Myriam Tanguy

A transcriptional network in polycystic kidney disease

Mutations in cystic kidney disease genes represent a major genetic cause of end‐stage renal disease. However, the molecular cascades controlling the expression of these genes are still poorly understood. Hepatocyte Nuclear Factor 1β (HNF1β) is a homeoprotein predominantly expressed in renal, pancreatic and hepatic epithelia. We report here that mice with renal‐specific inactivation of HNF1β develop polycystic kidney disease. We show that renal cyst formation is accompanied by a drastic defect in the transcriptional activation of Umod, Pkhd1 and Pkd2 genes, whose mutations are responsible for distinct cystic kidney syndromes. In vivo chromatin immunoprecipitation experiments demonstrated that HNF1β binds to several DNA elements in murine Umod, Pkhd1, Pkd2 and Tg737/Polaris genomic sequences. Our results uncover a direct transcriptional hierarchy between HNF1β and cystic disease genes. Interestingly, most of the identified HNF1β target gene products colocalize to the primary cilium, a crucial organelle that plays an important role in controlling the proliferation of tubular cells. This may explain the increased proliferation of cystic cells in MODY5 patients carrying autosomal dominant mutations in HNF1β.

Modulation of the host immune response by a transient intracellular stage of Mycobacterium ulcerans: the contribution of endogenous mycolactone toxin

Mycobacterium ulcerans (Mu), the aetiological agent of Buruli ulcer, is an extracellular pathogen producing the macrolide toxin mycolactone. Using a mouse model of intradermal infection, we found that Mu was initially captured by phagocytes and transported to draining lymph nodes (DLN) within host cells. Similar to Buruli ulcers in humans, the infection site eventually became ulcerated with tissue necrosis and extracellular bacteria, at later stages. In contrast to Mycobacterium bovis BCG (BCG), Mu did not disseminate to the spleen. However, mice infected with Mu or BCG developed comparable primary cellular responses to mycobacterial antigens in DLN and spleen. The role of mycolactone in this sequence of events was examined with a mycolactone‐deficient (mup045) mutant of Mu. Mup045 bacilli were better internalized than wild‐type (wt) bacteria by mouse phagocytes in vitro. Moreover, infection with wt but not mup045 Mu led to inhibition of TNF‐α expression, upregulation of MIP‐2 chemokine, and host cell death within 1 day. Our results suggest that mycolactone expression during the intracellular life of Mu may contribute to immune evasion by inhibiting phagocytosis, provoking apoptosis of antigen presenting cells and altering the establishment of an appropriate inflammatory reaction.

Secretory IgA-mediated neutralization of Shigella flexneri prevents intestinal tissue destruction by down-regulating inflammatory circuits.

Shigella, a Gram-negative invasive enteropathogenic bacterium responsible for bacillary dysentery, causes the rupture, invasion, and inflammatory destruction of the human colonic mucosa. We explored the mechanisms of protection mediated by Shigella LPS-specific secretory IgA (SIgA), the major mucosal Ab induced upon natural infection. Bacteria, SIgA, or SIgA-S. flexneri immune complexes were administered into rabbit ligated intestinal loops containing a Peyer’s patch. After 8 h, localizations of bacteria, SIgA, and SIgA-S. flexneri immune complexes were examined by immunohistochemistry and confocal microscopy imaging. We found that anti-Shigella LPS SIgA, mainly via immune exclusion, prevented Shigella-induced inflammation responsible for the destruction of the intestinal barrier. Besides this luminal trapping, a small proportion of SIgA-S. flexneri immune complexes were shown to enter the rabbit Peyer’s patch and were internalized by dendritic cells of the subepithelial dome region. Local inflammatory status was analyzed by quantitative RT-PCR using newly designed primers for rabbit pro- and anti-inflammatory mediator genes. In Peyer’s patches exposed to immune complexes, limited up-regulation of the expression of proinflammatory genes, including TNF-α, IL-6, Cox-2, and IFN-γ, was observed, consistent with preserved morphology. In contrast, in Peyer’s patches exposed to Shigella alone, high expression of the same mediators was measured, indicating that neutralizing SIgA dampens the proinflammatory properties of Shigella. These results show that in the form of immune complexes, SIgA guarantees both immune exclusion and neutralization of translocated bacteria, thus preserving the intestinal barrier integrity by preventing bacterial-induced inflammation. These findings add to the multiple facets of the noninflammatory properties of SIgA.

A synthetic carbohydrate-protein conjugate vaccine candidate against Shigella flexneri 2a infection.

The protective Ag of Shigella, the Gram-negative enteroinvasive bacterium causing bacillary dysentery, or shigellosis, is its O-specific polysaccharide (O-SP) domain of the LPS, the major bacterial surface component. As an alternative to the development of detoxified LPS-based conjugate vaccines, recent effort was put into the investigation of neoglycoproteins encompassing synthetic oligosaccharides mimicking the protective Ags of the O-SP. We previously reported that when coupled to tetanus toxoid via single point attachment, a synthetic pentadecasaccharide representing three biological repeating units of the O-SP of Shigella flexneri 2a (SF2a), one of the most common Shigella serotypes, elicits a better serum anti-LPS 2a Ab response in mice than shorter synthetic O-SP sequences. In this study, we show that the pentadecasaccharide-induced anti-LPS 2a Abs protect passively administered naive mice from Shigella infection. Therefore, this three repeating units sequence, which is recognized by anti-SF2a sera from infected patients, acts as a functional mimic of the native polysaccharide Ag. Analyses of parameters influencing immunogenicity revealed that an investigational SF2a vaccine displaying a pentadecasaccharide:tetanus toxoid molar loading of 14:1 triggers a high and sustained anti-LPS Ab response, without inducing anti-linker Ab, when administered four times at a dose corresponding to 1 μg of carbohydrate. In addition, the profile of the anti-LPS Ab response, dominated by IgG1 production (Th2-type response), mimics that observed in human upon natural SF2a infection. This synthetic carbohydrate-based conjugate may be a candidate for a SF2a vaccine.

Rational design and immunogenicity of liposome-based diepitope constructs: Application to synthetic oligosaccharides mimicking the Shigella flexneri 2a O-antigen

We have designed chemically defined diepitope constructs consisting of liposomes displaying at their surface synthetic oligosaccharides mimicking the O-antigen of the Shigella flexneri 2a lipopolysaccharide (B-cell epitope) and influenza hemagglutinin peptide HA 307-319 (Th epitope). Using well controlled and high-yielding covalent bioconjugation reactions, the two structurally independent epitopes were coupled to the lipopeptide Pam(3)CAG, i.e. a TLR2 ligand known for its adjuvant properties, anchored in preformed vesicles. The synthetic construct containing a pentadecasaccharide corresponding to three O-antigen repeating units triggered T-dependent anti-oligosaccharide and anti-S. flexneri 2a LPS antibody responses when administered i.m. to BALB/c mice. Moreover, the long-lasting anti-LPS antibody response afforded protection against a S. flexneri 2a challenge. These results show that liposome diepitope constructs could be attractive alternatives in the development of synthetic carbohydrate-based vaccines.