significantly higher circulating MIF compared to patients who had diarrhea but tested negative for C. difficile (non-CDI controls). Similarly, in a mouse model, C. difficile challenge significantly increased levels of plasma and tissue MIF. Antibody-mediated depletion of MIF decreased C. difficile-induced inflammatory responses, clinical disease, and mortality. Together, these results uncover a potential role for MIF in exacerbating CDI and suggest that use of anti-MIF antibodies may represent a therapeutic strategy to curb host inflammatory responses and improve disease outcomes in CDI.

Role of the leukocyte response in normal and immunocompromised host after Clostridium difficile infection.

Clostridium difficile is the leading cause of healthcare-associated infections in the United States. Clinically, C. difficile-associated disease can present as asymptomatic colonization, self-limited diarrheal illness or severe colitis (that may result in death). This variability in disease course and outcomes suggests that host factors play an important role as key determinants of disease severity. Currently, there are several scoring indices to estimate severity of C. difficile-associated disease. Leukocytosis and renal failure are considered to be the most important predictors of C. difficile disease severity in hosts with a normal immune system. The degree of leukocytosis which is considered significant for severe disease and how it is scored vary amongst scoring indices. None of the scores have been prospectively validated, and while total WBC count is useful to estimate the magnitude of the host response in most patient populations, in immune-compromised patients like those receiving chemotherapy, solid organ transplant patients or hematopoietic stem cell transplants the WBC response can be variable or even absent making this marker of severity difficult to interpret. Other cellular subsets like neutrophils, eosinophils and lymphocytes provide important information about the host immune status and play an important role in the immune response against C. difficile infection. However, under the current scoring systems the role of these cellular subsets have been underestimated and only total white blood cell counts are taken into account. In this review we highlight the role of host leukocyte response to C. difficile challenge in the normal and immunocompromised host, and propose possible ways that would allow for a better representation of the different immune cell subsets (neutrophils, lymphocytes and eosinophils) in the current scoring indices.

Neutrophil-mediated inflammation in the pathogenesis of Clostridium difficile infections.

Clostridium difficile is the most important cause of nosocomial infectious diarrhea in the western world. C. difficile infections are a major healthcare burden with approximately 500,000 new cases every year and an estimated annual cost of nearly $1 billion in the U.S. Furthermore, the infections are no longer restricted to health care facilities, and recent studies indicate spread of C. difficile infection to the community as well. The clinical spectrum of C. difficile infection ranges from asymptomatic colonization to severe diarrhea, fulminant colitis and death. This spectrum results from a complex interplay between bacterial virulence factors, the colonic microbiome and the host inflammatory response. The overall vigor of host inflammatory response is believed to be an important determinant of C. difficile disease severity, and a more robust immune response is associated with worse outcomes. Neutrophils are the primary cells that respond to C. difficile invasion and neutrophilic inflammation is the hallmark of C. difficile-associated disease. In this review, we will focus on the role of neutrophils (infiltration to infected tissue, pathogen clearance and resolution of inflammation) in the immuno-pathogenesis of C. difficile-associated disease (CDAD).

Role of obesity and adipose tissue-derived cytokine leptin during Clostridium difficile infection.

Obesity is among the most pressing health concerns in the world since it is increasingly common even in the developing world, and is clearly associated with increased risk for chronic debilitating diseases and death. Furthermore, obesity can influence the pathogenesis of infectious diseases by affecting the balance of pathogen clearance and pathological inflammation. The mechanisms that result in enhanced inflammation in obese individuals are poorly understood. Clostridium difficile is a major cause of nosocomial infections worldwide. Recent studies have shown that obesity is associated with increased risk of C. difficile infections. In this review, we will discuss our current knowledge of the role of obesity in determining risk of C. difficile infections, and focus on the role of the adipose tissue-derived cytokine leptin in C. difficile infections.

Leptin receptor mutation results in defective neutrophil recruitment to the colon during Entamoeba histolytica infection.

UNLABELLED: Amebiasis is an enteric infection caused by Entamoeba histolytica, with symptoms ranging in severity from asymptomatic colonization to dysentery. Humans with the Q223R leptin receptor mutation have increased susceptibility to amebiasis, but the mechanism has been unclear. Using a mouse model expressing the mutation, we tested the impact of the Q223R mutation on the innate immune response to E. histolytica infection. The 223R mutation resulted in delayed clearance of amebae from the cecum, as had been previously observed. We found that neutrophil influx to the site of the infection was reduced 12 h after infection in 223R mice. Depletion of neutrophils with anti-Ly6G monoclonal antibody increased susceptibility of wild-type mice to infection, supporting the importance of neutrophils in innate defense. Leptin expression was increased in the cecum by E. histolytica infection, suggesting that leptin could serve as a homing signal for neutrophils to the gut. Interestingly, neutrophils from mice with the 223R mutation had diminished chemotaxis toward leptin. This impaired chemotaxis likely explained the reduced gut infiltration of neutrophils. The newly recognized effect of the leptin receptor Q223R mutation on neutrophil chemotaxis and the impact of this mutation on multiple infectious diseases suggest a broader impact of this mutation on susceptibility to disease. IMPORTANCE: The Q223R leptin receptor mutation results in increased susceptibility of children and adults to E. histolytica, one of the leading causes of diarrhea morbidity and mortality in children of the developing world. Here we show that the mutation results in reduced neutrophil infiltration to the site of infection. This decreased infiltration is likely due to the mutation's impact on neutrophil chemotaxis toward leptin, an inflammatory agent upregulated in the cecum after infection. The significance of this work thus extends beyond understanding E. histolytica susceptibility by also providing insight into the potential impact of leptin on neutrophil function in other states of altered leptin signaling, which include both malnutrition and obesity.

