Elisabeth Billard

Western diet induces dysbiosis with increased E coli in CEABAC10 mice, alters host barrier function favouring AIEC colonisation

Objective Western diet is a risk factor for Crohns disease (CD). Carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6) is abnormally expressed in CD patients. This allows adherent-invasive Escherichia coli (AIEC) to colonise the gut mucosa and leads to inflammation. We assessed the effects of a high fat/high sugar (HF/HS) Western diet on gut microbiota composition, barrier integrity and susceptibility to infection in transgenic CEABAC10 mice expressing human CEACAMs. Design Colonic microbiota composition and susceptibility of CEABAC10 mice to AIEC LF82 bacteria infection were determined in mice fed a conventional or HF/HS diet. Barrier function and inflammatory response were assessed by studying intestinal permeability, tight junction protein and mucin expression and localisation, and by determining histological score and levels of cytokine release. Results HF/HS diet led to dysbiosis in WT and transgenic CEABAC10 mice, with a particular increase in E coli population in HF/HS-fed CEABAC10 mice. These mice showed decreased mucus layer thickness, increased intestinal permeability, induction of Nod2 and Tlr5 gene transcription, and increased TNFα secretion. These modifications led to a higher ability of AIEC bacteria to colonise the gut mucosa and to induce inflammation. Conclusions Western diet induces changes in gut microbiota composition, alters host homeostasis and promotes AIEC gut colonisation in genetically susceptible mice. These results support the multifactorial aetiology of CD and highlight the importance of diet in CD pathogenesis.

HIV-1 assembly in macrophages

The molecular mechanisms involved in the assembly of newly synthesized Human Immunodeficiency Virus (HIV) particles are poorly understood. Most of the work on HIV-1 assembly has been performed in T cells in which viral particle budding and assembly take place at the plasma membrane. In contrast, few studies have been performed on macrophages, the other major target of HIV-1. Infected macrophages represent a viral reservoir and probably play a key role in HIV-1 physiopathology. Indeed macrophages retain infectious particles for long periods of time, keeping them protected from anti-viral immune response or drug treatments. Here, we present an overview of what is known about HIV-1 assembly in macrophages as compared to T lymphocytes or cell lines.Early electron microscopy studies suggested that viral assembly takes place at the limiting membrane of an intracellular compartment in macrophages and not at the plasma membrane as in T cells. This was first considered as a late endosomal compartment in which viral budding seems to be similar to the process of vesicle release into multi-vesicular bodies. This view was notably supported by a large body of evidence involving the ESCRT (Endosomal Sorting Complex Required for Transport) machinery in HIV-1 budding, the observation of viral budding profiles in such compartments by immuno-electron microscopy, and the presence of late endosomal markers associated with macrophage-derived virions. However, this model needs to be revisited as recent data indicate that the viral compartment has a neutral pH and can be connected to the plasma membrane via very thin micro-channels. To date, the exact nature and biogenesis of the HIV assembly compartment in macrophages remains elusive. Many cellular proteins potentially involved in the late phases of HIV-1 cycle have been identified; and, recently, the list has grown rapidly with the publication of four independent genome-wide screens. However, their respective roles in infected cells and especially in macrophages remain to be characterized. In summary, the complete process of HIV-1 assembly is still poorly understood and will undoubtedly benefit from the ongoing explosion of new imaging techniques allowing better time-lapse and quantitative studies.

Western diet induces a shift in microbiota composition enhancing susceptibility to Adherent-Invasive E. coli infection and intestinal inflammation.

Recent advances have shown that the abnormal inflammatory response observed in CD involves an interplay among intestinal microbiota, host genetics and environmental factors. The escalating consumption of fat and sugar in Western countries parallels an increased incidence of CD during the latter 20th century. The impact of a HF/HS diet in mice was evaluated for the gut micro-inflammation, intestinal microbiota composition, function and selection of an E. coli population. The HF/HS diet created a specific inflammatory environment in the gut, correlated with intestinal mucosa dysbiosis characterized by an overgrowth of pro-inflammatory Proteobacteria such as E. coli, a decrease in protective bacteria, and a significantly decreased of SCFA concentrations. The expression of GPR43, a SCFA receptor was reduced in mice treated with a HF/HS diet and reduced in CD patients compared with controls. Interestingly, mice treated with an agonist of GPR43 were protected against DSS-induced colitis. Finally, the transplantation of feces from HF/HS treated mice to GF mice increased susceptibility to AIEC infection. Together, our results demonstrate that a Western diet could aggravate the inflammatory process and that the activation of the GPR43 receptor pathway could be used as a new strategy to treat CD patients.

