Molecular genetic evaluation of pediatric renovascular hypertension due to renal artery stenosis and abdominal aortic coarctation in neurofibromatosis type 1.

The etiology of renal artery stenosis (RAS) and abdominal aortic coarctation (AAC) causing the midaortic syndrome (MAS), often resulting in renovascular hypertension (RVH), remains ill-defined. Neurofibromatosis type 1 (NF-1) is frequently observed in children with RVH. Consecutive pediatric patients (N = 102) presenting with RVH secondary to RAS with and without concurrent AAC were prospectively enrolled in a clinical data base, and blood, saliva and operative tissue, when available, were collected. Among the 102 children, 13 were having a concurrent clinical diagnosis of NF-1 (12.5%). Whole exome sequencing was performed for germline variant detection, and RNA-Seq analysis of NF1, MAPK pathway genes and MCP1 levels were undertaken in five NF-1 stenotic renal arteries, as well as control renal and mesenteric arteries from children with no known vasculopathy or NF-1. In 11 unrelated children with sequencing data, 11 NF1 genetic variants were identified, of which 10 had not been reported in gnomAD. Histologic analysis of NF-1 RAS specimens consistently revealed intimal thickening, disruption of the internal elastic lamina and medial thinning. Analysis of transcript expression in arterial lesions documented an approximately 5-fold reduction in NF1 expression, confirming heterozygosity, MAPK pathway activation and increased MCP1 expression. In summary, NF-1-related RVH in children is rare but often severe and progressive and, as such, important to recognize. It is associated with histologic and molecular features consistent with an aggressive adverse vascular remodeling process. Further research is necessary to define the mechanisms underlying these findings.

Phospholipase Cε insufficiency causes ascending aortic aneurysm and dissection.

Phospholipase Cε (PLCε) is a phospholipase C isoform with a wide range of physiological functions. It has been implicated in aortic valve disorders, but its role in frequently associated aortic disease remains unclear. To determine the role of PLCε in thoracic aortic aneurysm and dissection (TAAD) we used PLCε-deficient mice, which develop aortic valve insufficiency and exhibit aortic dilation of the ascending thoracic aorta and arch without histopathological evidence of injury. Fourteen days of infusion of Plce1+/+ and Plce1-/- mice with angiotensin II (ANG II), which induces aortic dilation and dissection, led to sudden death secondary to ascending aortic dissection in 43% of Plce1-/- versus 5% of Plce1+/+ mice (P < 0.05). Medial degeneration and TAAD were detected in 80% of Plce1-/- compared with 10% of Plce1+/+ mice (P < 0.05) after 4 days of ANG II. Treatment with ANG II markedly increased PLCε expression within the ascending aortic adventitia. Total RNA sequencing demonstrated marked upregulation of inflammatory and fibrotic pathways mediated by interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In silico analysis of whole exome sequences of 258 patients with type A dissection identified 5 patients with nonsynonymous PLCE1 variants. Our data suggest that PLCε deficiency plays a role in the development of TAAD and aortic insufficiency.NEW & NOTEWORTHY We describe a novel phenotype by which PLCε deficiency predisposes to aortic valve insufficiency and ascending aortic aneurysm, dissection, and sudden death in the setting of ANG II-mediated hypertension. We demonstrate PLCE1 variants in patients with type A aortic dissection and aortic insufficiency, suggesting that PLCE1 may also play a role in human aortic disease. This finding is of very high significance because it has not been previously demonstrated that PLCε directly mediates aortic dissection.

A Novel Recurrent COL5A1 Genetic Variant Is Associated With a Dysplasia-Associated Arterial Disease Exhibiting Dissections and Fibromuscular Dysplasia.

