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.

Innate lymphoid cells promote lung-tissue homeostasis after infection with influenza virus.

Innate lymphoid cells (ILCs), a heterogeneous cell population, are critical in orchestrating immunity and inflammation in the intestine, but whether ILCs influence immune responses or tissue homeostasis at other mucosal sites remains poorly characterized. Here we identify a population of lung-resident ILCs in mice and humans that expressed the alloantigen Thy-1 (CD90), interleukin 2 (IL-2) receptor a-chain (CD25), IL-7 receptor a-chain (CD127) and the IL-33 receptor subunit T1-ST2. Notably, mouse ILCs accumulated in the lung after infection with influenza virus, and depletion of ILCs resulted in loss of airway epithelial integrity, diminished lung function and impaired airway remodeling. These defects were restored by administration of the lung ILC product amphiregulin. Collectively, our results demonstrate a critical role for lung ILCs in restoring airway epithelial integrity and tissue homeostasis after infection with influenza virus.

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.

Commensal bacteria calibrate the activation threshold of innate antiviral immunity.

Signals from commensal bacteria can influence immune cell development and susceptibility to infectious or inflammatory diseases. However, the mechanisms by which commensal bacteria regulate protective immunity after exposure to systemic pathogens remain poorly understood. Here, we demonstrate that antibiotic-treated (ABX) mice exhibit impaired innate and adaptive antiviral immune responses and substantially delayed viral clearance after exposure to systemic LCMV or mucosal influenza virus. Furthermore, ABX mice exhibited severe bronchiole epithelial degeneration and increased host mortality after influenza virus infection. Genome-wide transcriptional profiling of macrophages isolated from ABX mice revealed decreased expression of genes associated with antiviral immunity. Moreover, macrophages from ABX mice exhibited defective responses to type I and type II IFNs and impaired capacity to limit viral replication. Collectively, these data indicate that commensal-derived signals provide tonic immune stimulation that establishes the activation threshold of the innate immune system required for optimal antiviral immunity.

Rapid fucosylation of intestinal epithelium sustains host-commensal symbiosis in sickness.

Systemic infection induces conserved physiological responses that include both resistance and 'tolerance of infection' mechanisms. Temporary anorexia associated with an infection is often beneficial, reallocating energy from food foraging towards resistance to infection or depriving pathogens of nutrients. However, it imposes a stress on intestinal commensals, as they also experience reduced substrate availability; this affects host fitness owing to the loss of caloric intake and colonization resistance (protection from additional infections). We hypothesized that the host might utilize internal resources to support the gut microbiota during the acute phase of the disease. Here we show that systemic exposure to Toll-like receptor (TLR) ligands causes rapid α(1,2)-fucosylation of small intestine epithelial cells (IECs) in mice, which requires the sensing of TLR agonists, as well as the production of interleukin (IL)-23 by dendritic cells, activation of innate lymphoid cells and expression of fucosyltransferase 2 (Fut2) by IL-22-stimulated IECs. Fucosylated proteins are shed into the lumen and fucose is liberated and metabolized by the gut microbiota, as shown by reporter bacteria and community-wide analysis of microbial gene expression. Fucose affects the expression of microbial metabolic pathways and reduces the expression of bacterial virulence genes. It also improves host tolerance of the mild pathogen Citrobacter rodentium. Thus, rapid IEC fucosylation appears to be a protective mechanism that utilizes the host's resources to maintain host-microbial interactions during pathogen-induced stress.

Cooperativity between CD8+ T cells, non-neutralizing antibodies, and alveolar macrophages is important for heterosubtypic influenza virus immunity.

Seasonal epidemics of influenza virus result in ∼36,000 deaths annually in the United States. Current vaccines against influenza virus elicit an antibody response specific for the envelope glycoproteins. However, high mutation rates result in the emergence of new viral serotypes, which elude neutralization by preexisting antibodies. T lymphocytes have been reported to be capable of mediating heterosubtypic protection through recognition of internal, more conserved, influenza virus proteins. Here, we demonstrate using a recombinant influenza virus expressing the LCMV GP33-41 epitope that influenza virus-specific CD8+ T cells and virus-specific non-neutralizing antibodies each are relatively ineffective at conferring heterosubtypic protective immunity alone. However, when combined virus-specific CD8 T cells and non-neutralizing antibodies cooperatively elicit robust protective immunity. This synergistic improvement in protective immunity is dependent, at least in part, on alveolar macrophages and/or other lung phagocytes. Overall, our studies suggest that an influenza vaccine capable of eliciting both CD8+ T cells and antibodies specific for highly conserved influenza proteins may be able to provide heterosubtypic protection in humans, and act as the basis for a potential "universal" vaccine.

