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Background & Aims: Clostridium difficile toxin A (TcdA) and C difficile toxin toxin B (TcdB), the major virulence factors of the bacterium, cause intestinal tissue damage and inflammation. Although the 2 toxins are homologous and share a similar domain structure, TcdA is generally more inflammatory whereas TcdB is more cytotoxic. The functional domain of the toxins that govern the proinflammatory activities of the 2 toxins is unknown. Methods: Here, we investigated toxin domain functions that regulate the proinflammatory activity of C difficile toxins. By using a mouse ilea loop model, human tissues, and immune cells, we examined the inflammatory responses to a series of chimeric toxins or toxin mutants deficient in specific domain functions. Results: Blocking autoprocessing of TcdB by mutagenesis or chemical inhibition, while reducing cytotoxicity of the toxin, significantly enhanced its proinflammatory activities in the animal model. Furthermore, a noncleavable mutant TcdB was significantly more potent than the wild-type toxin in the induction of proinflammatory cytokines in human colonic tissues and immune cells. Conclusions: In this study, we identified a novel mechanism of regulating the biological activities of C difficile toxins in that cysteine protease-mediated autoprocessing regulates toxins’ proinflammatory activities. Our findings provide new insight into the pathogenesis of C difficile infection and the design of therapeutics against the disease. Keywords: C difficile, Toxins, Cysteine Protease, Autoprocessing, Inflammation

Enteric fevers, caused by the Salmonella enterica serovars Typhi (ST), Paratyphi A (PA) and Paratyphi B (PB), are life-threatening illnesses exhibiting very similar clinical symptoms but with distinct epidemiologies, geographical distributions and susceptibilities to antimicrobial treatment. Nevertheless, the mechanisms by which the host recognizes pathogens with high levels of homology, such as these bacterial serovars, remain poorly understood. Using a three-dimensional organotypic model of the human intestinal mucosa and PA, PB, and ST, we observed significant differences in the secretion patterns of pro-inflammatory cytokines and chemokines elicited by these serovars. These cytokines/chemokines were likely to be co-regulated and influenced the function of epithelial cells, such as the production of IL-8. We also found differing levels of polymorphonuclear leukocyte (PMN) migration among various infection conditions that either included or excluded lymphocytes and macrophages (Mϕ), strongly suggesting feedback mechanisms among these cells. Blocking experiments showed that IL-1β, IL-6, IL-8, TNF-α and CCL3 cytokines were involved in the differential regulation of migration patterns. We conclude that the crosstalk among the lymphocytes, Mϕ, PMN and epithelial cells is cytokine/chemokine-dependent and bacterial-serotype specific, and plays a pivotal role in orchestrating the functional efficiency of the innate cells and migratory characteristics of the leukocytes.

Infection by Salmonella enterica serovar Typhi (S. Typhi), the cause of enteric fevers, is low in high-income countries but persistent in low- and middle-income countries, resulting in 65,400-187,700 deaths yearly. Drug resistance, including in the United States, exacerbates this issue. Evidence indicates that innate lymphocytes (INLs), such as natural killer (NK) cells, and unconventional T lymphocytes (e.g., Mucosal-associated invariant T (MAIT) cells and T-cell receptor gamma delta (TCR-γδ) cells) can impact the intestinal epithelial barrier, the primary site of exposure to S. Typhi. Moreover, INL production of IFN-γ is central in controlling S. Typhi infection. However, the impact of epithelial cells (EC) on the secretion of IFN-γ by INLs and the relationship between these events and epigenetic changes remains unknown. Epigenetic modifications in host cells are fundamental for their differentiation and function, including IFN-γ production. Herein, using a human organoid-derived polarized intestinal epithelial cell monolayer, we investigated the role of H3K4me3 and H3K27me3 epigenetic marks in intestinal immunity, focusing on the function of EC, NK, MAIT, and TCR-γδ cells in response to S. Typhi. This study builds on our previous findings that MAIT subsets exhibiting specific IFN-γ pattern signatures were associated with protection against typhoid fever and that S. Typhi infection regulates changes in chromatin marks that depend on individual cell subsets. Here, we show that cultures exposed to S. Typhi without EC exhibit a significant increase in NK and MAIT cells, and, to a lesser extent, TCR-γδ cells, expressing IFN-γ and H3K4me3 but not H3K27me3 marks, contrasting with cultures where EC is present. The influence of EC on INL H3K4me3 marks might be indirectly mediated through the modulation of IL-18 secretion via the Histone Deacetylase 6 gene during S. Typhi infection.

