NKp46+ ILC3s promote early neutrophil defense against Clostridioides difficile infection through GM-CSF secretion.

Clostridioides difficile infection (CDI) is a common cause of antibiotic-associated colitis. C. difficile proliferates and produces toxins that damage the colonic epithelium, leading to symptoms ranging from mild diarrhea to severe pseudomembranous colitis. The host's innate response to CDI occurs in two phases: an early phase in which neutrophils reduce the bacterial load and a late phase involving repair mechanisms to restore epithelial integrity. Group 3 innate lymphoid cells (ILC3s) are crucial in protecting the gut from CDI. Previous studies have shown that ILC3-derived IL-22 is essential in the late phase of CDI for epithelial repair and maintaining an intestinal microbiota that competes with C. difficile, preventing its expansion. Our study finds that ILC3s also protect during the early stages of CDI by sustaining neutrophils through GM-CSF. Less neutrophil production, accumulation, and activation was evident in ILC3-deficient mice than in wild-type (WT) mice, which led to exacerbated symptoms, impaired pathogen clearance, a compromised epithelial barrier, and increased mortality. The adoptive transfer of ILC3s into ILC3-deficient mice restored neutrophil responses and improved disease outcomes. Both in vitro and in vivo experiments revealed that GM-CSF production by ILC3s is crucial for neutrophil production and effective resistance during CDI. Using mice lacking NKp46+ ILC3s, we found that this subset significantly contributes to GM-CSF production in CDI. These findings highlight the critical role of the ILC3-neutrophil connection in early innate responses to CDI. Enhancing ILC3 production of GM-CSF could be a promising strategy for improving host defense against CDI and other enteric infections.

Purified CDT toxins and a clean deletion within the CDT locus provide novel insights into the contribution of binary toxin in cellular inflammation and Clostridioides difficile infection.

Clostridioides difficile is a spore-forming pathogen and the most common cause of healthcare-associated diarrhea and colitis in the United States. Besides producing the main virulence factors, toxin A (TcdA) and toxin B (TcdB), many of the common clinical strains encode the C. difficile transferase (CDT) binary toxin. The role of CDT in the context of C. difficile infection (CDI) is poorly understood. Inflammation is a hallmark of CDI and multiple mechanisms of inflammasome activation have been reported for TcdA, TcdB, and the organism. Some studies have suggested that CDT contributes to this inflammation through a TLR2-dependent priming mechanism that leads to the suppression of protective eosinophils. Here, we show that CDT does not prime but instead activates the inflammasome in bone marrow-derived dendritic cells (BMDCs). In bone marrow-derived macrophages (BMDMs), the cell binding and pore-forming component of the toxin, CDTb, alone activates the inflammasome and is dependent on K+ efflux. The activation is not observed in the presence of CDTa and is not observed in BMDMs derived from Nlrp3-/- mice suggesting the involvement of the NLRP3 inflammasome. However, we did not observe evidence of CDT-dependent inflammasome priming or activation in vivo. Mice were infected with R20291 and an isogenic CRISPR/Cas9-generated R20291 ΔcdtB strain of C. difficile. While CDT contributes to increased weight loss and cecal edema at 2 days post infection, the relative levels of inflammasome-associated cytokines, IL-1ß and IL-18, in the cecum and distal colon are unchanged. We also saw CDT-dependent weightloss in Nlrp3-/- mice, suggesting that the increased weightloss associated with the presence of CDT is not a result of NLRP3-dependent inflammasome activation. This study highlights the importance of studying gene deletions in the context of otherwise fully isogenic strains and the challenge of translating toxin-specific cellular responses into a physiological context, especially when multiple toxins are acting at the same time.

Bacillus licheniformis suppresses Clostridium perfringens infection via modulating inflammatory response, antioxidant status, inflammasome activation and microbial homeostasis in broilers.

