Monoclonal antibody-mediated neutralization of Clostridioides difficile toxin does not diminish induction of the protective innate immune response to infection.

Clostridioides difficile infection causes pathology that ranges in severity from diarrhea to pseudomembranous colitis. Toxin A and Toxin B are the two primary virulence factors secreted by C. difficile that drive disease severity. The toxins damage intestinal epithelial cells leading to a loss of barrier integrity and induction of a proinflammatory host response. Monoclonal antibodies (mAbs) that neutralize Toxin A and Toxin B, actoxumab and bezlotoxumab, respectively, significantly reduce disease severity in a murine model of C. difficile infection. However, the impact of toxin neutralization on the induction and quality of the innate immune response following infection is unknown. The goal of this study was to define the quality of the host innate immune response in the context of anti-toxin mAbs therapy. At day 2 post-infection, C. difficile-infected, mAbs-treated mice had significantly less disease compared to isotype-treated mice despite remaining colonized with C. difficile. C. difficile-infected mAbs-treated mice still exhibited marked neutrophil infiltration and induction of a subset of proinflammatory cytokines within the intestinal lamina propria following infection that is comparable to isotype-treated mice. Furthermore, both mAbs and isotype-treated mice had an increase in IL-22-producing ILCs in the intestine following infection. MAbs-treated mice exhibited increased infiltration of eosinophils in the intestinal lamina propria, which has been previously reported to promote a protective host response following C. difficile infection. These findings show that activation of host protective mechanisms remain intact in the context of monoclonal antibody-mediated toxin neutralization.

Loss of Interleukin-10 (IL-10) Signaling Promotes IL-22-Dependent Host Defenses against Acute Clostridioides difficile Infection.

Infection with the bacterial pathogen Clostridioides difficile causes severe damage to the intestinal epithelium that elicits a robust inflammatory response. Markers of intestinal inflammation accurately predict clinical disease, however, the extent to which host-derived proinflammatory mediators drive pathogenesis versus promote host protective mechanisms remains elusive. In this report, we employed Il10-/- mice as a model of spontaneous colitis to examine the impact of constitutive intestinal immune activation, independent of infection, on C. difficile disease pathogenesis. Upon C. difficile challenge, Il10-/- mice exhibited significantly decreased morbidity and mortality compared to littermate Il10 heterozygote (Il10HET) control mice, despite a comparable C. difficile burden, innate immune response, and microbiota composition following infection. Similarly, antibody-mediated blockade of interleukin-10 (IL-10) signaling in wild-type C57BL/6 mice conveyed a survival advantage if initiated 3 weeks prior to infection. In contrast, no advantage was observed if blockade was initiated on the day of infection, suggesting that the constitutive activation of inflammatory defense pathways prior to infection mediated host protection. IL-22, a cytokine critical in mounting a protective response against C. difficile infection, was elevated in the intestine of uninfected, antibiotic-treated Il10-/- mice, and genetic ablation of the IL-22 signaling pathway in Il10-/- mice negated the survival advantage following C. difficile challenge. Collectively, these data demonstrate that constitutive loss of IL-10 signaling, via genetic ablation or antibody blockade, enhances IL-22-dependent host defense mechanisms to limit C. difficile pathogenesis.

Temporospatial shifts within commercial laboratory mouse gut microbiota impact experimental reproducibility.

BACKGROUND: Experimental reproducibility in mouse models is impacted by both genetics and environment. The generation of reproducible data is critical for the biomedical enterprise and has become a major concern for the scientific community and funding agencies alike. Among the factors that impact reproducibility in experimental mouse models is the variable composition of the microbiota in mice supplied by different commercial vendors. Less attention has been paid to how the microbiota of mice supplied by a particular vendor might change over time. RESULTS: In the course of conducting a series of experiments in a mouse model of malaria, we observed a profound and lasting change in the severity of malaria in mice infected with Plasmodium yoelii; while for several years mice obtained from a specific production suite of a specific commercial vendor were able to clear the parasites effectively in a relatively short time, mice subsequently shipped from the same unit suffered much more severe disease. Gut microbiota analysis of frozen cecal samples identified a distinct and lasting shift in bacteria populations that coincided with the altered response of the later shipments of mice to infection with malaria parasites. Germ-free mice colonized with cecal microbiota from mice within the same production suite before and after this change followed by Plasmodium infection provided a direct demonstration that the change in gut microbiota profoundly impacted the severity of malaria. Moreover, spatial changes in gut microbiota composition were also shown to alter the acute bacterial burden following Salmonella infection, and tumor burden in a lung tumorigenesis model. CONCLUSION: These changes in gut bacteria may have impacted the experimental reproducibility of diverse research groups and highlight the need for both laboratory animal providers and researchers to collaborate in determining the methods and criteria needed to stabilize the gut microbiota of animal breeding colonies and research cohorts, and to develop a microbiota solution to increase experimental rigor and reproducibility.