OBJECTIVE: While rare variants in the COL5A1 gene have been associated with classical Ehlers-Danlos syndrome and rarely with arterial dissections, recurrent variants in COL5A1 underlying a systemic arteriopathy have not been described. Monogenic forms of multifocal fibromuscular dysplasia (mFMD) have not been previously defined. Approach and Results: We studied 4 independent probands with the COL5A1 pathogenic variant c.1540G>A, p.(Gly514Ser) who presented with arterial aneurysms, dissections, tortuosity, and mFMD affecting multiple arteries. Arterial medial fibroplasia and smooth muscle cell disorganization were confirmed histologically. The COL5A1 c.1540G>A variant is predicted to be pathogenic in silico and absent in gnomAD. The c.1540G>A variant is on a shared 160.1 kb haplotype with 0.4% frequency in Europeans. Furthermore, exome sequencing data from a cohort of 264 individuals with mFMD were examined for COL5A1 variants. In this mFMD cohort, COL5A1 c.1540G>A and 6 additional relatively rare COL5A1 variants predicted to be deleterious in silico were identified and were associated with arterial dissections (P=0.005). CONCLUSIONS: COL5A1 c.1540G>A is the first recurring variant recognized to be associated with arterial dissections and mFMD. This variant presents with a phenotype reminiscent of vascular Ehlers-Danlos syndrome. A shared haplotype among probands supports the existence of a common founder. Relatively rare COL5A1 genetic variants predicted to be deleterious by in silico analysis were identified in ≈2.7% of mFMD cases, and as they were enriched in patients with arterial dissections, may act as disease modifiers. Molecular testing for COL5A1 should be considered in patients with a phenotype overlapping with vascular Ehlers-Danlos syndrome and mFMD.

N-Acetyl-L-cysteine and aminooxyacetic acid differentially modulate trichloroethylene reproductive toxicity via metabolism in Wistar rats.

Exposure to the industrial solvent trichloroethylene (TCE) has been associated with adverse pregnancy outcomes in humans and decreased fetal weight in rats. TCE kidney toxicity can occur through formation of reactive metabolites via its glutathione (GSH) conjugation metabolic pathway, largely unstudied in the context of pregnancy. To investigate the contribution of the GSH conjugation pathway and oxidative stress to TCE toxicity during pregnancy, we exposed rats orally to 480 mg TCE/kg/day from gestational day (GD) 6 to GD 16 with and without N-acetyl-L-cysteine (NAC) at 200 mg/kg/day or aminooxyacetic acid (AOAA) at 20 mg/kg/day as pre/co-treatments from GD 5-16. NAC is a reactive oxygen species scavenger that modifies the GSH conjugation pathway, and AOAA is an inhibitor of cysteine conjugate ß-lyase (CCBL) in the GSH conjugation pathway. TCE decreased fetal weight, and this was prevented by AOAA but not NAC pre/co-treatment to TCE. Although AOAA inhibited CCBL activity in maternal kidney, it did not inhibit CCBL activity in maternal liver and placenta, suggesting that AOAA prevention of TCE-induced decreased fetal weight was due to CCBL activity inhibition in the kidneys but not liver or placenta. Unexpectedly, NAC pre/co-treatment with TCE, relative to TCE treatment alone, altered placental morphology consistent with delayed developmental phenotype. Immunohistochemical staining revealed that the decidua basale, relative to basal and labyrinth zones, expressed the highest abundance of CCBL1, flavin-containing monooxygenase 3, and cleaved caspase-3. Together, the findings show the differential effects of NAC and AOAA on TCE-induced pregnancy outcomes are likely attributable to TCE metabolism modulation.

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

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.

Nanoparticle Digestion Simulator Reveals pH-Dependent Aggregation in the Gastrointestinal Tract.

