Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) α secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor κB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

International audienceExopolysaccharides (EPS) from Lactobacillus rhamnosus RW- 9595M have been prepared from bacterial cultures, isolated, concentrated, fractionated and tested in vitro for their possible modulating properties on mouse splenocytes from the C57Bl/6 and BALB/c strains, on the murine RAW 264.7 macrophage-like cell line and on human Peripheral Blood Mononuclear Cells (PBMC) from a total of 14 healthy donors. A first step of EPS fractionation was attempted, using membranes with different molecular weight cut-off. Fractions were as follows: F1: $> $1000 kg$\cdot$mol$^{-1}$; F2: 1000-100 kg$\cdot$mol$^{-1}$; F3: 100-10 kg$\cdot$mol$^{-1}$; F4: $< $10 kg$\cdot$mol$^{-1}$. Total EPS, as well as F1, appeared slightly mitogenic in both mouse splenocytes and human PBMC in 2-3 d cultures, and F3 also exhibited such a property on human PBMC. Unfractionated concentrated ("total" ) EPS, as well as F1, elicited TNF, IL-6 and IL-12 p40 both in the mouse and human cells, in 6 h and 24 h cultures, with important variability depending on the cell source. In 24 h cultures, total EPS or F1 elicited bio-active IFN-$\gamma$ in both C57Bl/6 and BALB/c splenocytes, and this IFN-$\gamma$ secretion was sustained until at least 3 d of culture. In human PBMC, no IFN-$\gamma$ production was observed despite high IL-12p40 secretion. These results suggest the possibility of enhancing the immune system through EPS from lactic acid bacteria, in individuals responsive to such a stimulus.Productions de TNF, IL-6, IL-12 et IFN-g chez des cellules immunocompétentes traitées avec des exopolysaccharides du Lactobacillus rhamnosus RW-9595M. Différence entre les réponses de cellules de sang périphérique humain et de splénocytes de souris. Les exopolysaccharides (EPS) du Lactobacillus rhamnosus RW- 9595M ont été préparés à partir de cultures bactériennes, isolés, concentrés, fractionnés et testés in vitro quant à leur potentiel immunomodulateur sur des splénocytes de souris C57Bl/6 et BALB/c, sur la lignée macrophagique murine RAW.264.7 et sur des cellules mononuclées du sang humain provenant de 14 donneurs. Nous avons fractionné ces EPS en fonction des tailles moléculaires, avec des membranes filtrantes de différentes porosités. Les fractions correspondent aux tailles suivantes : F1 : $> $ 1000 kg$\cdot$mol$^{-1}$, F2 : 1000-100 kg$\cdot$mol$^{-1}$, F3 : 100-10 kg$\cdot$mol$^{-1}$ et F4 : $< $ 10 kg$\cdot$mol$^{-1}$. Les EPS concentrés, non fractionnés, ainsi que F1, se sont avérés légèrement mitogènes sur des splénocytes de la souris, ou des cellules sanguines humaines, cultivées 2-3 jours. Dans le dernier cas, F3 manifeste également cette propriété. Les EPS non fractionnés, ainsi que F1, sont capables de provoquer la production de TNF, IL-6, IL-12p40 après 6 ou 24 h de culture, l'origine des cellules étant importante pour l'intensité de la réponse. Après 24, 48 et 72 h de culture, les EPS non fractionnés, ainsi que F1, ont provoqué la production d'interféron gamma par des splénocytes de souris C57Bl/6 et BALB/c. Au contraire, dans le ca...

We investigated the ability of compounds interfering with iron metabolism to inhibit the growth of Acinetobacter baumannii. Iron restriction with transferrin or 2,2-bipyridyl significantly inhibited A. baumannii growth in vitro. Gallium nitrate alone was moderately effective at reducing A. baumannii growth but became bacteriostatic in the presence of serum or transferrin. More importantly, gallium nitrate treatment reduced lung bacterial burdens in mice. The use of gallium-based therapies shows promise for the control of multidrug-resistant A. baumannii. Acinetobacter baumannii has emerged as a major cause of both community-associated and nosocomial infections worldwide. Infections have become increasingly difficult to treat because of the rapid development of A. baumannii antibiotic resistance. Counteracting the action of virulence factors represents a novel strategy of infection control with potentially high specificity and low impact on the host. Hence, identification of the critical factors necessary for the in vivo success of the pathogen might reveal novel therapeutic targets.One characteristic shared by virulent bacteria is their ability to acquire iron in the blood and tissues, where its availability is low. Conversely, several host factors exist whose role is to restrict iron and form a nutritional barrier (reviewed in reference 9). Pathogen iron acquisition could be further disrupted by using biologically compatible chelators (6-8) or by introducing gallium as a competitor (1).To examine the biological activity of iron-restricting compounds against A. baumannii, we first investigated the effect of a selection of iron chelators on A. baumannii ATCC 17978 growth in different media. 2,2-Bipyridyl (BiP) is a classical iron chelator and was chosen as a positive control for both metal depletion and growth inhibition of many organisms. Pyridoxal isonicotinyl hydrazone (PIH) is a potent cell-permeable chela-