Pathogenic bacteria infection, especially Clostridium perfringens (C. perfringens), markedly threatened the health of animals, and further caused huge economic loss. In this study, Bacillus licheniformis HJ0135 (BL) was used. Oxford cup bacteriostatic test and inhibitory rate test were conducted to evaluate the antibacterial ability of BL. Results showed the strongest inhibitory role of BL on C. perfringens (P < 0.05). Afterwards, 540 one-day-old yellow-feather broilers (32.7 ± 0.2 g) were randomly allocated into 3 groups, including CON group (basal diet), CP group (basal diet + 1 × 109 CFU C. perfringens in gavage), and BL + CP group (basal diet containing 7.5 × 106 CFU/g BL + 1 × 109 CFU C. perfringens in gavage). At d 70, broilers in the CP and BL + CP groups were treated with C. perfringens by continuously oral administration for 5 d. The experiment lasted for 75 d. The serum, immune organs, jejunal mucosa, and cecal contents were collected for analysis. In vivo experiment showed that BL supplementation markedly improved (P < 0.05) BW, ADG, thymus index, serum immunoglobins and antioxidases, reduced feed conversion ratio (FCR) and serum pro-inflammatory cytokines of C. perfringens-infected broilers. Furthermore, the increased jejunal injury and levels of pro-inflammatory cytokines, decreased gene expressions of tight junction proteins in the jejunal mucosa were significantly alleviated (P < 0.05) by BL. More importantly, the activation of NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome was inhibited (P < 0.05) by BL to further attenuate jejunal damage. Besides, BL supplementation markedly increased (P < 0.05) the cecal isobutyric acid and isovaleric acid. Microbial analysis showed that BL changed the composition and relative abundances of microbiota in the cecal contents (P < 0.05), especially the short chain fatty acids (SCFAs)-producing bacteria including Eubacterium_coprostanoligenes_group, Megamonas, Faecalibacterium, and Lactobacillus, which further protected against C. perfringens-induced jejunal inflammation in broilers. Our study laid a theoretical basis for the application of probiotics in lessening C. perfringens-related diseases in poultry farming.

Screening and selection of eubiotic compounds possessing immunomodulatory and anti-Clostridium perfringens properties.

Eubiotics are water and/or feed additives used in poultry to promote gut health and control enteric burden of pathogens, including Clostridium perfringens. While several eubiotic compounds (ECs) are being introduced commercially, it is essential to devise an in vitro model to screen these compounds to assess their immunomodulatory and antimicrobial properties prior to their testing in vivo. A chicken macrophage cell-line (MQ-NCSU) was used to develop an in vitro model to screen the immunological and anti-C. perfringens properties of 10 ECs: monobutyrin, monolaurin, calcium butyrate, tributyrin, carvacrol, curcumin, green tea extract, rosemary extract, monomyristate, and tartaric acid. An optimal concentration for each EC was selected by measuring the effect on viability of MQ-NCSU cells. Cells were then treated with ECs for 6, 12, and 24 h. and expression of interferon-gamma (IFNγ), interleukin (IL)-1ß, IL-6, IL-10, transforming growth factor-beta (TGFß) and cluster of differentiation (CD40) genes, as well as major histocompatibility complex (MHC)-II protein were evaluated. At 6 h post-stimulation, monobutyrin, calcium butyrate, and green tea extract treatments induced a significant downregulation of IFNγ, IL-6, or IL-1ß gene transcription and MHC-II expression, while the IL-10 or TGFß gene expression in these treatments as well as those receiving rosemary extract and tartaric acid was significantly upregulated, when compared to control, suggesting immunomodulatory properties of these ECs. Finally, pretreatment of macrophages with these selected 5 ECs for 24 h followed by C. perfringens infection showed that monobutyrin, green tea extract, rosemary extract, and calcium butyrate treatments can inhibit bacterial growth significantly at 12 and/or 24 h post-infection, when compared to the control. Collectively, our findings show that ECs possessing immunomodulatory and anti-C. perfringens properties can be selected using an in vitro avian macrophage cell-based model so that such ECs can further be tested in vivo for their disease prevention efficacy.

Absent in Melanoma 2 Mediates Inflammasome Signaling Activation against Clostridium perfringens Infection.