Determining the physicochemical properties of ingested nanoparticles within the gastrointestinal tract (GIT) is critical for evaluating the impact of environmental exposure and potential for nanoparticle drug delivery. However, it is challenging to predict nanoparticle physicochemical properties at the point of intestinal absorption due to the changing chemical environments within the GIT. Herein, a dynamic nanoparticle digestion simulator (NDS) was constructed to examine nanoparticle evolution due to changing pH and salt concentrations in the stomach and upper intestine. This multicompartment, flow-through system simulates digestion by transferring gastrointestinal fluids and digestive secretions at physiologically relevant time scales and flow rates. Pronounced differences in aggregation and aggregate stability were observed with silver nanoparticles (citrate-coated) with an initial hydrodynamic diameter (Dh) of 24.6 ± 0.4 nm examined under fasted (pH 2) and fed (pH 5) gastric conditions using nanoparticle tracking analysis (NTA) for size distributions and transmission electron microscopy with energy dispersive X-ray spectroscopy (TEM-EDX) for morphology and elemental composition. Under fasted stomach conditions, particles aggregated to Dh = 130 ± 10 nm and remained as large aggregates in the upper intestinal compartments (duodenum and jejunum) ending with Dh = 110 ± 20 nm and a smaller mode at 59 ± 8 nm. In contrast, under fed conditions, nanoparticles aggregated to 60 ± 10 nm in the stomach, then disaggregated to individual nanoparticles (26 ± 2 nm) in the intestinal compartments. The NDS provides an analytical approach for studying nanoparticle physicochemical modifications within the GIT and the impacts of intentionally and unintentionally ingested nanoparticles.

An Early Investigation into Possible Alternatives to Stapled Hysterotomy in Open Fetal Surgery.

PURPOSE: We hypothesized that surgical energy could be used to create hysterotomies in open fetal surgery. STUDY DESIGN: Initial studies compared the LigaSure Impact and Harmonic ACE + 7 Shears in the efficiency of hysterotomy and thermal damage. Pregnant ewes at an estimated gestational age (EGA) of 116 to 120 days (term = 145; n = 7) underwent hysterotomy using either device. Hysterotomy edges were resected, and thermal injury extent was determined by histopathological assessment. Upon determining a superior device, subsequent studies compared this to the AutoSuture Premium Poly CS*-57 Stapler in uterine healing. Pregnant ewes (n = 6) at an EGA of 87 to 93 days underwent 6-cm hysterotomy in each gravid horn with either the stapler (n = 5) or Harmonic (n = 5) followed by closure and animal recovery. After 37 to 42 days, uterine healing was assessed by evaluating tensile strength and histopathology. RESULTS: Thermal damage was more extensive with the LigaSure (n = 11 hysterotomies) than with the Harmonic (n = 11; 5.6 ± 1 vs. 3.1 ± 0.6 mm; p < 0.0001);therefore, the Harmonic was selected for healing studies. Gross scar appearance and tensile strength were the same between the Harmonic and stapler. The stapler caused more fibrosis (4/7 samples with "moderate" fibrosis vs. 0/8 with the Harmonic; p = 0.02). CONCLUSION: The Harmonic ACE + 7 caused less thermal injury than the LigaSure Impact and performed similar to the CS*-57 Stapler in uterine healing with continued gestation.

Impact of the Levonorgestrel-Releasing Intrauterine System on the Progression of Chlamydia trachomatis Infection to Pelvic Inflammatory Disease in a Baboon Model.

Background: Understanding the relationship between the levonorgestrel (LNG)-releasing intrauterine system (IUS) and sexually transmitted infections (STIs) is increasingly important as use of the LNG-IUS grows to include women at higher risk for STIs. This study assessed the impact of the LNG-IUS on development of Chlamydia trachomatis pelvic inflammatory disease, using a baboon model. Methods: Baboons with and those without the LNG-IUS were cervically inoculated with C. trachomatis and monitored daily, and cervical and fallopian tube swab specimens were collected weekly for C. trachomatis quantitation by nucleic acid amplification testing and culture. Vaginal swab specimens were collected for cytokine analysis, and serum samples were obtained for detection of C. trachomatis antibodies. Results: The LNG-IUS resulted in an increased C. trachomatis burden in the cervix, with the bacterial burden in the LNG-IUS group diverging from that in the non-LNG-IUS group by 6 weeks after infection. One of 7 baboons in the non-LNG-IUS group and 2 of 6 in the LNG-IUS group developed pelvic inflammatory disease, while 3 animals in each group met criteria suggestive of pelvic inflammatory disease. LNG-IUS increased baseline interleukin 8 levels but failed to further upregulate interleukin 8 during infection. In LNG-IUS recipients, early perturbations in the interleukin 1ß axis corresponded to decreased C. trachomatis clearance and increased T-helper type 2 immune responses. Conclusion: LNG-IUS use results in delayed clearance of C. trachomatis and might alter the reproductive tract immune environment.

