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Aging Dampens the Intestinal Innate Immune Response during Severe Clostridioides difficile Infection and Is Associated with Altered Cytokine Levels and Granulocyte Mobilization.

ABSTRACT: 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.

SUBMITTER: Abernathy-Close L

PROVIDER: S-EPMC7240091 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Aging Dampens the Intestinal Innate Immune Response during Severe Clostridioides difficile Infection and Is Associated with Altered Cytokine Levels and Granulocyte Mobilization.

Abernathy-Close Lisa L Dieterle Michael G MG Vendrov Kimberly C KC Bergin Ingrid L IL Rao Krishna K Young Vincent B VB

Infection and immunity 20200520 6

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 ...[more]

PMID: 32284366

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Project description:Clostridioides difficile infection (CDI) is the most common hospital-acquired infection in the United States. Antibiotic-induced dysbiosis is the primary cause of susceptibility, and fecal microbiota transplantation (FMT) has emerged as an effective therapy for recurrence. We previously demonstrated in the mouse model of CDI that antibiotic-induced dysbiosis reduced colonic expression of interleukin 25 (IL-25) and that FMT protected in part by restoring IL-25 signaling. Here, we conducted a prospective study in humans to test if FMT induced IL-25 expression in the colons of patients with recurrent CDI (rCDI). Colonic biopsy specimens and blood were collected at the time of FMT and 60 days later. Colon biopsy specimens were analyzed for IL-25 protein levels, total tissue transcriptome, and epithelium-associated microbiota before and after FMT, and peripheral immune cells were immunophenotyped. FMT increased alpha diversity of the colonic microbiota and levels of IL-25 in colonic tissue. In addition, FMT increased expression of homeostatic genes and repressed inflammatory genes. Finally, circulating Th17 cells were decreased post-FMT. The increase in levels of the cytokine IL-25 accompanied by decreased inflammation is consistent with FMT acting in part to protect from recurrent CDI via restoration of commensal activation of type 2 immunity. IMPORTANCE Fecal microbiota transplantation (FMT) is an effective treatment for C. difficile infection for most patients; however, introducing a complex mixture of microbes also has had unintended consequences for some patients. Attempts to create a standardized probiotic therapeutic that recapitulates the efficacy of FMT have been unsuccessful to date. We sought to understand what immune markers are changed in patients undergoing FMT to treat recurrent C. difficile infection and identified an immune signaling molecule, IL-25, that was restored by FMT. This finding indicates that adjunctive therapy with IL-25 could be useful in treating C. difficile infection.

Project description:Clostridioides difficile infection (CDI) creates an imbalance in the intestinal microbiota due to the interaction of the components making up this ecosystem, but little is known about the impact of this disease on other microbial members. This work has thus been aimed at evaluating the taxonomic composition, potential gene-associated functions, virulence factors, and antimicrobial resistance profiles of gut microbiomes. A total of 48 DNA samples obtained from patients with health care facility-acquired (HCFO) and community-onset (CO) diarrhea were distributed in the following four groups according to CDI status: HCFO/+ (n = 13), HCFO/- (n = 8), CO/+ (n = 13), and CO/- (n = 14). These samples were subjected to shotgun metagenomics sequencing. Although the CDI groups' microbiota had microbiome alterations, the greatest imbalance was observed in the in the HCFO+/- groups, with an increase in common pathogens and phage populations, as well as a decrease in beneficial microorganisms that leads to a negative impact on some intestinal homeostasis-related metabolic processes. A reduction in the relative abundance of butyrate metabolism-associated genes was also detected in the HCFO groups (P < 0.01), with an increase in some virulence factors and antibiotic-resistance markers. A set of 51 differentially abundant species in the groups with potential association to CDI enabled its characterization, leading to their spatial separation by onset. Strong correlations between phages and some archaeal and bacterial phyla were identified. This highlighted the need to study the microbiota's various components since their imbalance is multifactorial, with some pathogens contributing to a greater or lesser extent because of their interaction with the ecosystem they inhabit. IMPORTANCE Clostridioides difficile infection represents a serious public health problem in different countries due to its high morbi-mortality and the high costs it represents for health care systems. Studies have shown the impact of this infection on intestinal microbiome homeostasis, mainly on bacterial populations. Our research provides evidence of the impact of CDI at both the compositional (bacteria, archaea, and viruses), and functional levels, allowing us to understand that the alterations of the microbiota occur systemically and are caused by multiple perturbations generated by different members of the microbiota as well as by some pathogens that take advantage of the imbalance to proliferate. Likewise, the 51 differentially abundant species in the study groups with potential association to CDI found in this study could help us envisage future treatments against this and other inflammatory diseases, improving future therapeutic options for patients.