Absent in melanoma 2 (AIM2), a key component of the IFI20X/IFI16 (PYHIN) protein family, is characterized as a DNA sensor to detect cytosolic bacteria and DNA viruses. However, little is known about its immunological role during pathogenic Clostridium perfringens (C. perfringens) infection, an extracellular bacterial pathogen. In a pathogenic C. perfringens gas gangrene model, Aim2-/- mice are more susceptible to pathogenic C. perfringens soft tissue infection, revealing the importance of AIM2 in host protection. Notably, Aim2 deficiency leads to a defect in bacterial killing and clearance. Our in vivo and in vitro findings further establish that inflammasome signaling is impaired in the absence of Aim2 in response to pathogenic C. perfringens. Mechanistically, inflammasome signaling downstream of active AIM2 promotes pathogen control. Importantly, pathogenic C. perfringens-derived genomic DNA triggers inflammasome signaling activation in an AIM2-dependent manner. Thus, these observations uncover a central role for AIM2 in host defense and triggering innate immunity to combat pathogenic C. perfringens infections.

Supplementation with organic yeast-derived selenium provides immune protection against experimental necrotic enteritis in broiler chickens.

Necrotic enteritis (NE) is a potentially fatal poultry disease that causes enormous economic losses in the poultry industry worldwide. The study aimed to evaluate the effects of dietary organic yeast-derived selenium (Se) on immune protection against experimental necrotic enteritis (NE) in commercial broilers. Chickens were fed basal diets supplemented with different Se levels (0.25, 0.50, and 1.00 Se mg/kg). To induce NE, Clostridium perfringens (C. perfringens) was orally administered at 14 days of age post hatch. The results showed that birds fed 0.25 Se mg/kg exhibited significantly increased body weight gain compared with the non-supplemented/infected birds. There were no significant differences in gut lesions between the Se-supplemented groups and the non-supplemented group. The antibody levels against α-toxin and NetB toxin increased with the increase between 0.25 Se mg/kg and 0.50 Se mg/kg. In the jejunal scrapings and spleen, the Se-supplementation groups up-regulated the transcripts for pro-inflammatory cytokines IL-1ß, IL-6, IL-8, iNOS, and LITAF and avian ß-defensin 6, 8, and 13 (AvBD6, 8 and 13). In conclusion, supplementation with organic yeast-derived Se alleviates the negative consequences and provides beneficial protection against experimental NE.

Systemic neutrophil degranulation and emergency granulopoiesis in patients with Clostridioides difficile infection.

OBJECTIVES: Clostridioides difficile infection (CDI) is characterized by neutrophilia in blood, with a high leukocyte count accompanying severe infection. In this study, we characterized peripheral blood neutrophil activation and maturity in CDI by (i) developing a method to phenotype stored neutrophils for disease-related developmental alterations and (ii) assessing neutrophil-associated biomarkers. METHODS: We stored fixed leukocytes from blood collected within 24 h of diagnosis from a cohort of hospitalized patients with acute CDI. Additional study cohorts included recurrent CDI patients at time of and two months after FMT therapy and a control healthy cohort. We assessed levels of neutrophil surface markers CD66b, CD11b, CD16 and CD10 by flow cytometry. Plasma neutrophil elastase and lipocalin-2 were measured using ELISA, while G-CSF, GM-CSF and cytokines were measured using O-link Proteomic technology. RESULTS: CD66b+ neutrophil abundance assessed by flow cytometry correlated well with complete blood counts, establishing that neutrophils in stored blood are sufficiently well-preserved for phenotyping by flow cytometry. Neutrophil abundance was significantly increased in CDI patients compared to healthy controls. Emergency granulopoiesis in acute CDI patients was evidenced by lower neutrophil surface expression of CD10, CD11b and CD16. CD10+ staining of neutrophils started to recover within 3-7 days of CDI treatment. Neutrophil activation and degranulation were higher in acute CDI as assessed by plasma neutrophil elastase and lipocalin-2. Biomarker levels in immunocompetent subjects were associated with recurrence and fatal outcomes. CONCLUSIONS: Neutrophil activation and emergency granulopoiesis characterize the early immune response in acute CDI, with plasma degranulation biomarkers predictive of disease severity.

Effective definition of low humoral response to Clostridioides difficile infection.