An Expandable Mechanopharmaceutical Device (2): Drug Induced Granulomas Maximize the Cargo Sequestering Capacity of Macrophages in the Liver.

PURPOSE: Drug-induced liver injuries (DILI) comprise a significant proportion of adverse drug reactions leading to hospitalizations and death. One frequent DILI is granulomatous inflammation from exposure to harmful metabolites that activate inflammatory pathways of immune cells of the liver, which may act as a barrier to isolate the irritating stimulus and limit tissue damage. METHODS: Paralleling the accumulation of CFZ precipitates in the liver, granulomatous inflammation was studied to gain insight into its effect on liver structure and function. A structural analog that does not precipitate within macrophages was also studied using micro-analytical approaches. Depleting macrophages was used to inhibit granuloma formation and assess its effect on drug bioaccumulation and toxicity. RESULTS: Granuloma-associated macrophages showed a distinct phenotype, differentiating them from non-granuloma macrophages. Granulomas were induced by insoluble CFZ cargo, but not by the more soluble analog, pointing to precipitation being a factor driving granulomatous inflammation. Granuloma-associated macrophages showed increased activation of lysosomal master-regulator transcription factor EB (TFEB). Inhibiting granuloma formation increased hepatic necrosis and systemic toxicity in CFZ-treated animals. CONCLUSIONS: Granuloma-associated macrophages are a specialized cell population equipped to actively sequester and stabilize cytotoxic chemotherapeutic agents. Thus, drug-induced granulomas may function as drug sequestering "organoids" -an induced, specialized sub-compartment- to limit tissue damage.

Prevalence and geographical distribution of Papio hamadryas papillomavirus 1 (PhPV1) in Kenyan baboons.

Papio hamadryas papillomavirus (PhPV) 1, 2, and 3, are Alphapapillomaviruses that have been detected in Kenyan Olive baboons but the distribution is unknown. Therefore, cervical screening for PhPV1 was performed in baboons from various areas in Kenya using a nested polymerase chain reaction. The prevalence rate was 33%.

Dynamics and establishment of Clostridium difficile infection in the murine gastrointestinal tract.

Clostridium difficile infection (CDI) following antibiotic therapy is a major public health threat. While antibiotic disruption of the indigenous microbiota underlies the majority of cases of CDI, the early dynamics of infection in the disturbed intestinal ecosystem are poorly characterized. This study defines the dynamics of infection with C. difficile strain VPI 10463 throughout the gastrointestinal (GI) tract using a murine model of infection. After inducing susceptibility to C. difficile colonization via antibiotic administration, we followed the dynamics of spore germination, colonization, sporulation, toxin activity, and disease progression throughout the GI tract. C. difficile spores were able to germinate within 6 h postchallenge, resulting in the establishment of vegetative bacteria in the distal GI tract. Spores and cytotoxin activity were detected by 24 h postchallenge, and histopathologic colitis developed by 30 h. Within 36 h, all infected mice succumbed to infection. We correlated the establishment of infection with changes in the microbiota and bile acid profile of the small and large intestines. Antibiotic administration resulted in significant changes to the microbiota in the small and large intestines, as well as a significant shift in the abundance of primary and secondary bile acids. Ex vivo analysis suggested the small intestine as the site of spore germination. This study provides an integrated understanding of the timing and location of the events surrounding C. difficile colonization and identifies potential targets for the development of new therapeutic strategies.

Fecal Microbiota Transplantation Eliminates Clostridium difficile in a Murine Model of Relapsing Disease.

