Jean-Paul Motta

Hydrogen Sulfide Protects from Colitis and Restores Intestinal Microbiota Biofilm and Mucus Production

Background:Microbiota dysbiosis and impaired barrier function are among the most prominent features of inflammatory bowel disease. In the gastrointestinal tract, hydrogen sulfide (H2S) is an important regulator of mucosal homeostasis. We hypothesized that H2S promotes resolution of colonic inflammation through actions on microbiota biofilm and the mucus barrier. Methods:We used mice genetically deficient for a key enzyme for H2S production (cystathionine &ggr;-lyase) and pharmacologically inhibited that enzyme during colitis in wild-type mice. We tested the effects of administering an H2S donor (diallyl disulfide) to rodents during hapten-induced colitis. Colonic microbiota biofilm was visualized by fluorescent in situ hybridization, and mucus granules were quantified with periodic acid–alcian blue staining. We exposed human microbiota biofilms and planktonic bacteria to H2S donors ex vivo to determine changes in their growth, viability, and biomass. Results:Intestinal microbiota formed linear biofilms in the colon of healthy rodents. During colitis, microbiota biofilms were fragmented and mucus granule production decreased. Endogenous production of H2S had beneficial effects on establishment of microbiota biofilms and colonic mucus production. Therapeutic delivery of H2S into the colon reduced inflammation, restored the microbiota biofilm, and increased the production of mucus granules. In ex vivo human microbiota, H2S not only promoted biofilm formation but also reduced growth of planktonic bacteria. Conclusions:Our results suggest that H2S donors could be used therapeutically during colitis, facilitating correction of microbiota biofilm dysbiosis and mucus layer reconstitution.

Differential Induction of Antimicrobial REGIII by the Intestinal Microbiota and Bifidobacterium breve NCC2950

ABSTRACT The intestinal microbiota is a key determinant of gut homeostasis, which is achieved, in part, through regulation of antimicrobial peptide secretion. The aim of this study was to determine the efficiency by which members of the intestinal microbiota induce the antimicrobial peptide REGIII and to elucidate the underlying pathways. We showed that germfree mice have low levels of REGIII-γ in their ileum and colon compared to mice with different intestinal microbiota backgrounds. Colonization with a microbiota of low diversity (altered Schaedler flora) did not induce the expression of REGIII-γ as effectively as a complex community (specific pathogen free). Monocolonization with the probiotic Bifidobacterium breve, but not with the nonprobiotic commensal Escherichia coli JM83, upregulated REGIII-γ expression. Induction of REGIII-γ by B. breve was abrogated in mice lacking MyD88 and Ticam1 signaling. Both live and heat-inactivated B. breve but not spent culture medium from B. breve induced the expression of REGIII-α, the human ortholog and homolog of REGIII-γ, in human colonic epithelial cells (Caco-2). Taken together, the results suggest that REGIII-γ expression in the intestine correlates with the richness of microbiota composition. Also, specific bacteria such as Bifidobacterium breve NCC2950 effectively induce REGIII production in the intestine via the MyD88-Ticam1 pathway. Treatment with this probiotic may enhance the mucosal barrier and protect the host from infection and inflammation.

Proresolution effects of hydrogen sulfide during colitis are mediated through hypoxia-inducible factor-1α

During a course of colitis, production of the gaseous mediator hydrogen sulfide (H2S) is markedly up‐regulated at sites of mucosal damage and contributes significantly to healing and resolution of inflammation. The signaling mechanisms through which H2S promotes resolution of colitis are unknown. We hypothesized that the beneficial effects of H2S in experimental colitis are mediated via stabilization of hypoxia‐inducible factor (HIF)‐1α. The hapten dinitrobenzene sulfonic acid was used to induce colitis in rats and mice. This resulted in an elevated expression of the H2S‐producing enzyme, cystathionine γ‐lyase (CSE), and HIF‐1α at sites of mucosal ulceration, and the expression of these 2 enzymes followed a similar pattern throughout the course of colitis. This represented a functionally important relationship because the loss of CSE‐derived H2S production led to decreased HIF‐1α stabilization and exacerbation of colitis. Furthermore, application of an H2S‐releasing molecule, diallyl disulfide (DADS), stabilized colonic HIF‐1α expression, up‐regulated hypoxia‐responsive genes, and reduced the severity of disease during peak inflammation. Importantly, the ability of DADS to promote the resolution of colitis was abolished when coadministered with an inhibitor of HIF‐1α in vivo (PX‐478). DADS was also able to maintain HIF‐1α expression at a later point in colitis, when HIF‐1α levels would have normally returned to control levels, and to enhance resolution. Finally, we found that HIF‐1α stabilization inhibited colonic H2S production and may represent a negative feedback mechanism to prevent prolonged HIF‐1α stabilization. Our findings demonstrate an important link between H2S and HIF‐1α in the resolution of inflammation and injury during colitis and provide mechanistic insights into the therapeutic value of H2S donors.—Flannigan, K. L., Agbor, T. A., Motta, J. ‐P., Ferraz, J. G. P., Wang, R, Buret, A. G., Wallace, J. L. Proresolution effects of hydrogen sulfide during colitis are mediated through hypoxia‐inducible factor‐1α. FASEB J. 29, 1591‐1602 (2015). www.fasebj.org

Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn’s disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain Giardia infections may attenuate PMN accumulation by decreasing the expression of the mediators responsible for their recruitment.

Hydrogen sulphide protects against NSAID-enteropathy through modulation of bile and the microbiota.

Hydrogen sulphide is an important mediator of gastrointestinal mucosal defence. The use of non‐steroidal anti‐inflammatory drugs (NSAIDs) is significantly limited by their toxicity in the gastrointestinal tract. Particularly concerning is the lack of effective preventative or curative treatments for NSAID‐induced intestinal damage and bleeding. We evaluated the ability of a hydrogen sulphide donor to protect against NSAID‐induced enteropathy.

Giardia duodenalis induces pathogenic dysbiosis of human intestinal microbiota biofilms

Giardia duodenalis is a prevalent cause of acute diarrheal disease worldwide. However, recent outbreaks in Italy and Norway have revealed a link between giardiasis and the subsequent development of chronic post-infectious irritable bowel syndrome. While the mechanisms underlying the causation of post-infectious irritable bowel syndrome remain obscure, recent findings suggest that alterations in gut microbiota communities are linked to the pathophysiology of irritable bowel syndrome. In the present study, we use a laboratory biofilm system to culture and enrich mucosal microbiota from human intestinal biopsies. Subsequently, we show that co-culture with Giardia induces disturbances in biofilm species composition and biofilm structure resulting in microbiota communities that are intrinsically dysbiotic - even after the clearance of Giardia. These microbiota abnormalities were mediated in part by secretory-excretory Giardia cysteine proteases. Using in vitro cell culture and germ-free murine infection models, we show that Giardia-induced disruptions of microbiota promote bacterial invasion, resulting in epithelial apoptosis, tight junctional disruption, and bacterial translocation across an intestinal epithelial barrier. Additionally, these dysbiotic microbiota communities resulted in increased activation of the Toll-like receptor 4 signalling pathway, and overproduction of the pro-inflammatory cytokine IL-1beta in humanized germ-free mice. Previous studies that have sought explanations and risk factors for the development of post-infectious irritable bowel syndrome have focused on features of enteropathogens and attributes of the infected host. We propose that polymicrobial interactions involving Giardia and gut microbiota may cause persistent dysbiosis, offering a new interpretation of the reasons why those afflicted with giardiasis are predisposed to gastrointestinal disorders post-infection.

Giardia duodenalis induces paracellular bacterial translocation and causes postinfectious visceral hypersensitivity.

Irritable bowel syndrome (IBS) is the most frequent functional gastrointestinal disorder. It is characterized by abdominal hypersensitivity, leading to discomfort and pain, as well as altered bowel habits. While it is common for IBS to develop following the resolution of infectious gastroenteritis [then termed postinfectious IBS (PI-IBS)], the mechanisms remain incompletely understood. Giardia duodenalis is a cosmopolitan water-borne enteropathogen that causes intestinal malabsorption, diarrhea, and postinfectious complications. Cause-and-effect studies using a human enteropathogen to help investigate the mechanisms of PI-IBS are sorely lacking. In an attempt to establish causality between giardiasis and postinfectious visceral hypersensitivity, this study describes a new model of PI-IBS in neonatal rats infected with G. duodenalis At 50 days postinfection with G. duodenalis (assemblage A or B), long after the parasite was cleared, rats developed visceral hypersensitivity to luminal balloon distension in the jejunum and rectum, activation of the nociceptive signaling pathway (increased c-fos expression), histological modifications (villus atrophy and crypt hyperplasia), and proliferation of mucosal intraepithelial lymphocytes and mast cells in the jejunum, but not in the rectum. G. duodenalis infection also disrupted the intestinal barrier, in vivo and in vitro, which in turn promoted the translocation of commensal bacteria. Giardia-induced bacterial paracellular translocation in vitro correlated with degradation of the tight junction proteins occludin and claudin-4. The extensive observations associated with gut hypersensitivity described here demonstrate that, indeed, in this new model of postgiardiasis IBS, alterations to the gut mucosa and c-fos are consistent with those associated with PI-IBS and, hence, offer avenues for new mechanistic research in the field.