Project description:BACKGROUND & AIMS:Fecal microbiota transplantation (FMT) is effective for treating recurrent Clostridioides difficile infection (CDI), but there are concerns about its long-term safety. Understanding the mechanisms of the effects of FMT could help us design safer, targeted therapies. We aimed to identify microbial metabolites that are important for C difficile growth. METHODS:We used a CDI chemostat model as a tool to study the effects of FMT in vitro. The following analyses were performed: C difficile plate counts, 16S rRNA gene sequencing, proton nuclear magnetic resonance spectroscopy, and ultra-performance liquid chromatography and mass spectrometry bile acid profiling. FMT mixtures were prepared using fresh fecal samples provided by donors enrolled in an FMT program in the United Kingdom. Results from chemostat experiments were validated using human stool samples, C difficile batch cultures, and C57BL/6 mice with CDI. Human stool samples were collected from 16 patients with recurrent CDI and healthy donors (n = 5) participating in an FMT trial in Canada. RESULTS:In the CDI chemostat model, clindamycin decreased valerate and deoxycholic acid concentrations and increased C difficile total viable counts and valerate precursors, taurocholic acid, and succinate concentrations. After we stopped adding clindamycin, levels of bile acids and succinate recovered, whereas levels of valerate and valerate precursors did not. In the CDI chemostat model, FMT increased valerate concentrations and decreased C difficile total viable counts (94% decrease), spore counts (86% decrease), and valerate precursor concentrations; concentrations of bile acids were unchanged. In stool samples from patients with CDI, valerate was depleted before FMT but restored after FMT. Clostridioides difficile batch cultures confirmed that valerate decreased vegetative growth, and that taurocholic acid was required for germination but had no effect on vegetative growth. Clostridioides difficile total viable counts were decreased by 95% in mice with CDI given glycerol trivalerate compared with phosphate buffered saline. CONCLUSIONS:We identified valerate as a metabolite that is depleted with clindamycin and only recovered with FMT. Valerate is a target for a rationally designed recurrent CDI therapy.

Project description:Clostridioides difficile is an important nosocomial pathogen that produces toxins to cause life-threatening diarrhea and colitis. Toxins bind to epithelial receptors and promote the collapse of the actin cytoskeleton. C. difficile toxin activity is commonly studied in cancer-derived and immortalized cell lines. However, the biological relevance of these models is limited. Moreover, no model is available for examining C. difficile-induced enteritis, an understudied health problem. We hypothesized that human intestinal enteroids (HIEs) express toxin receptors and provide a new model to dissect C. difficile cytotoxicity in the small intestine. We generated biopsy-derived jejunal HIE and Vero cells, which stably express LifeAct-Ruby, a fluorescent label of F-actin, to monitor actin cytoskeleton rearrangement by live-cell microscopy. Imaging analysis revealed that toxins from pathogenic C. difficile strains elicited cell rounding in a strain-dependent manner, and HIEs were tenfold more sensitive to toxin A (TcdA) than toxin B (TcdB). By quantitative PCR, we paradoxically found that HIEs expressed greater quantities of toxin receptor mRNA and yet exhibited decreased sensitivity to toxins when compared with traditionally used cell lines. We reasoned that these differences may be explained by components, such as mucins, that are present in HIEs cultures, that are absent in immortalized cell lines. Addition of human-derived mucin 2 (MUC2) to Vero cells delayed cell rounding, indicating that mucus serves as a barrier to toxin-receptor binding. This work highlights that investigation of C. difficile infection in that HIEs can provide important insights into the intricate interactions between toxins and the human intestinal epithelium.NEW & NOTEWORTHY In this article, we developed a novel model of Clostridioides difficile-induced enteritis using jejunal-derived human intestinal enteroids (HIEs) transduced with fluorescently tagged F-actin. Using live-imaging, we identified that jejunal HIEs express high levels of TcdA and CDT receptors, are more sensitive to TcdA than TcdB, and secrete mucus, which delays toxin-epithelial interactions. This work also optimizes optically clear C. difficile-conditioned media suitable for live-cell imaging.

Project description:The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (760 g/l) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron transporters were found induced by Fur regulation during low iron (11 g/l) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were found significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some initial intermediates (phenylpyruvate, oxo-isocaproate, 3-hydroxy-butanoyl-CoA, crotonyl-CoA) were found accumulated, while some end product like isocaproate and butanoate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were found enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB,) of the F0F1-type was found induced while the formation of a V-type, mostly proton-pumping, ATP-consuming ATPase (atpDBAFCEKI, was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. An intensive remodeling of the cell wall was observed most likely to increase antibiotic resistance. Polyamine biosynthesis (spe) was found induced leading to an accumulation of spermine, spermidine and putrescine. The fur mutant lost most of its flagella. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, significant decrease in metabolic iron utilization and protection during the complex transition.

Dataset Information

Aging Dampens the Intestinal Innate Immune Response during Severe Clostridioides difficile Infection and Is Associated with Altered Cytokine Levels and Granulocyte Mobilization.

Publications

Aging Dampens the Intestinal Innate Immune Response during Severe Clostridioides difficile Infection and Is Associated with Altered Cytokine Levels and Granulocyte Mobilization.

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