Recurrent Clostridium difficile infection (CDI) is of particular concern among health care-associated infections. The role of the microbiota in disease recovery is apparent given the success of fecal microbiota transplantation (FMT) for recurrent CDI. Here, we present a murine model of CDI relapse to further define the microbiota recovery following FMT. Cefoperazone-treated mice were infected with C. difficile 630 spores and treated with vancomycin after development of clinical disease. Vancomycin treatment suppressed both C. difficile colonization and cytotoxin titers. However, C. difficile counts increased within 7 days of completing treatment, accompanied by relapse of clinical signs. The administration of FMT immediately after vancomycin cleared C. difficile and decreased cytotoxicity within 1 week. The effects of FMT on the gut microbiota community were detectable in recipients 1-day posttransplant. Conversely, mice not treated with FMT remained persistently colonized with high levels of C. difficile, and the gut microbiota in these mice persisted at low diversity. These results suggest that full recovery of colonization resistance against C. difficile requires the restoration of a specific community structure.

Interleukin-22 and CD160 play additive roles in the host mucosal response to Clostridium difficile infection in mice.

Our previous work has shown the significant up-regulation of Il22 and increased phosphorylation of signal transducer and activator of transcription 3 (STAT3) as part of the mucosal inflammatory response to Clostridium difficile infection in mice. Others have shown that phosphorylation of STAT3 at mucosal surfaces includes interleukin-22 (IL-22) and CD160-mediated components. The current study sought to determine the potential role(s) of IL-22 and/or CD160 in the mucosal response to C. difficile infection. Clostridium difficile-infected mice treated with anti-IL-22, anti-CD160 or a combination of the two showed significantly reduced STAT3 phosphorylation in comparison to C. difficile-infected mice that had not received either antibody. In addition, C. difficile-infected mice treated with anti-IL-22/CD160 induced a smaller set of genes, and at significantly lower levels than the untreated C. difficile-infected mice. The affected genes included pro-inflammatory chemokines and cytokines, and anti-microbial peptides. Furthermore, histopathological and flow cytometric assessments both showed a significantly reduced influx of neutrophils in C. difficile-infected mice treated with anti-IL-22/CD160. These data demonstrate that IL-22 and CD160 are together responsible for a significant fraction of the colonic STAT3 phosphorylation in C. difficile infection. They also underscore the additive effects of IL-22 and CD160 in mediating both the pro-inflammatory and pro-survival aspects of the host mucosal response in this infection.

The influence of quarantine on reproductive cycling in wild-caught Baboons (Papio anubis).

Stress impacts nonhuman primate menstrual cycle length but the impact of quarantine is unknown. A retrospective analysis was performed on cycle data from 31 wild-caught baboons during and following quarantine. Cycling initiated in 94 days (19-181) and length normalized within 4-6 cycles. Quarantine significantly impacts menstrual cycle length.

Clostridium difficile-induced colitis in mice is independent of leukotrienes.

Clostridium difficile is the major cause of antibiotic-associated diarrhea and pseudomembranous colitis in healthcare settings. However, the host factors involved in the intestinal inflammatory response and pathogenesis of C. difficile infection (CDI) are largely unknown. Here we investigated the role of leukotrienes (LTs), a group of pro-inflammatory lipid mediators, in CDI. Notably, the neutrophil chemoattractant LTB4, but not cysteinyl (cys) LTs, was induced in the intestine of C57BL/6 mice infected with either C. difficile strain VPI 10463 or strain 630. Genetic or pharmacological ablation of LT production did not ameliorate C. difficile colitis or clinical signs of disease in infected mice. Histological analysis demonstrated that intestinal neutrophilic inflammation, edema and tissue damage in mice during acute and severe CDI were not modulated in the absence of LTs. In addition, CDI induced a burst of cytokines in the intestine of infected mice in a LT-independent manner. Serum levels of anti-toxin A immunoglobulin (Ig) G levels were also not modulated by endogenous LTs. Collectively, our results do not support a role for LTs in modulating host susceptibility to CDI in mice.

Suppression of Clostridium difficile in the gastrointestinal tracts of germfree mice inoculated with a murine isolate from the family Lachnospiraceae.