BACKGROUND: Determination of the humoral response to Clostridioides difficile (CD) toxins could be of great value in the management of patients with CD infection (CDI). METHODS: A prospective study was conducted on the clinical characteristics and humoral response in patients with CDI. Determination of ELISA IgG CD anti-toxin B (tgcBiomics, Germany) was performed. The following dilutions were planned for each patient, 1:100, 1: 200, 1: 400, 1: 800: 1: 1600. A significant concentration of antibody was considered to be present in each dilution if an optical density 0.2 units higher than the negative control of the technique was evident. RESULTS: Eighty-five patients were included during the study period, November 2018-February 2020. The median age was 73 years (interquartile range: 62.5-85 years), with female predominance (45 patients, 52.9%). Thirty-nine patients (45.9%) had a severe infection. Seven patients (8.2%) had suffered an episode of CDI in the previous three months. Seventeen patients (20%) had one or more recurrent episodes during the three-month follow-up: No patient died during admission or required surgery for severe-complicated infection. The incidence of recurrence in patients with no antibody detected at 1:400 dilution was 25.4% (16 patients) while it was 4.3% (one patient) in patients with antibody present at that dilution (p = 0.03). Liver cirrhosis was associated with higher humoral response against CD. CONCLUSIONS: Antibodies IgG CD anti-toxin B detection at a dilution of 1:400, using a B ELISA technique, effectively identified patients at increased risk of recurrence. This information could help assist in the management of patients.

Blockade of T helper 17 cell function ameliorates recurrent Clostridioides difficile infection in mice.

Clostridioides difficile infection (CDI) is a common infection of the gastrointestinal tract. Typically, 20%-30% of CDI patients experience recurrent C.difficile infection (RCDI). Although the role of Th17 in infectious and inflammatory diseases including CDI has gained attention, reports on the correlation between Th17 and RCDI are scarce. In this study, CDI and RCDI mice models were challenged with C. difficile. Serum lactic acid dehydrogenase, inflammatory factor levels, reverse transcriptase-polymerase chain reaction, western blot analysis, hematoxylin and eosin staining, immunohistochemistry, flow cytometry analysis, and enzyme-linked immunosorbent assay were performed on the CDI, RCDI, and control group mice. The results showed more serious clinical manifestations in the RCDI group compared with those in the CDI group. More severe gut barrier disruption and higher degree of microbiota translocation were observed in the RCDI group compared with those in the CDI group. Moreover, extremely severe apoptosis was observed in HCT-116 cells incubated with the serum from RCDI mice model. In addition, higher levels of Th17 and IL-17 were detected in the blood or serum from the RCDI mouse model. Treatment with RORγt small molecule inhibitor SR1001 increased the expression of occludin, decreased the apoptotic rate of HCT-116 cells, and decreased the concentrations of Th17 and IL-17. Concisely, Th17 and IL-17 are potential indicators of RCDI and may serve as therapeutic targets for RCDI treatment. This study lays the foundation for future research on RCDI diagnosis and treatment.

Toward the development of defined microbial therapeutics.

The collection of micro-organisms living in the mammalian gastrointestinal tract, termed the gut microbiota, has been shown to have profound impacts on host health and increasingly is regarded as a viable therapeutic target. Clinical studies of fecal microbiota transplantation have demonstrated potential efficacy of microbiota-based therapies for diseases including Clostridioides difficile infections, inflammatory bowel disease, graft-versus-host disease and cancer. However, the lack of understanding of the active ingredients and potential risks of such therapies pose challenges for clinical application. Meanwhile, efforts are being made to identify effector microbes directly associated with a given phenotype, to establish causality and to devise well-characterized microbial therapeutics for clinical use. Strategies based on defined microbial components will likely enhance the potential of microbiota-targeted therapies.

Clostridioides difficile spores stimulate inflammatory cytokine responses and induce cytotoxicity in macrophages.