Antigen-independent differentiation and maintenance of effector-like resident memory T cells in tissues.

Differentiation and maintenance of recirculating effector memory CD8 T cells (T(EM)) depends on prolonged cognate Ag stimulation. Whether similar pathways of differentiation exist for recently identified tissue-resident effector memory T cells (T(RM)), which contribute to rapid local protection upon pathogen re-exposure, is unknown. Memory CD8αß(+) T cells within small intestine epithelium are well-characterized examples of T(RM), and they maintain a long-lived effector-like phenotype that is highly suggestive of persistent Ag stimulation. This study sought to define the sources and requirements for prolonged Ag stimulation in programming this differentiation state, including local stimulation via cognate or cross-reactive Ags derived from pathogens, microbial flora, or dietary proteins. Contrary to expectations, we found that prolonged cognate Ag stimulation was dispensable for intestinal T(RM) ontogeny. In fact, chronic antigenic stimulation skewed differentiation away from the canonical intestinal T cell phenotype. Resident memory signatures, CD69 and CD103, were expressed in many nonlymphoid tissues including intestine, stomach, kidney, reproductive tract, pancreas, brain, heart, and salivary gland and could be driven by cytokines. Moreover, TGF-ß-driven CD103 expression was required for T(RM) maintenance within intestinal epithelium in vivo. Thus, induction and maintenance of long-lived effector-like intestinal T(RM) differed from classic models of T(EM) ontogeny and were programmed through a novel location-dependent pathway that was required for the persistence of local immunological memory.

Starvation helps transition to abundance - a ferrosome story.

Iron is an essential nutrient for bacterial pathogenesis. In their study, Skaar and colleagues (Pi et al.) discovered and determined the detailed structure of ferrosomes within Clostridioides difficile, the iron-storage organelles that form under iron-limited conditions in anticipation of future iron overload.

Characterizing metabolic drivers of Clostridioides difficile infection with activity-based hydrazine probes.

Many enzymes require post-translational modifications or cofactor machinery for primary function. As these catalytically essential moieties are highly regulated, they act as dual sensors and chemical handles for context-dependent metabolic activity. Clostridioides difficile is a major nosocomial pathogen that infects the colon. Energy generating metabolism, particularly through amino acid Stickland fermentation, is central to colonization and persistence of this pathogen during infection. Here using activity-based protein profiling (ABPP), we revealed Stickland enzyme activity is a biomarker for C. difficile infection (CDI) and annotated two such cofactor-dependent Stickland reductases. We structurally characterized the cysteine-derived pyruvoyl cofactors of D-proline and glycine reductase in C. difficile cultures and showed through cofactor monitoring that their activity is regulated by their respective amino acid substrates. Proline reductase was consistently active in toxigenic C. difficile, confirming the enzyme to be a major metabolic driver of CDI. Further, activity-based hydrazine probes were shown to be active site-directed inhibitors of proline reductase. As such, this enzyme activity, via its druggable cofactor modality, is a promising therapeutic target that could allow for the repopulation of bacteria that compete with C. difficile for proline and therefore restore colonization resistance against C. difficile in the gut.

Antigen-specific memory Th17 cells promote cross-protection against nontypeable Haemophilus influenzae after mild influenza A virus infection.

