Presence Of Segmented Filamentous Bacteria Research Articles

P059 Diet controls segmented filamentous bacteria in driving Crohn’s disease-like inflammation in TNFdeltaARE mice

Abstract Background Crohn’s disease (CD)-like inflammation in TNFdeltaARE mice (ARE) mice is triggered by dysbiotic gut microbial communities. Similar to the therapeutic effect of exclusive enteral nutrition in CD, dietary intervention using semi-synthetic experimental diet prevents disease development in ARE mice. The aim of this study is to identify the causal microbial cues responsible for CD-like inflammation and to dissect the protective role of diet in ARE mice. Methods Bacterial communities in ARE and wildtype (WT) mice were profiled by 16S rRNA gene sequencing, FISH and qPCR. Germfree WT and ARE mice were colonized with segmented filamentous bacteria (SFB) from dysbiotic ARE mice. Disease activity, cytokine expression, mucosal immune cell infiltrates as well as Paneth and goblet cells phenotypes were assessed in murine ileal and colonic tissue. To assess the influence of diet on disease development, ARE mice were fed either chow, purified semi-synthetic experimental diet (SED) or fiber-rich diet. Impact of SFB on ileo-colonic inflammation was tested in additional mouse models (XIAP-/- and IL10 -/-). Mucosal biopsies from adult and pediatric IBD patients (N=407) were screened for the presence of human SFB using SFB-specific PCR primers. Results Under specific pathogen-free (SPF) conditions, ARE mice developed CD-like ileal inflammation which correlated strongly with increasing abundance of SFB. Mono-colonization of germfree ARE mice with SFB resulted in severe enterocolitis. Parallel to high tissue levels of TNF and IL-17, SPF-mediated inflammation was associated with neutrophil infiltration and the expansion of IFNγ expressing Th1 cells in the mucosa. Loss of Paneth and goblet cell function allowed SFB to penetrate mucus layers towards the epithelium. SED eradicated SFB and completely abolished inflammation in ARE mice. In contrast, fiber-rich diet partially restored SFB colonization and led to increased levels of inflammatory cytokines. SFB mono-colonization of GF XIAP-/- and IL10 -/- failed to induce inflammation, emphasizing host specificity to the mucosa-adherent SFB. Presence of SFB was confirmed in 12 ileal and 11 colonic mucosal biopsies from IBD patients with active or inactive disease. Conclusion We identified a novel pathogenic role of SFB in driving severe CD-like ileo-colonic inflammation characterized by loss of Paneth and goblet cell functions in ARE mice. Detection of SFB in mucosal biopsies of patients with IBD opens new perspectives about therapeutic strategies targeting SFB-mediated processes. Purified diet antagonized SFB colonization and prevented disease development in ARE mice in contrast to fiber-rich diet, clearly demonstrating the important role of diet in modulating a novel IBD-relevant pathobiont.

Presence of Segmented Filamentous Bacteria in Human Children and Its Potential Role in the Modulation of Human Gut Immunity.

Segmented filamentous bacteria (SFB) are commensal organisms that grow by anchoring a specialized holdfast structure to the intestinal walls of a variety of animals. Interaction of SFB with Peyer’s patches in mice promotes the post-natal maturation of the immune system. We previously reported that the colonization of SFB in humans mainly occurs by 36 months of age, and is difficult to be detected afterward. In this study, we measured the level of SFB in intestinal fluids of human children. SFB were found via qPCR to represent a small fraction of the whole SFB-positive microbiota (105 SFB in 1011 total bacteria). Bacteria with filamentous segmented morphology were observed in intestinal fluids via fluorescent in situ hybridization, and from gut biopsies via scanning electron microscopy. SFB-specific DNA and peptide fragments were also identified via multiple displacement amplification PCR and mass spectrometry. There was an overall positive correlation between the presence of SFB and the titer of total secretory immunoglobulin A (sIgA), which is more apparent in intestinal fluids of the age group of 8–36 months. Afterward there was a decline of SFB in numbers correlated with a reduction of total sIgA. RT-qPCR analysis of the terminal ileal biopsies revealed that the expression of Th17 pathway genes were induced in SFB-positive samples, while the markers of T and B cell receptor signaling pathways were also upregulated. Collectively, these data suggest that SFB is a rare member of microbiota, and may play an important role in the development of human gut immunity.

Modulatory Influence of Segmented Filamentous Bacteria on Transcriptomic Response of Gnotobiotic Mice Exposed to TCDD.