Composition of the gut microbiota modulates the severity of malaria.

Plasmodium infections result in clinical presentations that range from asymptomatic to severe malaria, resulting in ∼1 million deaths annually. Despite this toll on humanity, the factors that determine disease severity remain poorly understood. Here, we show that the gut microbiota of mice influences the pathogenesis of malaria. Genetically similar mice from different commercial vendors, which exhibited differences in their gut bacterial community, had significant differences in parasite burden and mortality after infection with multiple Plasmodium species. Germfree mice that received cecal content transplants from "resistant" or "susceptible" mice had low and high parasite burdens, respectively, demonstrating the gut microbiota shaped the severity of malaria. Among differences in the gut flora were increased abundances of Lactobacillus and Bifidobacterium in resistant mice. Susceptible mice treated with antibiotics followed by yogurt made from these bacterial genera displayed a decreased parasite burden. Consistent with differences in parasite burden, resistant mice exhibited an elevated humoral immune response compared with susceptible mice. Collectively, these results identify the composition of the gut microbiota as a previously unidentified risk factor for severe malaria and modulation of the gut microbiota (e.g., probiotics) as a potential treatment to decrease parasite burden.

Malaria and the Microbiome: A Systematic Review.

Background: The microbiome influences malaria parasite fitness and transmission efficiency in mosquitoes and appears to affect malaria dynamics in mammalian hosts as well. Nascent research examining the interrelationship of malaria and the mammalian microbiome has yielded interesting insights inviting further study. Methods: We conducted a systematic review of the literature examining associations between the microbiome and malaria in mammalian hosts. An electronic search algorithm was adapted to PubMed, MEDLINE, Scopus, Embase, and Web of Science, and reference lists of relevant sources were manually searched. Identified studies were screened and assessed independently by 2 authors, and results were compiled in a qualitative synthesis of the evidence. Results: Ten relevant studies were identified. They demonstrate associations between certain intestinal communities and protection against Plasmodium infection and modulation of disease severity. Plasmodium infection acutely and reversibly reshapes gut microbial composition in mice. The makeup of human skin microbial communities may influence mosquito attraction and thus disease transmission. Conclusions: Early research supports a relationship between malaria and the microbiome. The evidence is incomplete, but the observed associations are evocative and signal a promising avenue of inquiry. Microbiome-based studies of malaria can be readily integrated into field-based research.

Antimalarial activity of tulathromycin in a murine model of malaria.

There is an urgent need for new antimalarial agents and strategies to treat and control malaria. This study shows an antiplasmodium effect of tulathromycin in mice infected with Plasmodium yoelii. The administration of tulathromycin around the time of infection prevented the progression of disease in 100% of the animals. In addition, highly parasitized mice treated with tulathromycin showed a decreased parasite burden and cleared the parasite faster than did untreated infected mice.

Pheromone responsiveness is regulated by components of the Gpr1p-mediated glucose sensing pathway in Saccharomyces cerevisiae.