Secondary bacterial pneumonia after influenza A virus (IAV) infection is the leading cause of hospitalization and death associated with IAV infection worldwide. Nontypeable Haemophilus influenzae (NTHi) is one of the most common causes of secondary bacterial pneumonia. Current efforts to develop vaccines against NTHi infection focus on inducing antibodies but are hindered by antigenic diversity among NTHi strains. Therefore, we investigated the contribution of the memory T helper type 17 (Th17) response in protective immunity against IAV/NTHi coinfection. We observed that even a mild IAV infection impaired the NTHi-specific Th17 response and increased morbidity and mortality compared with NTHi monoinfected mice. However, pre-existing memory NTHi-specific Th17 cells induced by a previous NTHi infection overcame IAV-driven Th17 inhibition and were cross-protective against different NTHi strains. Last, mice immunized with a NTHi protein that induced a strong Th17 memory response were broadly protected against diverse NTHi strains after challenge with coinfection. These results indicate that vaccination that limits IAV infection to mild disease may be insufficient to eliminate the risk of a lethal secondary bacterial pneumonia. However, NTHi-specific memory Th17 cells provide serotype-independent protection despite an ongoing IAV infection and demonstrate the advantage of developing broadly protective Th17-inducing vaccines against secondary bacterial pneumonia.

Immunological consequences of microbiome-based therapeutics.

The complex network of microscopic organisms living on and within humans, collectively referred to as the microbiome, produce wide array of biologically active molecules that shape our health. Disruption of the microbiome is associated with susceptibility to a range of diseases such as cancer, diabetes, allergy, obesity, and infection. A new series of next-generation microbiome-based therapies are being developed to treat these diseases by transplanting bacteria or bacterial-derived byproducts into a diseased individual to reset the recipient's microbiome and restore health. Microbiome transplantation therapy is still in its early stages of being a routine treatment option and, with a few notable exceptions, has had limited success in clinical trials. In this review, we highlight the successes and challenges of implementing these therapies to treat disease with a focus on interactions between the immune system and microbiome-based therapeutics. The immune activation status of the microbiome transplant recipient prior to transplantation has an important role in supporting bacterial engraftment. Following engraftment, microbiome transplant derived signals can modulate immune function to ameliorate disease. As novel microbiome-based therapeutics are developed, consideration of how the transplants will interact with the immune system will be a key factor in determining whether the microbiome-based transplant elicits its intended therapeutic effect.

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.

Monocyte Reconstitution and Gut Microbiota Composition after Hematopoietic Stem Cell Transplantation.

BACKGROUND: Monocytes are an essential cellular component of the innate immune system that support the host's effectiveness to combat a range of infectious pathogens. Hemopoietic cell transplantation (HCT) results in transient monocyte depletion, but the factors that regulate recovery of monocyte populations are not fully understood. In this study, we investigated whether the composition of the gastrointestinal microbiota is associated with the recovery of monocyte homeostasis after HCT. METHODS: We performed a single-center, prospective, pilot study of 18 recipients of either autologous or allogeneic HCT. Serial blood and stool samples were collected from each patient during their HCT hospitalization. Analysis of the gut microbiota was done using 16S rRNA gene sequencing, and flow cytometric analysis was used to characterize the phenotypic composition of monocyte populations. RESULTS: Dynamic fluctuations in monocyte reconstitution occurred after HCT, and large differences were observed in monocyte frequency among patients over time. Recovery of absolute monocyte counts and subsets showed significant variability across the heterogeneous transplant types and conditioning intensities; no relationship to the microbiota composition was observed in this small cohort. CONCLUSION: In this pilot study, a relationship between the microbiota composition and monocyte homeostasis could not be firmly established. However, we identify multivariate associations between clinical factors and monocyte reconstitution post-HCT. Our findings encourage further longitudinal surveillance of the intestinal microbiome and its link to immune reconstitution.

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.

The intestinal microbiota in health and disease: the influence of microbial products on immune cell homeostasis.

PURPOSE OF REVIEW: A vast and diverse array of microbes colonizes the mammalian gastrointestinal tract. These microorganisms are integral in shaping the development and function of the immune system. Metagenomic sequencing analysis has revealed alterations in intestinal microbiota in patients suffering from chronic inflammatory diseases, including inflammatory bowel disease and asthma. This review will discuss the mechanisms through which the innate immune system recognizes and responds to the intestinal microbiota as well as the effect of specific microbiota-derived signals on immune cell homeostasis. RECENT FINDINGS: Recent studies in murine model systems have demonstrated that manipulation of the intestinal microbiota can alter mammalian immune cell homeostasis. Specific microbial signals have been identified that can impact immune cell function both within the intestinal tract and in peripheral tissues. These microbiota-derived signals can either have an immunoregulatory effect, creating an immune state that is refractory to inflammation, or conversely, act as an adjuvant, aiding in the propagation of an immune response. SUMMARY: Associations between alterations in the microbiota and human disease implicate intestinal microbial signals in shaping immune responses. These signals are recognized by innate immune cells and influence the ability of these cells to modulate both the local and systemic immune response.