Clostridioides difficile is a gram-positive, spore-forming anaerobic bacterium, and the leading cause of antibiotic-associated diarrhea worldwide. During C. difficile infection, spores germinate in the presence of bile acids into vegetative cells that subsequently colonize the large intestine and produce toxins. In this study, we demonstrated that C. difficile spores can universally adhere to, and be phagocytosed by, murine macrophages. Only spores from toxigenic strains were able to significantly stimulate the production of inflammatory cytokines by macrophages and subsequently induce significant cytotoxicity. Spores from the isogenic TcdA and TcdB double mutant induced significantly lower inflammatory cytokines and cytotoxicity in macrophages, and these activities were restored by pre-exposure of the spores to either toxins. These findings suggest that during sporulation, spores might be coated with C. difficile toxins from the environment, which could affect C. difficile pathogenesis in vivo.

Immunomodulatory effect of short-term supplementation with Bacillus toyonensis BCT-7112T and Saccharomyces boulardii CNCM I-745 in sheep vaccinated with Clostridium chauvoei.

The bacterium Clostridium chauvoei is the causative agent of blackleg in livestock, and vaccination is the most effective means of prevention. The aim of this study was to assess the effect of short-term supplementation with Bacillus toyonensis and Saccharomyces boulardii on the immune response to a C. chauvoei vaccine in sheep. Sheep were vaccinated subcutaneously on day 0 and received a booster dose on day 21, with 2 mL of a commercial vaccine formulated with inactivated C. chauvoei bacterin adsorbed on aluminum hydroxide. Probiotics were orally administered B. toyonensis (3 × 108 cfu) and S. boulardii (3 × 108 cfu) over five days prior to the first and second doses of the vaccine. Sheep supplemented with B. toyonensis and S. boulardii showed significantly higher specific IgG, IgG1, and IgG2 titers (P<0.05), with approximately 24- and 14-fold increases in total IgG levels, respectively, than the nonsupplemented group. Peripheral blood mononuclear cells from the supplemented group had increased mRNA transcription levels of the IFN-γ, IL2, and Bcl6 genes. These results demonstrate an adjuvant effect of short-term supplementation with B. toyonensis and S. boulardii on the immune response against the C. chauvoei vaccine in sheep.

Identification of linear epitopes on the flagellar proteins of Clostridioides difficile.

Clostridioides difficile (C. difficile) is an opportunistic anaerobic bacterium that causes severe diseases of the digestive tract of humans and animals. One of the possible methods of preventing C. difficile infection is to develop a vaccine. The most promising candidates for vaccine antigens are the proteins involved in the adhesion phenomena. Among them, the FliC and FliD are considered to be suitable candidates. In this paper, the FliC and FliD protein polypeptide epitopes were mapped in silico and by using PEPSCAN procedure. We identified four promising epitopes: 117QRMRTLS123, 205MSKAG209 of FliC and 226NKVAS230, 306TTKKPKD312 of FliD protein. We showed that 117QRMRTLS123 sequence is not only located in TLR5-binding and activating region, as previously shown, but forms an epitope recognized by C. difficile-infected patients' antibodies. 205MSKAG209 is a C. difficile-unique, immunogenic sequence that forms an exposed epitope on the polymerized flagella structure which makes it a suitable vaccine antigen. 226NKVAS230 and 306TTKKPKD312 are well exposed and possess potential protective properties according to VaxiJen analysis. Our results open the possibility to use these epitopes as suitable anti-C. difficile vaccine antigens.

Immunosuppression and Clostridioides (Clostridium) difficile Infection Risk in Metabolic and Bariatric Surgery Patients.

BACKGROUND: Immunosuppressant use increases risk of Clostridioides (Clostridium) difficile infection. To date, no studies have analyzed the relationship between immunosuppressant use and C difficile infections after metabolic and bariatric surgery (MBS). METHODS: A retrospective analysis of the 2015-2018 MBSAQIP data was conducted. The MBSAQIP data include information from 854 affiliated practices in the US and Canada. Initial sample size was 760,076 MBS patients. After excluding participants due to missing variables (n = 188,106) and the use of surgical procedures other than Roux-en-Y gastric bypass and sleeve gastroplasty (n = 129,712), final analyses were performed on 442,258 participants. Logistic regression models generated the odds of C difficile infection developing post MBS, according to immunosuppressant status (positive or negative). RESULTS: Unadjusted logistic regression analysis showed that patients using immunosuppressants were 95% more likely to have postoperative C difficile infection (odds ratio 1.945; 95% CI, 1.230 to 3.075; p < 0.001) vs MBS patients not taking immunosuppressants. After adjusting for age, sex, ethnicity, preoperative BMI, diabetes status, and surgical procedure type, the association remained unaffected (adjusted odds ratio 1.956; 95% CI, 1.236 to 3.095; p < 0.01). Patients who completed the laparoscopic Roux-en-Y gastric bypass procedure had more than double the odds of C difficile infection developing compared with those who completed the laparoscopic sleeve gastrectomy procedure (odds ratio 2.183; 95% CI, 1.842 to 2.587; p < 0.0001). CONCLUSIONS: Our results using a population-based sample of MBS patients showed that those taking immunosuppressants have a significantly higher risk of developing Clostridioides (Clostridium) difficile infection postoperatively. These findings suggest that patients using immunosuppressants should be closely monitored both pre and post procedure.