Brucella suis Prevents Human Dendritic Cell Maturation and Antigen Presentation through Regulation of Tumor Necrosis Factor Alpha Secretion

ABSTRACT Brucella is a facultative intracellular pathogen and the etiological agent of brucellosis. In some cases, human brucellosis results in a persistent infection that may reactivate years after the initial exposure. The mechanisms by which the parasite evades clearance by the immune response to chronically infect its host are unknown. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection and are a preferential niche for the development of the bacteria. Here, we report that in contrast to several intracellular bacteria, Brucella prevented the infected DCs from engaging in their maturation process and impaired their capacities to present antigen to naïve T cells and to secrete interleukin-12. Moreover, Brucella-infected DCs failed to release tumor necrosis factor alpha (TNF-α), a defect involving the bacterial protein Omp25. Exogenous TNF-α addition to Brucella-infected DCs restored cell maturation and allowed them to present antigens. Two avirulent mutants of B. suis, B. suis bvrR and B. suis omp25 mutants, which do not express the Omp25 protein, triggered TNF-α production upon DC invasion. Cells infected with these mutants subsequently matured and acquired the ability to present antigens, two properties which were dramatically impaired by addition of anti-TNF-α antibodies. In light of these data, we propose a model in which virulent Brucella alters the maturation and functions of DCs through Omp25-dependent control of TNF-α production. This model defines a specific evasion strategy of the bacteria by which they can escape the immune response to chronically infect their host.

Replication of Crohn's disease-associated AIEC within macrophages is dependent on TNF- α secretion

Adherent and invasive Escherichia coli (AIEC) associated with Crohns disease are able to survive and to replicate extensively in active phagolysosomes within macrophages. AIEC-infected macrophages release large amounts of tumour necrosis factor-alpha (TNF-α) and do not undergo cell death. The aim of the present study was to determine what benefit AIEC bacteria could gain from inducing the release of large amounts of TNF-α by infected macrophages and to what extent the neutralization of TNF-α could affect AIEC intramacrophagic replication. Our results showed that the amount of TNF-α released by infected macrophages is correlated with the load of intramacrophagic AIEC bacteria and their intracellular replication. TNF-α secretion was not related to the number of bacteria entering host cells because when the number of bacteria internalized in macrophage was decreased by blocking lipid raft-dependent and clathrin-coated pits-dependent endocytosis, the amount of TNF-α secreted by infected macrophages was not modified. Interestingly, dose-dependent increases in the number of intracellular AIEC LF82 bacteria were observed when infected macrophages were stimulated with exogenous TNF-α, and neutralization of TNF-α secreted by AIEC-infected macrophages using anti-TNF-α antibodies induced a significant decrease in the number of intramacrophagic bacteria. These results indicate that AIEC bacteria use TNF-α as a Trojan horse to ensure their intracellular replication because replication of AIEC bacteria within macrophages induces the release of TNF-α, which in turn increases the intramacrophagic replication of AIEC. Neutralizing TNF-α secreted by infected macrophages may represent an effective strategy to control AIEC intracellular replication.

Interaction of Brucella suis and Brucella abortus Rough Strains with Human Dendritic Cells

ABSTRACT Brucella is a facultative intracellular pathogen of various mammals and the etiological agent of brucellosis. We recently demonstrated that dendritic cells (DCs), which are critical components of adaptive immunity, are highly susceptible to Brucella infection. Furthermore, Brucella prevented the infected DCs from engaging in maturation processes and impaired their capacity to present antigen to naive T cells and to secrete interleukin-12 (IL-12). The lipopolysaccharide (LPS) phenotype is largely associated with the virulence of Brucella. Depending on whether they express the O-side chain of LPS or not, the bacteria display a smooth or rough phenotype. Rough Brucella mutants are attenuated and induce a potent protective T-cell-dependent immune response. Due to the essential role of DCs in the initiation of T-cell-dependent adaptive immune responses, it seemed pertinent to study the interaction between rough Brucella strains and human DCs. In the present paper, we report that, in contrast to smooth bacteria, infection of DCs with rough mutants of Brucella suis or Brucella abortus leads to both phenotypic and functional maturation of infected cells. Rough mutant-infected DCs then acquire the capacity to produce IL-12 and to stimulate naive CD4+ T lymphocytes. Experiments with rough and smooth purified LPS of Brucella supported the hypothesis of an indirect involvement of the O-side chain. These results provide new data concerning the role of LPS in Brucella virulence strategy and illuminate phenomena contributing to immune protection conferred by rough vaccine strains.

Understanding Host-Adherent-Invasive Escherichia coli Interaction in Crohn’s Disease: Opening Up New Therapeutic Strategies

A trillion of microorganisms colonize the mammalian intestine. Most of them have coevolved with the host in a symbiotic relationship and some of them have developed strategies to promote their replication in the presence of competing microbiota. Recent evidence suggests that perturbation of the microbial community favors the emergence of opportunistic pathogens, in particular adherent-invasive Escherichia coli (AIEC) that can increase incidence and severity of gut inflammation in the context of Crohns disease (CD). This review will report the importance of AIEC as triggers of intestinal inflammation, focusing on their impact on epithelial barrier function and stimulation of mucosal inflammation. Beyond manipulation of immune response, restoration of gut microbiota as a new treatment option for CD patients will be discussed.