An Additive Sugar Helps the C. diff Go Round.

Outbreaks of hypervirulent strains of Clostridium difficile began to be reported in healthcare facilities worldwide around 20 years ago. Concurrently, trehalose became a common additive used by the global food industry. A new study provides evidence that these two observations are a linked phenomenon (Collins et al., 2018).

Acute Gastroenteritis Leaves a Lasting Impression.

Host immunity shapes intestinal microbiota composition, influencing health and disease. In this issue of Cell Host & Microbe,Kamdar et al. (2016) demonstrate that an aberrant acute inflammatory response to Yersinia enterocolitica infection leads to long-lasting shifts in commensal communities and renders the host susceptible to chronic inflammation despite pathogen clearance.

Clostridium difficile colitis: pathogenesis and host defence.

Clostridium difficile is a major cause of intestinal infection and diarrhoea in individuals following antibiotic treatment. Recent studies have begun to elucidate the mechanisms that induce spore formation and germination and have determined the roles of C. difficile toxins in disease pathogenesis. Exciting progress has also been made in defining the role of the microbiome, specific commensal bacterial species and host immunity in defence against infection with C. difficile. This Review will summarize the recent discoveries and developments in our understanding of C. difficile infection and pathogenesis.

TLR-7 activation enhances IL-22-mediated colonization resistance against vancomycin-resistant enterococcus.

Antibiotic administration can disrupt the intestinal microbiota and down-regulate innate immune defenses, compromising colonization resistance against orally acquired bacterial pathogens. Vancomycin-resistant Enterococcus faecium (VRE), a major cause of antibiotic-resistant infections in hospitalized patients, thrives in the intestine when colonization resistance is compromised, achieving extremely high densities that can lead to bloodstream invasion and sepsis. Viral infections, by mechanisms that remain incompletely defined, can stimulate resistance against invading bacterial pathogens. We report that murine norovirus infection correlates with reduced density of VRE in the intestinal tract of mice with antibiotic-induced loss of colonization resistance. Resiquimod (R848), a synthetic ligand for Toll-like receptor 7 (TLR-7) that stimulates antiviral innate immune defenses, restores expression of the antimicrobial peptide Reg3γ and reestablishes colonization resistance against VRE in antibiotic-treated mice. Orally administered R848 triggers TLR-7 on CD11c(+) dendritic cells, inducing interleukin-23 (IL-23) expression followed by a burst of IL-22 secretion by innate lymphoid cells, leading to Reg3γ expression and restoration of colonization resistance against VRE. Our findings reveal that an orally bioavailable TLR-7 ligand that stimulates innate antiviral immune pathways in the intestine restores colonization resistance against a highly antibiotic-resistant bacterial pathogen.

Innate Immune Defenses Mediated by Two ILC Subsets Are Critical for Protection against Acute Clostridium difficile Infection.

Infection with the opportunistic enteric pathogen Clostridium difficile is an increasingly common clinical complication that follows antibiotic treatment-induced gut microbiota perturbation. Innate lymphoid cells (ILCs) are early responders to enteric pathogens; however, their role during C. difficile infection is undefined. To identify immune pathways that mediate recovery from C. difficile infection, we challenged C57BL/6, Rag1(-/-) (which lack T and B cells), and Rag2(-/-)Il2rg(-/-) (Ragγc(-/-)) mice (which additionally lack ILCs) with C. difficile. In contrast to Rag1(-/-) mice, ILC-deficient Ragγc(-/-) mice rapidly succumbed to infection. Rag1(-/-) but not Ragγc(-/-) mice upregulate expression of ILC1- or ILC3-associated proteins following C. difficile infection. Protection against infection was restored by transferring ILCs into Ragγc(-/-) mice. While ILC3s made a minor contribution to resistance, loss of IFN-γ or T-bet-expressing ILC1s in Rag1(-/-) mice increased susceptibility to C. difficile. These data demonstrate a critical role for ILC1s in defense against C. difficile.