Environmental toxicants such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an aryl hydrocarbon receptor (AhR), are known to induce host toxicity and structural shifts in the gut microbiota. Key bacterial populations with similar or opposing functional responses to AhR ligand exposure may potentially help regulate expression of genes associated with immune dysfunction. To examine this question and the mechanisms for AhR ligand-induced bacterial shifts, C57BL/6 gnotobiotic mice were colonized with and without segmented filamentous bacteria (SFB) – an immune activator. Mice were also colonized with polysaccharide A producing Bacteroides fragilis – an immune suppressor to serve as a commensal background. Following colonization, mice were administered TCDD (30 μg/kg) every 4 days for 28 days by oral gavage. Quantified with the nCounter® mouse immunology panel, opposing responses in ileal gene expression (e.g., genes associated with T-cell differentiation via the class II major histocompatibility complex) as a result of TCDD dosing and SFB colonization were observed. Genes that responded to TCDD in the presence of SFB did not show a significant response in the absence of SFB, and vice versa. Regulatory T-cells examined in the mesenteric lymph-nodes, spleen, and blood were also less impacted by TCDD in mice colonized with SFB. TCDD-induced shifts in abundance of SFB and B. fragilis compared with previous studies in mice with a traditional gut microbiome. With regard to the mouse model colonized with individual populations, results indicate that TCDD-induced host response was significantly modulated by the presence of SFB in the gut microbiome, providing insight into therapeutic potential between AhR ligands and key commensals.

T-bet expression by Th cells promotes type 1 inflammation but is dispensable for colitis.

The transcription factor T-bet is highly expressed by Th cells isolated from the inflamed intestine of Crohn's disease patients, and has been regarded a critical driver of murine T cell-induced colitis. However, we show here that T-bet expression by Th cells is not required for the manifestation of T-cell-induced colitis in the presence of segmented filamentous bacteria and Helicobacter hepaticus. T-bet expression by Th cells controls their survival and localization, their repertoire of chemokine and chemokine receptor expression, the accumulation of monocytes and macrophages in the inflamed colon, and their differentiation to the M1 type, i.e., type 1 inflammation. Nevertheless, T-bet-deficient Th cells efficiently induce colitis, as reflected by weight loss, diarrhea, and colon histopathology. T-bet-deficient Th cells differentiate into Th1/17 cells, able to express IFN-γ and IL-17A upon restimulation. While neutralization of IL-17A exacerbated colitis induced by wild-type or T-bet-deficient Th cells, neutralization of IFN-γ completely abolished colitis.

Intestinal Microbiota of Mice Influences Resistance to Staphylococcus aureus Pneumonia.

Th17 immunity in the gastrointestinal tract is regulated by the intestinal microbiota composition, particularly the presence of segmented filamentous bacteria (sfb), but the role of the intestinal microbiota in pulmonary host defense is not well explored. We tested whether altering the gut microbiota by acquiring sfb influences the susceptibility to staphylococcal pneumonia via induction of type 17 immunity. Groups of C57BL/6 mice which differed in their intestinal colonization with sfb were challenged with methicillin-resistant Staphylococcus aureus in an acute lung infection model. Bacterial burdens, bronchoalveolar lavage fluid (BALF) cell counts, cell types, and cytokine levels were compared between mice from different vendors, mice from both vendors after cohousing, mice given sfb orally prior to infection, and mice with and without exogenous interleukin-22 (IL-22) or anti-IL-22 antibodies. Mice lacking sfb developed more severe S. aureus pneumonia than mice colonized with sfb, as indicated by higher bacterial burdens in the lungs, lung inflammation, and mortality. This difference was reduced when sfb-negative mice acquired sfb in their gut microbiota through cohousing with sfb-positive mice or when given sfb orally. Levels of type 17 immune effectors in the lung were higher after infection in sfb-positive mice and increased in sfb-negative mice after acquisition of sfb, as demonstrated by higher levels of IL-22 and larger numbers of IL-22(+) TCRβ(+) cells and neutrophils in BALF. Exogenous IL-22 protected mice from S. aureus pneumonia. The murine gut microbiota, particularly the presence of sfb, promotes pulmonary type 17 immunity and resistance to S. aureus pneumonia, and IL-22 protects against severe pulmonary staphylococcal infection.

