Raphaëlle Bourdet-Sicard

Anti-inflammatory effect of Lactobacillus casei on Shigella-infected human intestinal epithelial cells.

Shigella invades the human intestinal mucosa, thus causing bacillary dysentery, an acute recto-colitis responsible for lethal complications, mostly in infants and toddlers. Conversely, commensal bacteria live in a mutualistic relationship with the intestinal mucosa that is characterized by homeostatic control of innate responses, thereby contributing to tolerance to the flora. Cross-talk established between commensals and the intestinal epithelium mediate this active process, the mechanisms of which remain largely uncharacterized. Probiotics such as Lactobacillus casei belong to a subclass of these commensals that modulate mucosal innate responses and possibly display anti-inflammatory properties. We analyzed whether L. casei could attenuate the pro-inflammatory signaling induced by Shigella flexneri after invasion of the epithelial lining. Cultured epithelial cells were infected with L. casei, followed by a challenge with S. flexneri. Using macroarray DNA chips, we observed that L. casei down-regulated the transcription of a number of genes encoding pro-inflammatory effectors such as cytokines and chemokines and adherence molecules induced by invasive S. flexneri. This resulted in an anti-inflammatory effect that appeared mediated by the inhibition of the NF-κB pathway, particularly through stabilization of I-κBα. In a time-course experiment using GeneChip hybridization analysis, the expression of many genes involved in ubiquitination and proteasome processes were modulated during L. casei treatment. Thus, L. casei has developed a sophisticated means to maintain intestinal homeostasis through a process that involves manipulation of the ubiquitin/proteasome pathway upstream of I-κBα.

Binding of the Shigella protein IpaA to vinculin induces F-actin depolymerization

Shigella flexneri, the causative agent of bacillary dysentery, enters into epithelial cells by a macropinocytic process. IpaA, a Shigella protein secreted upon cell contact, binds to the focal adhesion protein vinculin and is required for efficient bacterial uptake. IpaA was shown here to bind with high affinity to the N‐terminal residues 1–265 of vinculin. Using co‐sedimentation and solid‐phase assays, we demonstrated that binding of IpaA to vinculin strongly increases the association of vinculin with F‐actin. We also characterized a depolymerizing activity on actin filaments associated with the vinculin–IpaA complex both in vitro and in microinjected cells. We propose that the conformational change of vinculin induced by IpaA binding allows interaction of the vinculin–IpaA complex with F‐actin and subsequent depolymerization of actin filaments.

Bacterial signals and cell responses during Shigella entry into epithelial cells

Shigella invades epithelial cells by inducing cytoskeletal reorganization localized at the site of bacterial–host cell interaction. During entry, the Shigella type III secretion apparatus allows the insertion of a pore that contains the IpaB and IpaC proteins into cell membranes. Insertion of this complex is thought to allow translocation of the carboxy‐terminus moiety of IpaC, but also of other Shigella effectors, such as IpaA, into the cell cytosol. IpaC triggers actin polymerization and the formation of filopodial and lamellipodial extensions dependent on the Cdc42 and Rac GTPases. IpaA, on the other hand, binds to the focal adhesion protein vinculin and induces depolymerization of actin filaments. IpaA and the GTPase Rho are not required for actin polymerization at the site of bacterial contact with the cell membrane, but allow the transformation of the IpaC‐induced extensions into a structure that is productive for bacterial entry. Rho is required for the recruitment at entry foci of ezrin, a cytoskeletal linker required for Shigella entry, and also of the Src tyrosine kinase. The Src tyrosine kinase activity, which is required for Shigella‐induced actin polymerization, also appears to be involved in a negative regulatory loop that downregulates Rho at the site of entry.

Commensal bacteria trigger a full dendritic cell maturation program that promotes the expansion of non-Tr1 suppressor T cells

Dendritic cells (DCs) orchestrate the immune response establishing immunity versus tolerance. These two opposite functions may be dictated by DC maturation status with maturity linked to immunogenicity. DCs directly interact with trillions of noninvasive intestinal bacteria in vivo, a process that contributes to gut homeostasis. We here evaluated the maturation program elicited in human DCs by direct exposure to commensal‐related bacteria (CB) in the absence of inflammatory signals. We showed that eight gram+ and gram− CB strains up‐regulated costimulatory molecule expression in DCs and provoked a chemokine receptor switch similar to that activated by gram+ pathogens. CB strains may be classified into three groups according to DC cytokine release: high IL‐12 and low IL‐10; low IL‐12 and high IL‐10; and low IL‐12 and IL‐10. All CB‐treated DCs produced IL‐1β and IL‐6 and almost no TGF‐β. Yet, CB instructed DCs to convert naive CD4+ T cells into hyporesponsive T cells that secreted low or no IFN‐γ, IL‐10, and IL‐17 and instead, displayed suppressor function. These data demonstrate that phenotypic DC maturation combined to an appropriate cytokine profile is insufficient to warrant Th1, IL‐10‐secreting T regulatory Type 1 (Tr1), or Th17 polarization. We propose that commensal flora and as such, probiotics manipulate DCs by a yet‐unidentified pathway to enforce gut tolerance.

