Aleh Bobr

Microbiota transplantation restores normal fecal bile acid composition in recurrent Clostridium difficile infection

Fecal microbiota transplantation (FMT) has emerged as a highly effective therapy for refractory, recurrent Clostridium difficile infection (CDI), which develops following antibiotic treatments. Intestinal microbiota play a critical role in the metabolism of bile acids in the colon, which in turn have major effects on the lifecycle of C. difficile bacteria. We hypothesized that fecal bile acid composition is altered in patients with recurrent CDI and that FMT results in its normalization. General metabolomics and targeted bile acid analyses were performed on fecal extracts from patients with recurrent CDI treated with FMT and their donors. In addition, 16S rRNA gene sequencing was used to determine the bacterial composition of pre- and post-FMT fecal samples. Taxonomic bacterial composition of fecal samples from FMT recipients showed rapid change and became similar to the donor after the procedure. Pre-FMT fecal samples contained high concentrations of primary bile acids and bile salts, while secondary bile acids were nearly undetectable. In contrast, post-FMT fecal samples contained mostly secondary bile acids, as did non-CDI donor samples. Therefore, our analysis showed that FMT resulted in normalization of fecal bacterial community structure and metabolic composition. Importantly, metabolism of bile salts and primary bile acids to secondary bile acids is disrupted in patients with recurrent CDI, and FMT corrects this abnormality. Since individual bile salts and bile acids have pro-germinant and inhibitory activities, the changes suggest that correction of bile acid metabolism is likely a major mechanism by which FMT results in a cure and prevents recurrence of CDI.

Dynamic changes in short- and long-term bacterial composition following fecal microbiota transplantation for recurrent Clostridium difficile infection

BackgroundFecal microbiota transplantation (FMT) is an effective treatment for recurrent Clostridium difficile infection (CDI) that often fails standard antibiotic therapy. Despite its widespread recent use, however, little is known about the stability of the fecal microbiota following FMT.ResultsHere we report on short- and long-term changes and provide kinetic visualization of fecal microbiota composition in patients with multiply recurrent CDI that were refractory to antibiotic therapy and treated using FMT. Fecal samples were collected from four patients before and up to 151 days after FMT, with daily collections until 28 days and weekly collections until 84 days post-FMT. The composition of fecal bacteria was characterized using high throughput 16S rRNA gene sequence analysis, compared to microbiota across body sites in the Human Microbiome Project (HMP) database, and visualized in a movie-like, kinetic format. FMT resulted in rapid normalization of bacterial fecal sample composition from a markedly dysbiotic state to one representative of normal fecal microbiota. While the microbiome appeared most similar to the donor implant material 1 day post-FMT, the composition diverged variably at later time points. The donor microbiota composition also varied over time. However, both post-FMT and donor samples remained within the larger cloud of fecal microbiota characterized as healthy by the HMP.ConclusionsDynamic behavior is an intrinsic property of normal fecal microbiota and should be accounted for in comparing microbial communities among normal individuals and those with disease states. This also suggests that more frequent sample analyses are needed in order to properly assess success of FMT procedures.

Acute Ablation of Langerhans Cells Enhances Skin Immune Responses

Understanding the function of Langerhans cells (LCs) in vivo has been complicated by conflicting results from LC-deficient mice. Human Langerin-DTA mice constitutively lack LCs and develop exaggerated contact hypersensitivity (CHS) responses. Murine Langerin-diptheria toxin receptor (DTR) mice allow for the inducible elimination of LCs and Langerin+ dermal dendritic cells (dDCs) after administration of diphtheria toxin, which results in reduced CHS. When Langerin+ dDCs have partially repopulated the skin but LCs are still absent, CHS returns to normal. Thus, LCs appear to be suppressive in human Langerin-DTA mice and redundant in murine Langerin-DTR mice. To determine whether inducible versus constitutive LC ablation explains these results, we engineered human Langerin-DTR mice in which diphtheria toxin ablates LCs without affecting Langerin+ dDCs. The inducible ablation of LCs in human Langerin-DTR mice resulted in increased CHS. Thus, LC-mediated suppression does not require their absence during ontogeny or during the steady-state and is consistent with a model in which LCs actively suppress Ag-specific CHS responses.

Autocrine/paracrine TGF-β1 inhibits Langerhans cell migration

Langerhans cells (LCs) are skin-resident dendritic cells (DC) located in the epidermis that migrate to skin-draining lymph nodes during the steady state and in response to inflammatory stimuli. TGF-β1 is a critical immune regulator that is highly expressed by LCs. The ability to test the functional importance of LC-derived TGF-β1 is complicated by the requirement of TGF-β1 for LC development and by the absence of LCs in mice with an LC-specific ablation of TGF-β1 or its receptor. To overcome these problems, we have engineered transgenic huLangerin-CreERT2 mice that allow for inducible LC-specific excision. Highly efficient and LC-specific expression was confirmed in mice bred onto a YFP Cre reporter strain. We next generated huLangerin-CreERT2 × TGF-βRIIfl and huLangerin-CreERT2 × TGF-β1fl mice. Excision of the TGFβRII or TGFβ1 genes induced mass migration of LCs to the regional lymph node. Expression of costimulatory markers and inflammatory cytokines was unaffected, consistent with homeostatic migration. In addition, levels of p-SMAD2/3 were decreased in LCs from wild-type mice before inflammation-induced migration. We conclude that TGF-β1 acts directly on LCs in an autocrine/paracrine manner to inhibit steady-state and inflammation-induced migration. This is a readily targetable pathway with potential therapeutic implications for skin disease.