Homeostatic IL-23R signaling limits TH17 response through IL-22-mediated containment of commensal microbiota (MUC9P.818)

Abstract Mammalian hosts are colonized with commensal microbes in various mucosal and epithelial tissues, including the intestinal tract. In mice, the presence of segmented filamentous bacteria (SFB) is required for TH17 differentiation and the development of autoimmune disease. Here we demonstrate that the IL-23 pathway dynamically regulates the abundance of SFB as well as mucosal barrier function in the adult animal. Genetic or pharmacological inactivation of the pathway selectively perturbs abundance of a small group of commensals, including SFB, and results in an impaired mucosal barrier, leading to systemic microbial dissemination. Conversely, microbial dissemination leads to induction of the IL-23 pathway with dual consequences: IL-23 drives IL-22 production to reinforce mucosal barrier function and elicit antimicrobial activities. It also drives the differentiation of TH17 cells in an attempt to combat escaped microbes in the lamina propria and in distal tissues. Thus barrier defects generate a systemic environment that facilitates TH17 development.

Role of SFB in autoimmune arthritis

A key role for segmented filamentous bacteria (SFB) has recently been demonstrated in several mouse models of autoimmune diseases, including autoimmune arthritis and multiple sclerosis. The mechanism governing the activation of systemic autoreactive T cell responses by such commensals in the gut, however, remained elusive. In this addendum, we discuss recent results addressing the local regulation of autoreactive T cell sensitivity by these unique bacteria. We found that the presence of SFB in the gut microbiota was sufficient to promote a local inflammatory microenvironment altering the T cell-intrinsic desensitization process normally occurring in response to chronic self-antigen stimulation. In the absence of this key tolerance checkpoint, sustained chronic T cell proliferation, IFNγ production, and B cell activation eventually led to the development of enhanced pathologies in a Th1-driven T cell-transfer model of autoimmune arthritis.

Comparative analysis of the distribution of segmented filamentous bacteria in humans, mice and chickens

Segmented filamentous bacteria (SFB) are indigenous gut commensal bacteria. They are commonly detected in the gastrointestinal tracts of both vertebrates and invertebrates. Despite the significant role they have in the modulation of the development of host immune systems, little information exists regarding the presence of SFB in humans. The aim of this study was to investigate the distribution and diversity of SFB in humans and to determine their phylogenetic relationships with their hosts. Gut contents from 251 humans, 92 mice and 72 chickens were collected for bacterial genomic DNA extraction and subjected to SFB 16S rRNA-specific PCR detection. The results showed SFB colonization to be age-dependent in humans, with the majority of individuals colonized within the first 2 years of life, but this colonization disappeared by the age of 3 years. Results of 16S rRNA sequencing showed that multiple operational taxonomic units of SFB could exist in the same individuals. Cross-species comparison among human, mouse and chicken samples demonstrated that each host possessed an exclusive predominant SFB sequence. In summary, our results showed that SFB display host specificity, and SFB colonization, which occurs early in human life, declines in an age-dependent manner.

A comparative molecular study of the presence of “Candidatus arthromitus” in the digestive system of rainbow trout, Oncorhynchus mykiss (Walbaum), healthy and affected with rainbow trout gastroenteritis

Observations were made using histopathological techniques in conjunction with a nested polymerase chain reaction (PCR) protocol for the specific detection of "Candidatus arthromitus" on DNA extracted from wax-embedded tissues and fresh digestive contents of rainbow trout. Samples positive for "Candidatus arthromitus" DNA included fish with rainbow trout gastroenteritis (RTGE), clinically normal cohabiting fish, and apparently healthy controls from RTGE positive and RTGE negative sites. The results obtained from the PCR were confirmed by nucleotide sequencing. "Candidatus arthromitus" DNA was found in distal intestine as well as in sections of pyloric caeca, suggesting that both these locations are appropriate for molecular detection of "Candidatus arthromitus" DNA in trout. Furthermore, rainbow trout fry distal intestinal samples from two different hatcheries where RTGE had not been reported were also positive. Differences in "Candidatus arthromitus" DNA detection between paraffin wax-embedded and fresh digestive content samples from the same fish suggested that it may be predominantly epithelium-associated in healthy trout. Parallel histopathological observations indicated that pyloric caeca are the preferred site for visualizing segmented filamentous bacteria (SFB) in trout with RTGE. The results of this study showed that the presence of SFB was not invariably associated with clinical disease and that more information is required to understand the role of these organisms.