The chromosomal region 10p13 has been linked to paucibacillary leprosy in two independent studies. The MRC1 gene, encoding the human mannose receptor (MR), is located in the 10p13 region and non-synonymous SNPs in exon 7 of the gene have been suggested as leprosy susceptibility factors. We determined that G396S is the only non-synonymous exon 7-encoded polymorphism in 396 unrelated Vietnamese subjects. This SNP was genotyped in 490 simplex and 90 multiplex leprosy families comprising 704 patients (47% paucibacillary; 53% multibacillary). We observed significant under-transmission of the serine allele of the G396S polymorphism with leprosy per se (P = 0.036) and multibacillary leprosy (P = 0.034). In a sample of 384 Brazilian leprosy cases (51% paucibacillary; 49% multibacillary) and 399 healthy controls, we observed significant association of the glycine allele of the G396S polymorphism with leprosy per se (P = 0.016) and multibacillary leprosy (P = 0.023). In addition, we observed a significant association of exon 7 encoded amino acid haplotypes with leprosy per se (P = 0.012) and multibacillary leprosy (P = 0.004). Next, we tested HEK293 cells over-expressing MR constructs (293-MR) with three exon 7 haplotypes of MRC1 for their ability to bind and internalize ovalbumin and zymosan, two classical MR ligands. No difference in uptake was measured between the variants. In addition, 293-MR failed to bind and internalize viable Mycobacterium leprae and BCG. We propose that the MR–M. leprae interaction is modulated by an accessory host molecule of unknown identity.

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Leprosy is a persistent infectious disease caused by Mycobacterium leprae that still affects over 200,000 new patients annually. The host genetic background is an important risk factor for leprosy susceptibility and the PARK2 gene is a replicated leprosy susceptibility candidate gene. The protein product of PARK2, Parkin, is an E3 ubiquitin ligase that is involved in the development of various forms of Parkinsonism. The human macrophage is both a natural host cell of M. leprae as well as a primary mediator of natural immune defenses, in part by secreting important pro-inflammatory cytokines and chemokines. Here, we report that down-regulation of Parkin in THP-1 macrophages, human monocyte-derived macrophages and human Schwann cells resulted in a consistent and specific decrease in interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1/CCL2) production in response to mycobacteria or LPS. Interestingly, production of IL-6 at 6 hours by THP-1 cells stimulated with live M. leprae and M. bovis BCG was dependent on pretreatment with 1,25-dihydroxyvitamin D3 (VD). Parkin knockdown in VD-treated cells blocked IL-6 induction by mycobacteria. However, IκB-α phosphorylation and levels of IκB-ξ, a nuclear protein required for IL-6 expression, were not affected by Parkin silencing. Phosphorylation of MAPK ERK1/2 and p38 was unaffected by Parkin silencing while JNK activation was promoted but did not explain the altered cytokine production. In a final set of experiments we found that genetic risk factors of leprosy located in the PARK2 promoter region were significantly correlated with M. leprae sonicate triggered CCL2 and IL6 transcript levels in whole blood assays. These results associated genetically controlled changes in the production of MCP-1/CCL2 and IL-6 with known leprosy susceptibility factors.

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The orphan nuclear receptor Nur77 has been described as a mediator of apoptosis and has also been associated with growth promotion and apoptotic resistance. This study aimed at evaluating the contribution of Nur77 to different apoptotic stimuli. Nur77 overexpression in the fibroblastic cell line HEK293 promoted resistance to programmed cell death induced by death receptor engagement, DNA-damaging agents and endoplasmic reticulum stress. Nur77 overexpression led to enhanced NF-jB activity, and DNA-binding inhibitors confirmed the contribution of NF-jB to Nur77 antiapoptotic activity. Nur77 overexpression leads to NF-jBdependent induction of the antiapoptotic gene cIAP1. Paradoxically, while dominant-negative Nur77 expression sensitised cells to Fas ligand-induced cell death, it protected cells from endoplasmic reticulum stress apoptosis in a manner similar to wild-type Nur77. These results show that nuclear crosstalk between Nur77 and other transcription factors contribute to cell fate in response to different apoptosis-inducing agents.