Salmonella enterica serovar Typhi (S. Typhi), a human-restricted pathogen, invades the host through the gut to cause typhoid fever. Recent calculations of the typhoid fever burden estimated that more than 10 million new typhoid fever cases occur in low and middle-income countries, resulting in 65,400-187,700 deaths yearly. Interestingly, if not antibiotic-treated, upon the resolution of acute disease, 1%-5% of patients become asymptomatic chronic carriers. Chronically infected hosts are not only critical reservoirs of infection that transmit the disease to naive individuals but are also predisposed to developing gallbladder carcinoma. Nevertheless, the molecular mechanisms involved in the early interactions between gallbladder epithelial cells and S. Typhi remain largely unknown. Based on our previous studies showing that closely related S. Typhi strains elicit distinct innate immune responses, we hypothesized that host molecular pathways activated by S. Typhi strains derived from acutely and chronically infected patients would differ. To test this hypothesis, we used a novel human organoid-derived polarized gallbladder monolayer model, and S. Typhi strains derived from acutely and chronically infected patients. We found that S. Typhi strains derived from acutely and chronically infected patients differentially regulate host mitogen-activated protein kinase (MAPK) and S6 transcription factors. These variations might be attributed to differential cytokine signaling, predominantly via TNF-α and IL-6 production and appear to be influenced by the duration the isolate was subjected to selective pressures in the gallbladder. These findings represent a significant leap in understanding the complexities behind chronic S. Typhi infections in the gallbladder and may uncover potential intervention targets.

Pectins, a class of dietary fibers abundant in vegetables and fruits, have drawn considerable interest due to their potential anti-inflammatory properties. Numerous studies have indicated that incorporating pectins into infant formula could be a safe strategy for alleviating infant regurgitation and diarrhea. Moreover, pectins have been shown to modulate cytokine production, macrophage activity, and NF-kB expression, all contributing to their anti-inflammatory effects. Despite this promising evidence, the exact mechanisms through which pectins exert these functions and how their structural characteristics influence these processes remain largely unexplored. This knowledge is particularly significant in the context of gut inflammation in developing preterm babies, a critical aspect of necrotizing enterocolitis (NEC), and in children and adults dealing with inflammatory bowel disease (IBD). Our mini review aims to provide an up-to-date compilation of relevant research on the effects of pectin on gut immune responses, specifically focusing on preterms and newborns. By shedding light on the underlying mechanisms and implications of pectin-mediated anti-inflammatory properties, this review seeks to advance our knowledge in this area and pave the way for future research and potential therapeutic interventions.