Many fungi have evolved mechanisms to assess environmental nutrient availability prior to the energy-intensive process of mating. In this study, we examined one such system in Saccharomyces cerevisiae, involving a glucose-sensing pathway mediated by Gpr1p and the pheromone-induced mating pathway. Initially we observed that the mating pathway in MATa cells is sensitive to environmental glucose depletion. This phenomenon can be partially reversed with a high glucose spike, but not with the addition of low levels of glucose. Deletion of the low-affinity glucose receptor, Gpr1p, eliminated this glucose-induced recovery of pheromone responsiveness. We then determined the impact of GPR1 deletion on the mating pathway and observed that, in all end points studied, the mating pathway response to pheromone is reduced in the absence of Gpr1p. Similarly, elimination of the Gα for Gpr1p, Gpa2p, resulted in reduction in pheromone sensitivity in all assays studied. The negative effect of removing Gpr1p on mating pathway activation could be recovered by overexpressing the mating receptor, Ste2p. Furthermore, Ste2p levels are reduced in the absence of glucose and GPR1. These data suggest that activity of the GPCR-mediated mating pathway in S. cerevisiae is modulated by extracellular glucose concentrations through the only other GPCR in MATa cells, Gpr1p.

Gut Bacteroides act in a microbial consortium to cause susceptibility to severe malaria.

Malaria is caused by Plasmodium species and remains a significant cause of morbidity and mortality globally. Gut bacteria can influence the severity of malaria, but the contribution of specific bacteria to the risk of severe malaria is unknown. Here, multiomics approaches demonstrate that specific species of Bacteroides are causally linked to the risk of severe malaria. Plasmodium yoelii hyperparasitemia-resistant mice gavaged with murine-isolated Bacteroides fragilis develop P. yoelii hyperparasitemia. Moreover, Bacteroides are significantly more abundant in Ugandan children with severe malarial anemia than with asymptomatic P. falciparum infection. Human isolates of Bacteroides caccae, Bacteroides uniformis, and Bacteroides ovatus were able to cause susceptibility to severe malaria in mice. While monocolonization of germ-free mice with Bacteroides alone is insufficient to cause susceptibility to hyperparasitemia, meta-analysis across multiple studies support a main role for Bacteroides in susceptibility to severe malaria. Approaches that target gut Bacteroides present an opportunity to prevent severe malaria and associated deaths.

Dynamic modulation of spleen germinal center reactions by gut bacteria during Plasmodium infection.

Gut microbiota educate the local and distal immune system in early life to imprint long-term immunological outcomes while maintaining the capacity to dynamically modulate the local mucosal immune system throughout life. It is unknown whether gut microbiota provide signals that dynamically regulate distal immune responses following an extra-gastrointestinal infection. We show here that gut bacteria composition correlated with the severity of malaria in children. Using the murine model of malaria, we demonstrate that parasite burden and spleen germinal center reactions are malleable to dynamic cues provided by gut bacteria. Whereas antibiotic-induced changes in gut bacteria have been associated with immunopathology or impairment of immunity, the data demonstrate that antibiotic-induced changes in gut bacteria can enhance immunity to Plasmodium. This effect is not universal but depends on baseline gut bacteria composition. These data demonstrate the dynamic communications that exist among gut bacteria, the gut-distal immune system, and control of Plasmodium infection.

Host immunity modulates the efficacy of microbiota transplantation for treatment of Clostridioides difficile infection.

Fecal microbiota transplantation (FMT) is a successful therapeutic strategy for treating recurrent Clostridioides difficile infection. Despite remarkable efficacy, implementation of FMT therapy is limited and the mechanism of action remains poorly understood. Here, we demonstrate a critical role for the immune system in supporting FMT using a murine C. difficile infection system. Following FMT, Rag1 heterozygote mice resolve C. difficile while littermate Rag1-/- mice fail to clear the infection. Targeted ablation of adaptive immune cell subsets reveal a necessary role for CD4+ Foxp3+ T-regulatory cells, but not B cells or CD8+ T cells, in FMT-mediated resolution of C. difficile infection. FMT non-responsive mice exhibit exacerbated inflammation, impaired engraftment of the FMT bacterial community and failed restoration of commensal bacteria-derived secondary bile acid metabolites in the large intestine. These data demonstrate that the host's inflammatory immune status can limit the efficacy of microbiota-based therapeutics to treat C. difficile infection.

Evidence of Microbiome-Drug Interaction between the Antimalarial Lumefantrine and Gut Microbiota in Mice.