Role of leptin-mediated colonic inflammation in defense against Clostridium difficile colitis.

The role of leptin in the mucosal immune response to Clostridium difficile colitis, a leading cause of nosocomial infection, was studied in humans and in a murine model. Previously, a mutation in the receptor for leptin (LEPR) was shown to be associated with susceptibility to infectious colitis and liver abscess due to Entamoeba histolytica as well as to bacterial peritonitis. Here we discovered that European Americans homozygous for the same LEPR Q223R mutation (rs1137101), known to result in decreased STAT3 signaling, were at increased risk of C. difficile infection (odds ratio, 3.03; P = 0.015). The mechanism of increased susceptibility was studied in a murine model. Mice lacking a functional leptin receptor (db/db) had decreased clearance of C. difficile from the gut lumen and diminished inflammation. Mutation of tyrosine 1138 in the intracellular domain of LepRb that mediates signaling through the STAT3/SOCS3 pathway also resulted in decreased mucosal chemokine and cell recruitment. Collectively, these data support a protective mucosal immune function for leptin in C. difficile colitis partially mediated by a leptin-STAT3 inflammatory pathway that is defective in the LEPR Q223R mutation. Identification of the role of leptin in protection from C. difficile offers the potential for host-directed therapy and demonstrates a connection between metabolism and immunity.

Role of interleukin 23 signaling in Clostridium difficile colitis.

Clostridium difficile is currently the leading cause of hospital-acquired infections in the United States. Here, we observed increased interleukin 23 (IL-23) protein levels in human colon biopsy specimens positive for C. difficile toxins, compared with levels in negative controls (P = .008) We also investigated the role of IL-23 during C. difficile infection, using 2 distinct murine models. Mice lacking IL-23 signaling had a significant increase in survival (100% [12 mice]), compared with control mice (16.7%-50% [12 mice]). These data suggest a new potential drug target for human C. difficile treatment and indicate the first link between IL-23 and disease severity during murine infection.

Immune responses to Clostridium difficile infection.

Clostridium difficile is the causal agent of antibiotic-associated diarrhea and is a leading cause of hospital-acquired infections in the US. C. difficile has been known to cause severe diarrhea and colitis for more than 30 years, but the emergence of a newer, hypervirulent strain of C. difficile (BI/NAP1) has further compounded the problem, and recently both the number of cases and mortality associated with C. difficile-associated diarrhea have been increasing. One of the major drivers of disease pathogenesis is believed to be an excessive host inflammatory response. A better understanding of the host inflammation and immune mechanisms that modulate the course of disease and control host susceptibility to C. difficile could lead to novel (host-targeted) strategies for combating the challenges posed by this deadly infection. This review summarizes our current knowledge of the host inflammatory response during C. difficile infection.

Functional specializations of intestinal dendritic cell and macrophage subsets that control Th17 and regulatory T cell responses are dependent on the T cell/APC ratio, source of mouse strain, and regional localization.

Although several subsets of intestinal APCs have been described, there has been no systematic evaluation of their phenotypes, functions, and regional localization to date. In this article, we used 10-color flow cytometry to define the major APC subsets in the small and large intestine lamina propria. Lamina propria APCs could be subdivided into CD11c(+)CD11b(-), CD11c(+)CD11b(+), and CD11c(dull)CD11b(+) subsets. CD11c(+)CD11b(-) cells were largely CD103(+)F4/80(-) dendritic cells (DCs), whereas the CD11c(+)CD11b(+) subset comprised CD11c(+)CD11b(+)CD103(+)F4/80(-) DCs and CD11c(+)CD11b(+)CD103(-)F4/80(+) macrophage-like cells. The majority of CD11c(dull)CD11b(+) cells were CD103(-)F4/80(+) macrophages. Although macrophages were more efficient at inducing Foxp3(+) regulatory T (T(reg)) cells than DCs, at higher T cell/APC ratios, all of the DC subsets efficiently induced Foxp3(+) T(reg) cells. In contrast, only CD11c(+)CD11b(+)CD103(+) DCs efficiently induced Th17 cells. Consistent with this, the regional distribution of CD11c(+)CD11b(+)CD103(+) DCs correlated with that of Th17 cells, with duodenum > jejunum > ileum > colon. Conversely, CD11c(+)CD11b(-)CD103(+) DCs, macrophages, and Foxp3(+) T(reg) cells were most abundant in the colon and scarce in the duodenum. Importantly, however, the ability of DC and macrophage subsets to induce Foxp3(+) T(reg) cells versus Th17 cells was strikingly dependent on the source of the mouse strain. Thus, DCs from C57BL/6 mice from Charles River Laboratories (that have segmented filamentous bacteria, which induce robust levels of Th17 cells in situ) were more efficient at inducing Th17 cells and less efficient at inducing Foxp3(+) T(reg) cells than DCs from B6 mice from The Jackson Laboratory. Thus, the functional specializations of APC subsets in the intestine are dependent on the T cell/APC ratio, regional localization, and source of the mouse strain.