Giardia duodenalis: New Research Developments in Pathophysiology, Pathogenesis, and Virulence Factors

Giardia duodenalis is a very common, ubiquitous, intestinal protozoan parasite infecting animals and humans. Of the eight distinct genetic assemblages known to date, assemblages A and B are infectious to humans. Giardia is the most commonly recognized cause of traveller’s diarrhea. Giardiasis impairs weight gain and is responsible for a variety of extra-intestinal and post-infectious complications, including post-infectious irritable bowel syndrome, chronic fatigue, failure to thrive, and cognitive impairment. Giardiasis occurs in the absence of invasion of the intestinal tissues by the trophozoites and in the absence of any overt inflammatory cell infiltration, with the exception of a modest increase in intraepithelial lymphocytes and mast cells. In endemic parts of the World where the infection is often concurrent with bacterial enteritis causing inflammation-driven diarrheal disease, giardiasis appears to be protective against diarrhea. Recent observations have demonstrated that this effect may be due to a direct immuno-modulating effect of the parasite via its cathepsin B cysteine protease which cleaves pro-inflammatory CXCL8. No known toxin has yet been directly implicated in the pathophysiology of giardiasis. Diarrhea in giardiasis is mostly malabsorptive in nature, rather than hypersecretory. Findings from ongoing research indicate that the post-infectious effects of giardiasis may be due to microbiota dysbiosis induced by the parasite during the acute phase of infection.

Interactions of Giardia sp. with the intestinal barrier: Epithelium, mucus, and microbiota

ABSTRACT Understanding how intestinal enteropathogens cause acute and chronic alterations has direct animal and human health perspectives. Significant advances have been made on this field by studies focusing on the dynamic crosstalk between the intestinal protozoan parasite model Giardia duodenalis and the host intestinal mucosa. The concept of intestinal barrier function is of the highest importance in the context of many gastrointestinal diseases such as infectious enteritis, inflammatory bowel disease, and post-infectious gastrointestinal disorders. This crucial function relies on 3 biotic and abiotic components, first the commensal microbiota organized as a biofilm, then an overlaying mucus layer, and finally the tightly structured intestinal epithelium. Herein we review multiple strategies used by Giardia parasite to circumvent these 3 components. We will summarize what is known and discuss preliminary observations suggesting how such enteropathogen directly and/ or indirectly impairs commensal microbiota biofilm architecture, disrupts mucus layer and damages host epithelium physiology and survival.

Giardia co-infection promotes the secretion of antimicrobial peptides beta-defensin 2 and trefoil factor 3 and attenuates attaching and effacing bacteria-induced intestinal disease

Our understanding of polymicrobial gastrointestinal infections and their effects on host biology remains incompletely understood. Giardia duodenalis is an ubiquitous intestinal protozoan parasite infecting animals and humans. Concomitant infections with Giardia and other gastrointestinal pathogens commonly occur. In countries with poor sanitation, Giardia infection has been associated with decreased incidence of diarrheal disease and fever, and reduced serum inflammatory markers release, via mechanisms that remain obscure. This study analyzed Giardia spp. co-infections with attaching and effacing (A/E) pathogens, and assessed whether and how the presence of Giardia modulates host responses to A/E enteropathogens, and alters intestinal disease outcome. In mice infected with the A/E pathogen Citrobacter rodentium, co-infection with Giardia muris significantly attenuated weight loss, macro- and microscopic signs of colitis, bacterial colonization and translocation, while concurrently enhancing the production and secretion of antimicrobial peptides (AMPs) mouse β-defensin 3 and trefoil factor 3 (TFF3). Co-infection of human intestinal epithelial cells (Caco-2) monolayers with G. duodenalis trophozoites and enteropathogenic Escherichia coli (EPEC) enhanced the production of the AMPs human β-defensin 2 (HBD-2) and TFF3; this effect was inhibited with treatment of G. duodenalis with cysteine protease inhibitors. Collectively, these results suggest that Giardia infections are capable of reducing enteropathogen-induced colitis while increasing production of host AMPs. Additional studies also demonstrated that Giardia was able to directly inhibit the growth of pathogenic bacteria. These results reveal novel mechanisms whereby Giardia may protect against gastrointestinal disease induced by a co-infecting A/E enteropathogen. Our findings shed new light on how microbial-microbial interactions in the gut may protect a host during concomitant infections.