The indigenous microbial community of the gastrointestinal (GI) tract determines susceptibility to Clostridium difficile colonization and disease. Previous studies have demonstrated that antibiotic-treated mice challenged with C. difficile either developed rapidly lethal C. difficile infection or were stably colonized with mild disease. The GI microbial community of animals with mild disease was dominated by members of the bacterial family Lachnospiraceae, while the gut community in moribund animals had a predominance of Escherichia coli. We investigated the roles of murine Lachnospiraceae and E. coli strains in colonization resistance against C. difficile in germfree mice. Murine Lachnospiraceae and E. coli isolates were cultured from wild-type mice. The ability of each of these isolates to interfere with C. difficile colonization was tested by precolonizing germfree mice with these bacteria 4 days prior to experimental C. difficile challenge. Mice precolonized with a murine Lachnospiraceae isolate, but not those colonized with E. coli, had significantly decreased C. difficile colonization, lower intestinal cytotoxin levels and exhibited less severe clinical signs and colonic histopathology. Infection of germfree mice or mice precolonized with E. coli with C. difficile strain VPI 10463 was uniformly fatal by 48 h, but only 20% mortality was seen at 2 days in mice precolonized with the Lachnospiraceae isolate prior to challenge with VPI 10463. These findings confirm that a single component of the GI microbiota, a murine Lachnospiraceae isolate, could partially restore colonization resistance against C. difficile. Further study of the members within the Lachnospiraceae family could lead to a better understanding of mechanisms of colonization resistance against C. difficile and novel therapeutic approaches for the treatment and prevention of C. difficile infection.

E-prostanoid 2 receptor signaling suppresses lung innate immunity against Streptococcus pneumoniae.

Pneumonia is a major global health problem. Prostaglandin (PG) E(2) is an immunomodulatory lipid with anti-inflammatory, immunosuppressive, and pro-resolving actions. Data suggest that the E-prostanoid (EP) 2 receptor mediates immunomodulatory effects of PGE(2), but the extent to which this occurs in Streptococcus pneumoniae infection is unknown. Intratracheal lung infection of C57BL/6 mice possessing (EP2(+/+)) or lacking (EP2(-/-)) the EP2 receptor was performed, as were in vitro studies of alveolar macrophage (AM) host defense functions. Bacterial clearance and survival were significantly improved in vivo in EP2(-/-) mice and it correlated with greater neutrophilic inflammation and higher lung IL-12 levels. Upon ex vivo challenge with pneumococcus, EP2(-/-)cells expressed greater amounts of TNF-α and MIP-2 than did EP2(+/+) AMs, and had improved phagocytosis, intracellular killing, and reactive oxygen intermediate generation. These data suggest that PGE(2)-EP2 signaling may provide a novel pharmacological target for treating pneumococcal pneumonia in combination with antimicrobials.

The effects of a single cervical inoculation of Chlamydia trachomatis on the female reproductive tract of the baboon (Papio anubis).

BACKGROUND: The baboon (Papio hamadryas anubis) can be transcervically instrumented, facilitating studies of intrauterine contraception and reproductive tract infection. We sought to determine if the baboon could become infected with a single cervical inoculation of Chlamydia trachomatis. METHODS: Ten female baboons were randomized and inoculated cervically with C. trachomatis serovar E (or buffer alone). Animals underwent weekly clinical and laparoscopic evaluations for four weeks and at post-inoculation week 8, to monitor upper tract infection. Cervical culture and nucleic acid amplification testing (NAAT) were completed weekly throughout the study. Animals were euthanized at week 16 and the reproductive tracts were examined histologically. RESULTS: All inoculated animals developed cervical infection. The average duration of positive NAAT results was 6.8 weeks (range 2-16). Two of eight (25%) animals tested positive from fallopian tube samples. Infected animals showed histological findings consistent with chlamydial infection, such as germinal centers. Five of ten animals seroconverted to C. trachomatis. CONCLUSIONS: Baboons cervically inoculated once with C. trachomatis develop infection similar to humans, with a low incidence of upper tract infection. This novel model of Chlamydia infection closely resembles human disease and opens new avenues for studying the pathogenesis of sexually transmitted infections and contraceptive safety.

The Gut Bacterial Community Potentiates Clostridioides difficile Infection Severity.