Stromal cells control the epithelial residence of DCs and memory T cells by regulated activation of TGF-β.

Cells of the immune system that reside in barrier epithelia provide a first line of defense against pathogens. Langerhans cells (LCs) and CD8+ tissue-resident memory T cells (TRM cells) require active transforming growth factor-β1 (TGF-β) for epidermal residence. Here we found that integrins αvβ6 and αvβ8 were expressed in non-overlapping patterns by keratinocytes (KCs) and maintained the epidermal residence of LCs and TRM cells by activating latent TGF-β. Similarly, the residence of dendritic cells and TRM cells in the small intestine epithelium also required αvβ6. Treatment of the skin with ultraviolet irradiation decreased integrin expression on KCs and reduced the availability of active TGF-β, which resulted in LC migration. Our data demonstrated that regulated activation of TGF-β by stromal cells was able to directly control epithelial residence of cells of the immune system through a novel mechanism of intercellular communication.

Langerhans Cells Require MyD88-Dependent Signals for Candida albicans Response but Not for Contact Hypersensitivity or Migration

Langerhans cells (LC) are a subset of skin-resident dendritic cells (DC) that reside in the epidermis as immature DC, where they acquire Ag. A key step in the life cycle of LC is their activation into mature DC in response to various stimuli, including epicutaneous sensitization with hapten and skin infection with Candida albicans. Mature LC migrate to the skin-draining LN, where they present Ag to CD4 T cells and modulate the adaptive immune response. LC migration is thought to require the direct action of IL-1β and IL-18 on LC. In addition, TLR ligands are present in C. albicans, and hapten sensitization produces endogenous TLR ligands. Both could contribute to LC activation. We generated Langerin-Cre MyD88fl mice in which LC are insensitive to IL-1 family members and most TLR ligands. LC migration in the steady state, after hapten sensitization and postinfection with C. albicans, was unaffected. Contact hypersensitivity in Langerin-Cre MyD88fl mice was similarly unaffected. Interestingly, in response to C. albicans infection, these mice displayed reduced proliferation of Ag-specific CD4 T cells and defective Th17 subset differentiation. Surface expression of costimulatory molecules was intact on LC, but expression of IL-1β, IL-6, and IL-23 was reduced. Thus, sensitivity to MyD88-dependent signals is not required for LC migration, but is required for the full activation and function of LC in the setting of fungal infection.

Early colon screening of adult patients with cystic fibrosis reveals high incidence of adenomatous colon polyps.

Background and Goals: Cystic fibrosis (CF) is associated with increased incidence of gastrointestinal cancer. Increasing overall life expectancy of CF patients predicts emergence of colon cancer as a significant clinical problem in the adult CF population. The primary aim of this study was to estimate the incidence of adenomatous colon polyps in patients with CF during systematic screening by colonoscopy. Study: This is a single-center series of 45 CF patients aged 40 years and above (mean age, 47 y) undergoing colonoscopic screening. A fraction of these patients (9/45) had history of organ transplantation. Results from transplant and nontransplant patients were analyzed separately. Results: Adult CF patients have a high incidence of adenomatous polyps identified by colonoscopy. In addition, positive examinations are characterized by multiple polyps and common features of advanced pathology. The incidence of adenomatous colon polyps is greater in male patients, although the 1 patient in this cohort found to have colorectal cancer was female. Conclusions: CF has features of a hereditary colon cancer syndrome. Increasing life expectancy of CF patients suggests that earlier colon screening in this population may be warranted. Optimal criteria for initiation of screening and frequency of surveillance should be subject of further studies.

Environmental Contamination in Households of Patients with Recurrent Clostridium difficile Infection

ABSTRACT Recurrent Clostridium difficile infection (R-CDI) is common and difficult to treat, potentially necessitating fecal microbiota transplantation (FMT). Although C. difficile spores persist in the hospital environment and cause infection, little is known about their potential presence or importance in the household environment. Households of R-CDI subjects in the peri-FMT period and of geographically matched and age-matched controls were analyzed for the presence of C. difficile. Household environmental surfaces and fecal samples from humans and pets in the household were examined. Households of post-FMT subjects were also examined (environmental surfaces only). Participants were surveyed regarding their personal history and household cleaning habits. Species identity and molecular characteristics of presumptive C. difficile isolates from environmental and fecal samples were determined by using the Pro kit (Remel, USA), Gram staining, PCR, toxinotyping, tcdC gene sequencing, and pulsed-field gel electrophoresis (PFGE). Environmental cultures detected C. difficile on ≥1 surface in 8/8 (100%) peri-FMT households, versus 3/8 (38%) post-FMT households and 3/8 (38%) control households (P = 0.025). The most common C. difficile-positive sites were the vacuum (11/27; 41%), toilet (8/30; 27%), and bathroom sink (5/29; 17%). C. difficile was detected in 3/36 (8%) fecal samples (two R-CDI subjects and one household member). Nine (90%) of 10 households with multiple C. difficile-positive samples had a single genotype present each. In conclusion, C. difficile was found in the household environment of R-CDI patients, but whether it was found as a cause or consequence of R-CDI is unknown. If household contamination leads to R-CDI, effective decontamination may be protective.