GipA Factor Supports Colonization of Peyer's Patches by Crohn's Disease-associated Escherichia Coli.

Background:Adherent-invasive Escherichia coli (AIEC) associated with Crohns disease target M cells lining Peyers patches (PPs) through the expression of long polar fimbriae (LPF) and survive macrophage killing. Invasion of PPs constitutes a way to colonize the mucosa for bacteria able to escape or resist killing of underlying immune cells. We aimed to identify new virulence factors involved in PPs colonization by AIEC. Methods:The presence of gipA (Growth in PPs) gene was determined by polymerase chain reaction. In vivo experiments were performed using CEABAC10 transgenic mice. Intramacrophagic behavior of AIEC was assessed in murine bone marrow-derived macrophages and human monocyte-derived macrophages. Cytokines production was quantified by ELISA. Results:A higher prevalence of gipA-positive E. coli was observed in patients with Crohns disease (27.3%) compared with controls (17.2%). Unlike non-AIEC strains, all gipA-positive AIEC strains also harbored lpfA. GipA deletion impaired AIEC translocation across M cells and their replication inside macrophages. GipA expression was induced by gastrointestinal (bile salts) and phagolysosomal (reactive oxygen species and acid pH) conditions. GipA deletion decreased lpfA mRNA level in AIEC bacteria. Survival of AIEC-&Dgr;gipA bacteria was reduced in medium containing H2O2 or acidic pH. GipA deletion impaired AIEC colonization of PPs and dissemination to mesenteric lymph nodes in mice. Conclusions:GipA is required for optimal colonization of mouse PPs and survival within macrophages by AIEC, suggesting that this factor plays a role in AIEC promotion of Crohns disease. Detection of gipA and lpfA could be a predictor for the presence of AIEC.

Microbial markers in colorectal cancer detection and/or prognosis

Colorectal cancer (CRC) is the second leading cause of cancer worldwide. CRC is still associated with a poor prognosis among patients with advanced disease. On the contrary, due to its slow progression from detectable precancerous lesions, the prognosis for patients with early stages of CRC is encouraging. While most robust methods are invasive and costly, actual patient-friendly screening methods for CRC suffer of lack of sensitivity and specificity. Therefore, the development of sensitive, non-invasive and cost-effective methods for CRC detection and prognosis are necessary for increasing the chances of a cure. Beyond its beneficial functions for the host, increasing evidence suggests that the intestinal microbiota is a key factor associated with carcinogenesis. Many clinical studies have reported a disruption in the gut microbiota balance and an alteration in the faecal metabolome of CRC patients, suggesting the potential use of a microbial-based test as a non-invasive diagnostic and/or prognostic tool for CRC screening. This review aims to discuss the microbial signatures associated with CRC known to date, including dysbiosis and faecal metabolome alterations, and the potential use of microbial variation markers for non-invasive early diagnosis and/or prognostic assessment of CRC and advanced adenomas. We will finally discuss the possible use of these markers as predicators for treatment response and their limitations.

Unveiling and Characterizing Early Bilateral Interactions between Biofilm and the Mouse Innate Immune System

A very substantial progress has been made in our understanding of infectious diseases caused by invasive bacteria. Under their planktonic forms, bacteria transiently reside in the otherwise sterile mammal body tissues, as the physiological inflammation insures both their clearance and repair of any tissue damage. Yet, the bacteria prone to experience planktonic to biofilm developmental transition still need to be studied. Of note, sessile bacteria not only persist but also concur preventing the effectors and regulators of the physiological inflammation to operate. Thus, it is urgent to design biologically sound experimental approaches aimed to extract, at the earliest stage, immune signatures of mono-bacteria planktonic to biofilm developmental transition in vivo and ex vivo. Indeed, the transition is often the first event to which succeeds the “chronicization” process whereby classical bacteria-targeting therapies are no more efficacious. An in vivo model of micro-injection of Staphylococcus aureus planktonic or biofilm cells in the ear pinna dermis of laboratory transgenic mice with fluorescent immune cells is proposed. It allows visualizing, in real time, the range of the early interactions between the S. aureus and myeloid cell subsets- the resident macrophages and dendritic cells, the recruited neutrophil granulocytes/polymorphonuclear neutrophils, monocytes otherwise known to differentiate as macrophages or dendritic cells. One main objective is to extract contrasting immune signatures of the modulation of the physiological inflammation with respect to the two bacterial lifestyles.