A microscopical view of the intestine of Onychochaeta borincana (Oligochaeta: Glossoscolecidae)The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Summary Scanning electron microscopy was used to detect the presence of microorganisms in the intestinal surface of Onychochaeta borincana . The intestines of fourteen adult specimens were extracted, washed, and prepared for microscopy protocol. The presence of segmented filamentous bacteria (SFB) and rod-shaped bacteria (RSB) were observed in the fore, mid, and hindgut wall of all samples. SFBs and RSBs appeared to be more abundant in the midgut. Spore-like segments were only present in the hindgut. A non-characterized network of filaments was also observed exclusively in the posterior part of the gut. SFBs appear to be attached to gut walls and are probably indigenous.

A microscopical view of the intestine of Onychochaeta borincana (Oligochaeta: Glossoscolecidae) : The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Scanning electron microscopy was used to detect the presence of microorganisms in the intestinal surface of Onychochaeta borincana . The intestines of fourteen adult specimens were extracted, washed, and prepared for microscopy protocol. The presence of segmented filamentous bacteria (SFB) and rod-shaped bacteria (RSB) were observed in the fore, mid, and hindgut wall of all samples. SFBs and RSBs appeared to be more abundant in the midgut. Spore-like segments were only present in the hindgut. A non-characterized network of filaments was also observed exclusively in the posterior part of the gut. SFBs appear to be attached to gut walls and are probably indigenous.

Segmented filamentous bacteria prevent colonization of enteropathogenic Escherichia coli O103 in rabbits.

Despite their distribution in the intestines of many mammals, including man, segmented filamentous bacteria (SFB) have not been found in rabbits, nor has any function been identified for these uncultivable microbes. New Zealand White rabbits were infected with rabbit enteropathogenic Escherichia coli O103 (REPEC O103) derivatives, followed up clinically, and randomly killed 1-4 days after inoculation. Intestinal tissue samples were examined by electron and light microscopy to search for SFB and to evaluate REPEC O103 colonization. Twelve of 21 rabbits showed SFB colonization on ileal absorptive villi. The presence of SFB was correlated with lack of REPEC 0103 ileal colonization (P<.01) and disease. Rabbits without SFB were always colonized by this pathogen. SFB appear to inhibit intestinal colonization by REPEC O103 and thus protect against REPEC 0103 disease. SFB colonization in rabbits is also described for the first time.

Influence of Antimicrobial Drugs on Segmented Filamentous Bacteria in the Ileum of Mice

The effects of various types of 3 d antibiotic treatment of mice on the presence of segmented filamentous bacteria (SFBs) in the ileum were measured in order to further characterise these microorganisms. To assess any specific effects of the antimicrobial drugs on SFBs, relative caecal weight, relative number of fusiform-shaped bacteria in the caecum and the number of faecal Enterobacteriaceae were also determined. All drugs tested, i.e. amoxycillin, doxycyclin, gentamicin, vancomycin, ciprofloxacin, trimethoprim, metronidazole, clindamycin, streptomycin and cefotaxim, reduced the presence of SFBs in the ileum, although to different degrees. None of the drugs affected body weight of the mice. There was no correlation of the drug effects on SFBs and those on either relative caecal weight, percentage of caecal fusiforms or faecal Enterobacteriaceae. Thus, the effects of the antimicrobial drugs on SFBs can be considered rather specific. The sensitivity pattern of SFBs suggests that they are facultatively anaerobic bacteria with relatively high sensitivity to antimicrobial drugs. Keywords: Segmented filamentous bacteria; Intestinal bacteria; Small intestine; Mouse; Antibiotics.

The influence of stress and cheese-whey on intestinal parameters in mice.

In a mouse model the effects of environmental stress on gastrointestinal parameters and the effects of cheese-whey on the changes induced by stress were studied. Mice were subjected to overcrowding, lack of bedding, overcrowding together with lack of bedding, continuous light, and housing at 30 degrees C. The influence of stress on relative caecal weight, faecal enterobacteriaceae, colonisation resistance (CR), filamentous segmented bacteria in the small intestine, fusiform bacteria in the faeces, and concentration of faecal bile acids was studied. Stress had no influence on relative caecal weight, faecal enterobacteriaceae, and faecal bile acids. Stress did decrease colonisation resistance, presence of segmented filamentous bacteria in the small intestine, and fusiform bacteria in the faeces. Cheese-whey had no positive effect on the microbiological disturbances caused by stress. The number of filamentous segmented bacteria in the small intestine and fusiforms in the faeces decreased by giving cheese-whey.