The severity of Clostridioides difficile infections (CDI) has increased over the last few decades. Patient age, white blood cell count, and creatinine levels as well as C. difficile ribotype and toxin genes have been associated with disease severity. However, it is unclear whether specific members of the gut microbiota are associated with variations in disease severity. The gut microbiota is known to interact with C. difficile during infection. Perturbations to the gut microbiota are necessary for C. difficile to colonize the gut. The gut microbiota can inhibit C. difficile colonization through bile acid metabolism, nutrient consumption, and bacteriocin production. Here, we sought to demonstrate that members of the gut bacterial communities can also contribute to disease severity. We derived diverse gut communities by colonizing germfree mice with different human fecal communities. The mice were then infected with a single C. difficile ribotype 027 clinical isolate, which resulted in moribundity and histopathologic differences. The variation in severity was associated with the human fecal community that the mice received. Generally, bacterial populations with pathogenic potential, such as Enterococcus, Helicobacter, and Klebsiella, were associated with more-severe outcomes. Bacterial groups associated with fiber degradation and bile acid metabolism, such as Anaerotignum, Blautia, Lactonifactor, and Monoglobus, were associated with less-severe outcomes. These data indicate that, in addition to the host and C. difficile subtype, populations of gut bacteria can influence CDI disease severity. IMPORTANCE Clostridioides difficile colonization can be asymptomatic or develop into an infection ranging in severity from mild diarrhea to toxic megacolon, sepsis, and death. Models that predict severity and guide treatment decisions are based on clinical factors and C. difficile characteristics. Although the gut microbiome plays a role in protecting against CDI, its effect on CDI disease severity is unclear and has not been incorporated into disease severity models. We demonstrated that variation in the microbiome of mice colonized with human feces yielded a range of disease outcomes. These results revealed groups of bacteria associated with both severe and mild C. difficile infection outcomes. Gut bacterial community data from patients with CDI could improve our ability to identify patients at risk of developing more severe disease and improve interventions that target C. difficile and the gut bacteria to reduce host damage.

Intestinal Inflammation Reversibly Alters the Microbiota to Drive Susceptibility to Clostridioides difficile Colonization in a Mouse Model of Colitis.

Susceptibility to Clostridioides difficile infection (CDI) typically follows the administration of antibiotics. Patients with inflammatory bowel disease (IBD) have increased incidence of CDI, even in the absence of antibiotic treatment. However, the mechanisms underlying this susceptibility are not well understood. To explore the intersection between CDI and IBD, we recently described a mouse model where colitis triggered by the murine gut bacterium, Helicobacter hepaticus, in IL-10-/- mice led to susceptibility to C. difficile colonization without antibiotic administration. The current work disentangles the relative contributions of inflammation and gut microbiota in colonization resistance to C. difficile in this model. We show that inflammation drives changes in microbiota composition, which leads to CDI susceptibility. Decreasing inflammation with an anti-p40 monoclonal antibody promotes a shift of the microbiota back toward a colonization-resistant state. Transferring microbiota from susceptible and resistant mice to germfree animals transfers the susceptibility phenotype, supporting the primacy of the microbiota in colonization resistance. These findings shine light on the complex interactions between the host, microbiota, and C. difficile in the context of intestinal inflammation, and may form a basis for the development of strategies to prevent or treat CDI in IBD patients. IMPORTANCE Patients with inflammatory bowel disease (IBD) have an increased risk of developing C. difficile infection (CDI), even in the absence of antibiotic treatment. Yet, mechanisms regulating C. difficile colonization in IBD patients remain unclear. Here, we use an antibiotic-independent mouse model to demonstrate that intestinal inflammation alters microbiota composition to permit C. difficile colonization in mice with colitis. Notably, treating inflammation with an anti-p40 monoclonal antibody, a clinically relevant IBD therapeutic, restores microbiota-mediated colonization resistance to the pathogen. Through microbiota transfer experiments in germfree mice, we confirm that the microbiota shaped in the setting of IBD is the primary driver of susceptibility to C. diffiicile colonization. Collectively, our findings provide insight into CDI pathogenesis in the context of intestinal inflammation, which may inform methods to manage infection in IBD patients. More broadly, this work advances our understanding of mechanisms by which the host-microbiota interface modulates colonization resistance to C. difficile.