Systemic but not local infections elicit immunosuppressive IL-10 production by natural killer cells.

Surviving infection represents a balance between the proinflammatory responses needed to eliminate the pathogen, and anti-inflammatory signals limiting damage to the host. IL-10 is a potent immunosuppressive cytokine whose impact is determined by the timing and localization of release. We show that NK cells rapidly express IL-10 during acute infection with diverse rapidly disseminating pathogens. The proinflammatory cytokine IL-12 was necessary and sufficient for NK cell induction of IL-10. NK cells from mice with systemic parasitic infection inhibited dendritic cell release of IL-12 in an IL-10-dependent manner, and NK cell depletion resulted in elevated serum IL-12. These data suggest an innate, negative feedback loop in which IL-12 limits its own production by eliciting IL-10 from NK cells. In contrast to disseminating pathogens, locally restricted infections did not elicit NK cell IL-10. Thus systemic infections uniquely engage NK cells in an IL-10-mediated immunoregulatory circuit that functions to alleviate inflammation.

Interleukin 10 acts on regulatory T cells to maintain expression of the transcription factor Foxp3 and suppressive function in mice with colitis.

Regulatory T cells (T(reg) cells) that express the transcription factor Foxp3 suppress the activity of other cells. Here we show that interleukin 10 (IL-10) produced by CD11b(+) myeloid cells in recombination-activating gene 1-deficient (Rag1(-/-)) recipient mice was needed to prevent the colitis induced by transferred CD4(+)CD45RB(hi) T cells. In Il10(-/-)Rag1(-/-) mice, T(reg) cells failed to maintain Foxp3 expression and regulatory activity. The loss of Foxp3 expression occurred only in recipients with colitis, which indicates that the requirement for IL-10 is manifested in the presence of inflammation. IL-10 receptor-deficient (Il10rb(-/-)) T(reg) cells also failed to maintain Foxp3 expression, which suggested that host IL-10 acted directly on the T(reg) cells. Our data indicate that IL-10 released from myeloid cells acts in a paracrine manner on T(reg) cells to maintain Foxp3 expression.

Cutting edge: IL-27 induces the transcription factor c-Maf, cytokine IL-21, and the costimulatory receptor ICOS that coordinately act together to promote differentiation of IL-10-producing Tr1 cells.

IL-27 has recently been identified as a differentiation factor for the generation of IL-10-producing regulatory type 1 (Tr1) T cells. However, how IL-27 induces the expansion of Tr1 cells has not been elucidated. In this study we demonstrate that IL-27 drives the expansion and differentiation of IL-10-producing murine Tr1 cells by inducing three key elements: the transcription factor c-Maf, the cytokine IL-21, and the costimulatory receptor ICOS. IL-27-driven c-Maf expression transactivates IL-21 production, which acts as an autocrine growth factor for the expansion and/or maintenance of IL-27-induced Tr1 cells. ICOS further promotes IL-27-driven Tr1 cells. Each of those elements is essential, because loss of c-Maf, IL-21-signaling, or ICOS decreases the frequency of IL-27-induced differentiation of IL-10-producing Tr1 cells.

Nonredundant roles for B cell-derived IL-10 in immune counter-regulation.

IL-10 plays a central role in restraining the vigor of inflammatory responses, but the critical cellular sources of this counter-regulatory cytokine remain speculative in many disease models. Using a novel IL-10 transcriptional reporter mouse, we found an unexpected predominance of B cells (including plasma cells) among IL-10-expressing cells in peripheral lymphoid tissues at baseline and during diverse models of in vivo immunological challenge. Use of a novel B cell-specific IL-10 knockout mouse revealed that B cell-derived IL-10 nonredundantly decreases virus-specific CD8(+) T cell responses and plasma cell expansion during murine cytomegalovirus infection and modestly restrains immune activation after challenge with foreign Abs to IgD. In contrast, no role for B cell-derived IL-10 was evident during endotoxemia; however, although B cells dominated lymphoid tissue IL-10 production in this model, myeloid cells were dominant in blood and liver. These data suggest that B cells are an underappreciated source of counter-regulatory IL-10 production in lymphoid tissues, provide a clear rationale for testing the biological role of B cell-derived IL-10 in infectious and inflammatory disease, and underscore the utility of cell type-specific knockouts for mechanistic limning of immune counter-regulation.