Mucosal-associated invariant T (MAIT) cells are an innate-like population of T cells that display a TCR Vα7.2+ CD161+ phenotype and are restricted by the nonclassical MHC-related molecule 1 (MR1). Although B cells control MAIT cell development and function, little is known about the mechanisms underlying their interaction(s). Here, we report, for the first time, that during Salmonella enterica serovar Typhi (S. Typhi) infection, HLA-G expression on B cells downregulates IFN-γ production by MAIT cells. In contrast, blocking HLA-G expression on S. Typhi-infected B cells increases IFN-γ production by MAIT cells. After interacting with MAIT cells, kinetic studies show that B cells upregulate HLA-G expression and downregulate the inhibitory HLA-G receptor CD85j on MAIT cells resulting in their loss. These results provide a new role for HLA-G as a negative feedback loop by which B cells control MAIT cell responses to antigens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Although immunity induced by typhoid fever is moderated and short-lived, typhoid vaccination with the attenuated Ty21a oral vaccine generates long-lasting protection rates reaching up to 92%. Thus, there are important differences on how wild-type Salmonella and typhoid vaccine strains stimulate host immunity. We hypothesize that vaccine strains with different mutations might affect gut inflammation and intestinal permeability by different mechanisms. To test this hypothesis, we used an in vitro organotypic model of the human intestinal mucosa composed of human intestinal epithelial cells, lymphocytes/monocytes, endothelial cells, and fibroblasts. We also used six Salmonella enterica serovar Typhi (S. Typhi) strains: the licensed Ty21a oral vaccine, four typhoid vaccine candidates (i.e., CVD 908, CVD 909, CVD 910, and CVD 915) and the wild-type Ty2 strain. We found that genetically engineered S. Typhi vaccine strains elicit differential host changes not only in the intestinal permeability and secretion of inflammatory cytokines, but also in the phenotype and activation pathways of innate cells. These changes were distinct from those elicited by the parent wild-type S. Typhi and depended on the genetic manipulation. In sum, these results emphasize the importance of carefully selecting specific manipulations of the Salmonella genome in the development of typhoid vaccines.

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of the typhoid fever, is a pathogen of great public health importance. Typhoid vaccines have the potential to be cost-effective measures towards combating this disease, yet the antigens triggering host protective immune responses are largely unknown. Given the key role of cellular-mediated immunity in S. Typhi protection, it is crucial to identify S. Typhi proteins involved in T-cell responses. Here, cells from individuals immunized with Ty21a typhoid vaccine were collected before and after immunization and used as effectors. We also used an innovative antigen expressing system based on the infection of B-cells with recombinant Escherichia coli (E. coli) expressing one of four S. Typhi gene products (i.e., SifA, OmpC, FliC, GroEL) as targets. Using flow cytometry, we found that the pattern of response to specific S. Typhi proteins was variable. Some individuals responded to all four proteins while others responded to only one or two proteins. We next evaluated whether T-cells responding to recombinant E. coli also possess the ability to respond to purified proteins. We observed that CD4+ cell responses, but not CD8+ cell responses, to recombinant E. coli were significantly associated with the responses to purified proteins. Thus, our results demonstrate the feasibility of using an E. coli expressing system to uncover the antigen specificity of T-cells and highlight its applicability to vaccine studies. These results also emphasize the importance of selecting the stimuli appropriately when evaluating CD4+ and CD8+ cell responses.

BACKGROUND:The emergence of antibiotic-resistant strains of Salmonella enterica serovar Typhi (S. Typhi), the etiologic agent of typhoid fever, has aggravated an already important public health problem and added new urgency to the development of more effective typhoid vaccines. To this end it is critical to better understand the induction of immunity to S. Typhi. CD8(+) T cells are likely to play an important role in host defense against S. Typhi by several effector mechanisms, including killing of infected cells and IFN-gamma secretion. However, how S. Typhi regulates the development of specific CD8(+) responses in humans remains unclear. Recent studies in mice have shown that dendritic cells (DC) can either directly (upon uptake and processing of Salmonella) or indirectly (by bystander mechanisms) elicit Salmonella-specific CD8(+) T cells. METHODOLOGY/PRINCIPAL FINDINGS:We report here that upon infection with live S. Typhi, human DC produced high levels of pro-inflammatory cytokines IL-6, IL-8 and TNF-alpha, but low levels of IL-12 p70 and IFN-gamma. In contrast, DC co-cultured with S. Typhi-infected cells, through suicide cross-presentation, uptake S. Typhi-infected human cells and release high levels of IFN-gamma and IL-12p70, leading to the subsequent presentation of bacterial antigens and triggering the induction of memory T cells, mostly CD3(+)CD8(+)CD45RA(-)CD62L(-) effector/memory T cells. CONCLUSIONS/SIGNIFICANCE:This study is the first to demonstrate the effect of S. Typhi on human DC maturation and on their ability to prime CD8(+) cells and highlights the significance of these phenomena in eliciting adaptive immunity to S. Typhi.