MicroRNA-21-5p targets PDCD4 to modulate apoptosis and inflammatory response to Clostridium perfringens beta2 toxin infection in IPEC-J2 cells.

Clostridium perfringens (C. perfringens), a toxin-producing enteric pathogen, causes a variety of intestinal infections in humans and animals. C. perfringens beta2 (CPB2) toxin has been considered to be a strong virulence factor for C. perfringens infectious enteric diseases (CPED). Altered levels and functions of microRNA-21-5p (miR-21-5p) have been associated with apoptosis and inflammation response in pathological processes. However, little is known about its functional mechanism in CPED. Here, we found that miR-21-5p expressed in multiple tissues of pig, had a highest level in jejunum, and significantly upregulated in intestinal porcine epithelial cells (IPEC-J2) exposed to CPB2 toxin. Noteworthily, transfection of CPB2-treated IPEC-J2 cells with miR-21-5p mimic increased cell viability and Bcl2 expression, as well as reduced cytotoxicity, apoptosis rates and Bax level. Moreover, overexpression of miR-21-5p significantly suppressed the levels of interleukin (IL)-6, IL-8, TNF-α, IL-1ß and nuclear factor-kappa B (NF-κB p65) activity induced by CPB2 toxin, whereas that of the IL-10 was increased in IPEC-J2 cells. On the contrary, transfection of miR-21-5p inhibitor promoted CPB2-induced cell apoptosis and inflammation. Furthermore, we validated that programmed cell death 4 (PDCD4) was strikingly downregulated in CPB2-treated IPEC-J2 cells. PDCD4 exhibited opposing effects to those of miR-21-5p mimic on IPEC-J2 cells, and restoration of PDCD4 expression counteracted the suppressive effect of miR-21-5p on CPB2-induced apoptosis and inflammatory response. Collectively, our findings demonstrated that miR-21-5p was involved in regulating the immune response triggered by CPB2 toxin and contributed to protective effects in CPB2-induced CPED cell model by targeting PDCD4.

A standardised model for stool banking for faecal microbiota transplantation: a consensus report from a multidisciplinary UEG working group.

BACKGROUND: Faecal microbiota transplantation is an emerging therapeutic option, particularly for the treatment of recurrent Clostridioides difficile infection. Stool banks that organise recruitment and screening of faeces donors are being embedded within the regulatory frameworks described in the European Union Tissue and Cells Directive and the technical guide to the quality and safety of tissue and cells for human application, published by the European Council. OBJECTIVE: Several European and international consensus statements concerning faecal microbiota transplantation have been issued. While these documents provide overall guidance, we aim to provide a detailed description of all processes that relate to the collection, handling and clinical application of human donor stool in this document. METHODS: Collaborative subgroups of experts on stool banking drafted concepts for all domains pertaining to stool banking. During a working group meeting in the United European Gastroenterology Week 2019 in Barcelona, these concepts were discussed and finalised to be included in our overall guidance document about faecal microbiota transplantation. RESULTS: A guidance document for all domains pertaining to stool banking was created. This document includes standard operating manuals for several processes involved with stool banking, such as handling of donor material, storage and donor screening. CONCLUSION: The implementation of faecal microbiota transplantation by stool banks in concordance with our guidance document will enable quality assurance and guarantee the availability of donor faeces preparations for patients.