Lactic acid bacteria as adjuvants for sublingual allergy vaccines.

We compared immunomodulatory properties of 11 strains of lactic acid bacteria as well as their capacity to enhance sublingual immunotherapy efficacy in a murine asthma model. Two types of bacterial strains were identified, including: (i) potent inducers of IL-12p70 and IL-10 in dendritic cells, supporting IFN-gamma and IL-10 production in CD4+ T cells such as Lactobacillus helveticus; (ii) pure Th1 inducers such as L. casei. Sublingual administration in ovalbumin-sensitized mice of L. helveticus, but not L. casei, reduced airways hyperresponsiveness, bronchial inflammation and proliferation of specific T cells in cervical lymph nodes. Thus, probiotics acting as a Th1/possibly Treg, but not Th1 adjuvant, potentiate tolerance induction via the sublingual route.

Capping of actin filaments by vinculin activated by the Shigella IpaA carboxyl-terminal domain.

Shigella, the causative agent of bacillary dysentery, invades epithelial cells. Upon bacterial–cell contact, the type III bacterial effector IpaA binds to the cytoskeletal protein vinculin to promote actin reorganization required for efficient bacterial uptake. We show that the last 74 C‐terminal residues of IpaA (A559) bind to human vinculin (HV) and promotes its association with actin filaments. Polymerisation experiments demonstrated that A559 was sufficient to induce HV‐dependent partial capping of the barbed ends of actin filaments. These results suggest that IpaA regulates actin polymerisation/depolymerisation at sites of Shigella invasion by modulating the barbed end capping activity of vinculin.

Selected commensal-related bacteria and Toll-like receptor 3 agonist combinatorial codes synergistically induce interleukin-12 production by dendritic cells to trigger a T helper type 1 polarizing programme

Enteric infections remain a major health problem causing millions of deaths in developing countries. The interplay among the host intestinal epithelium, the mucosa‐associated immune system and microbiota performs an essential role in gut homeostasis and protection against infectious diseases. Dendritic cells (DCs) play a key role in orchestrating protective immunity and tolerance in the gut. The mechanisms by which DCs adapt their responses and discriminate between virulent microbes and trillions of innocuous bacteria remain ill‐defined. Here we investigated the effect of cross‐talk between commensal‐related bacteria (CB) and Toll‐like receptor (TLR) agonists on DC activation and the outcome of the in vitro T helper response. Human monocyte‐derived DCs were exposed to eight different Gram‐positive or Gram‐negative CB strains prior to activation with five different TLR agonists. The key polarizing cytokines interleukin (IL)‐12p70, IL‐10, IL‐1β and IL‐6 were quantified and the fate of naïve T‐cell differentiation was evaluated. We identified a unique combination of Lactobacillus casei and TLR3 signals that acted in synergy to selectively increase IL‐12p70 secretion. Exposure to poly(I:C) converted L. casei‐treated DCs into potent promoters of T helper type 1 (Th1) responses. We propose that DCs can integrate harmless and dangerous non‐self signals delivered by viral products, to mount robust Th1 responses. Thus, in vivo DC targeting with selective probiotics may improve strategies for the management of enteric diseases.

Diversion of cytoskeletal processes by Shigella during invasion of epithelial cells

Shigella, the causative agent of bacillar dysentery, invades colonic epithelial cells and moves intracellularly to spread from cell to cell. The processes of Shigella entry, determined by the Ipa proteins, and of actin-based motility, dependent on the IcsA/VirG protein, represent different levels of bacterial manipulation of the cell cytoskeleton.

Individual strains of Lactobacillus paracasei differentially inhibit human basophil and mouse mast cell activation.

The microbiota controls a variety of biological functions, including immunity, and alterations of the microbiota in early life are associated with a higher risk of developing allergies later in life. Several probiotic bacteria, and particularly lactic acid bacteria, were described to reduce both the induction of allergic responses and allergic manifestations. Although specific probiotic strains were used in these studies, their protective effects on allergic responses also might be common for all lactobacilli.