The antimalarial drug lumefantrine exhibits erratic pharmacokinetics. Intersubject variability might be attributed, in part, to differences in gut microbiome-mediated drug metabolism. We assessed lumefantrine disposition in healthy mice stratified by enterotype to explore associations between the gut microbiota and lumefantrine pharmacokinetics. Gut microbiota enterotypes were classified according to abundance and diversity indices from 16S rRNA sequencing. Pharmacokinetic parameters were computed using noncompartmental analysis. Two distinct enterotypes were identified. Maximal concentration (C max) and total drug exposure measured as the area under the drug concentration-time curve (AUC0-24) differed significantly between the groups. The mean and standard deviation of C max were 660 ± 220 ng/mL versus 390 ± 59 ng/mL (P = 0.02), and AUC0-24 was 9,600 ± 2,800 versus 5,800 ± 810 ng × h/mL (P = 0.01). In healthy mice intragastrically dosed with the antimalarial drug lumefantrine in combination with artemether, lumefantrine exposure was associated with gut bacterial community structure. Studies of xenobiotic-microbiota interactions can inform drug posology and elucidate mechanisms of drug disposition.

Detection and elimination of a novel non-toxigenic Clostridioides difficile strain from the microbiota of a mouse colony.

Clostridioides difficile is an enteric bacterial pathogen that can a cause nosocomial infection leading to debilitating colitis. The development of a murine model of C. difficile infection has led to fundamental discoveries in disease pathogenesis and the host immune response to infection. Recently, C. difficile endogenously present in the microbiota of mice has been reported and was found to complicate interpretation of mouse studies. Here, we report a novel C. difficile strain, named NTCD-035, isolated from the microbiota of our mouse colony. The presence of NTCD-035 in mice prior to challenge with a highly pathogenic C. difficile strain (VPI10463) led to significantly reduced disease severity. Phylogenetic characterization derived from whole genome sequencing and PCR ribotyping identified the isolate as a novel clade 1, ribotype 035 strain that lacks the pathogenicity locus required to produce toxins. Deficiency in toxin production along with sporulation capacity and secondary bile acid sensitivity was confirmed using in vitro assays. Inoculation of germ-free mice with NTCD-035 did not cause morbidity despite the strain readily colonizing the large intestine. Implementation of a culture-based screening procedure enabled the identification of mice harboring C. difficile in their microbiota, the establishment of a C. difficile-free mouse colony, and a monitoring system to prevent future contamination. Taken together, these data provide a framework for screening mice for endogenously harbored C. difficile and support clinical findings that demonstrate the therapeutic potential of non-toxigenic strains in preventing C. difficile associated disease. Abbreviations: PaLoc - Pathogenicity locus, CFUs - Colony forming units, TcdA - toxin-A, TcdB - toxin-B, CdtA - binary toxin A, CdtB - binary toxin B, CdtR - binary toxin R, NTCD - non-toxigenic C. difficile.

Gut Microbiota Composition Modulates the Magnitude and Quality of Germinal Centers during Plasmodium Infections.

Gut microbiota composition is associated with human and rodent Plasmodium infections, yet the mechanism by which gut microbiota affects the severity of malaria remains unknown. Humoral immunity is critical in mediating the clearance of Plasmodium blood stage infections, prompting the hypothesis that mice with gut microbiota-dependent decreases in parasite burden exhibit better germinal center (GC) responses. In support of this hypothesis, mice with a low parasite burden exhibit increases in GC B cell numbers and parasite-specific antibody titers, as well as better maintenance of GC structures and a more targeted, qualitatively different antibody response. This enhanced humoral immunity affects memory, as mice with a low parasite burden exhibit robust protection against challenge with a heterologous, lethal Plasmodium species. These results demonstrate that gut microbiota composition influences the biology of spleen GCs as well as the titer and repertoire of parasite-specific antibodies, identifying potential approaches to develop optimal treatments for malaria.

Oral Administration of Clinically Relevant Antimalarial Drugs Does Not Modify the Murine Gut Microbiota.

Malaria is an infectious disease responsible for the death of around 450,000 people annually. As an effective vaccine against the parasite that causes malaria is not available, antimalarial drug treatments are critical in fighting the disease. Previous data has shown that the gut microbiota is important in modulating the severity of malaria. Although it is well appreciated that antibiotics substantially alter the gut microbiota, it is largely unknown how antimalarial drugs impact the gut microbiota. We show here that the two commonly used artemisinin combination therapies of artesunate plus amodiaquine and artemether plus lumefantrine do not change the gut microbiota. The overall relative species abundance and alpha diversity remained stable after treatment, while beta diversity analysis showed minimal changes due to drug treatment, which were transient and quickly returned to baseline. Additionally, treatment with antimalarial drugs did not change the kinetics of later Plasmodium infection. Taken together, antimalarial drug administration does not affect the gut microbiota.