Saccharomyces boulardii attenuates inflammatory response induced by Clostridium perfringens via TLR4/TLR15-MyD8 pathway in HD11 avian macrophages.

Macrophages are professional phagocytic cells that play a critical role in initiating immune responses by presenting antigen and phagocytic clearance. The macrophages can be targeted for immunomodulation by beneficial microbes, such as probiotics. The aim of this study is to investigate the protective effect of Saccharomyces boulardii against Clostridium perfringens infection in avian macrophage cell line HD11. In this study, HD11 macrophages were prestimulated with S. boulardii for 6 h and then infected with C. perfringens for 3 h. Results showed that S. boulardii enhanced phagocytosis and bactericidal capacity against C. perfringens by HD11 cells. The S. boulardii effectively promoted the mRNA expression of CD80, CD83, and CD197 cell-surface molecules in C. perfringens-infected HD11 cells. Moreover, we found that prestimulation with S. boulardii reduced the mRNA expression of CD40, toll-like receptor [TLR] 4, and TLR15 induced by C. perfringens and thereby downregulated the mRNA expression of myeloid differentiation primary response 88, TNF receptor associated factor 6, nuclear factor kappa-B p65 subunit, and c-Jun N-terminal kinase genes in HD11 cells. The upregulation of cytokines (interleukin [IL]-6, tumor necrosis factor alpha, and IL-10) and inducible nitric oxide synthase mRNA expression in C. perfringens-infected HD11 cells were noticeably inhibited by S. boulardii pretreatment. Conclusively, these results might provide a new insight into the role of S. boulardii in regulating avian immune defense against C. perfringens invasion and immune escape.

Niche-Selective Inhibition of Pathogenic Th17 Cells by Targeting Metabolic Redundancy.

Targeting glycolysis has been considered therapeutically intractable owing to its essential housekeeping role. However, the context-dependent requirement for individual glycolytic steps has not been fully explored. We show that CRISPR-mediated targeting of glycolysis in T cells in mice results in global loss of Th17 cells, whereas deficiency of the glycolytic enzyme glucose phosphate isomerase (Gpi1) selectively eliminates inflammatory encephalitogenic and colitogenic Th17 cells, without substantially affecting homeostatic microbiota-specific Th17 cells. In homeostatic Th17 cells, partial blockade of glycolysis upon Gpi1 inactivation was compensated by pentose phosphate pathway flux and increased mitochondrial respiration. In contrast, inflammatory Th17 cells experience a hypoxic microenvironment known to limit mitochondrial respiration, which is incompatible with loss of Gpi1. Our study suggests that inhibiting glycolysis by targeting Gpi1 could be an effective therapeutic strategy with minimum toxicity for Th17-mediated autoimmune diseases, and, more generally, that metabolic redundancies can be exploited for selective targeting of disease processes.

Microbiota in vitro modulated with polyphenols shows decreased colonization resistance against Clostridioides difficile but can neutralize cytotoxicity.

While the knowledge on gut microbiota - C. difficile interactions has improved over the years, the understanding of the underlying mechanisms providing colonization resistance as well as preventative measures against the infection remain incomplete. In this study the antibiotic clindamycin and polyphenol extracts from pomegranate and blueberries were used individually and in combination to modulate fecal microbial communities in minibioreactor arrays (MBRA). Modulated communities were inoculated with C. difficile (ribotype 027). Subsequent 7-day periodical monitoring included evaluation of C. difficile growth and activity of toxins TcdA and TcdB as well as analysis of MBRA bacterial community structure (V3V4 16 S metagenomics). Polyphenols affected multiple commensal bacterial groups and showed different synergistic and antagonistic effects in combination with clindamycin. Exposure to either clindamycin or polyphenols led to the loss of colonization resistance against C. difficile. The successful growth of C. difficile was most significantly correlated with the decrease in Collinsella and Lachnospiraceae. Additionally, we demonstrated that Clostridium sporogenes decreased the activity of both C. difficile toxins TcdA and TcdB. The feature was shown to be common among distinct C. sporogenes strains and could potentially be applicable as a non-antibiotic agent for the alleviation of C. difficile infection.