Differential Sensitivity to Plasmodium yoelii Infection in C57BL/6 Mice Impacts Gut-Liver Axis Homeostasis.

Experimental models of malaria have shown that infection with specific Plasmodium species in certain mouse strains can transiently modulate gut microbiota and cause intestinal shortening, indicating a disruption of gut homeostasis. Importantly, changes in gut homeostasis have not been characterized in the context of mild versus severe malaria. We show that severe Plasmodium infection in mice disrupts homeostasis along the gut-liver axis in multiple ways compared to mild infection. High parasite burden results in a larger influx of immune cells in the lamina propria and mice with high parasitemia display specific metabolomic profiles in the ceca and plasma during infection compared to mice with mild parasitemia. Liver damage was also more pronounced and longer lasting during severe infection, with concomitant changes in bile acids in the gut. Finally, severe Plasmodium infection changes the functional capacity of the microbiota, enhancing bacterial motility and amino acid metabolism in mice with high parasite burden compared to a mild infection. Taken together, Plasmodium infections have diverse effects on host gut homeostasis relative to the severity of infection that may contribute to enteric bacteremia that is associated with malaria.

Local and Long-Distance Calling: Conversations between the Gut Microbiota and Intra- and Extra-Gastrointestinal Tract Infections.

Preservation of health from infectious diseases depends upon both mucosal and systemic immunity via the collaborative effort of innate and adaptive immune responses. The proficiency of host immunity stems from robust defense mechanisms--physical barriers and specialized immune cells--and a failure of these mechanisms leads to pathology. Intriguingly, immunocompetence to pathogens can be shaped by the gut microbiome as recent publications highlight a dynamic interplay between the gut microbiome and host susceptibility to infection. Modulation of host immunity to enteric pathogens has long been studied where gut bacteria shape multiple facts of both innate and adaptive immunity. Conversely, the impact of gut commensals on host immunity to extra-gastrointestinal (GI) tract infections has only recently been recognized. In this context, the gut microbiome can augment host immunity to extra-GI tract bacterial, viral, and parasitic pathogens. This review explores the research that affords insight into the role of the gut microbiome in various infectious diseases, with a particular emphasis on extra-GI tract infections. A better understanding of the link between the gut microbiome and infectious disease will be critical for improving global health in the years ahead.

Functional Characteristics of the Gut Microbiome in C57BL/6 Mice Differentially Susceptible to Plasmodium yoelii.

C57BL/6 mice are widely used for in vivo studies of immune function and metabolism in mammals. In a previous study, it was observed that when C57BL/6 mice purchased from different vendors were infected with Plasmodium yoelii, a causative agent of murine malaria, they exhibited both differential immune responses and significantly different parasite burdens: these patterns were reproducible when gut contents were transplanted into gnotobiotic mice. To gain insight into the mechanism of resistance, we removed whole ceca from mice purchased from two vendors, Taconic Biosciences (low parasitemia) and Charles River Laboratories (high parasitemia), to determine the combined host and microflora metabolome and metatranscriptome. With the exception of two Charles River samples, we observed ≥90% similarity in overall bacterial gene expression within vendors and ≤80% similarity between vendors. In total 33 bacterial genes were differentially expressed in Charles River mice (p-value < 0.05) relative to the mice purchased from Taconic. Included among these, fliC, ureABC, and six members of the nuo gene family were overrepresented in microbiomes susceptible to more severe malaria. Moreover, 38 mouse genes were differentially expressed in these purported genetically identical mice. Differentially expressed genes included basigin, a cell surface receptor required for P. falciparum invasion of red blood cells. Differences in metabolite pools were detected, though their relevance to malaria infection, microbial community activity, or host response is not yet understood. Our data have provided new targets that may connect gut microbial activity to malaria resistance and susceptibility phenotypes in the C57BL/6 model organism.