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Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, we identify and chemically optimize pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through inhibition of its primary RND transporter, AcrAB-TolC. Characterisation of resistant E. coli mutants and structural biology analyses indicate that the compounds bind to a unique site on the transmembrane domain of the AcrB L protomer, lined by key catalytic residues involved in proton relay. Molecular dynamics simulations suggest that the inhibitors access this binding pocket from the cytoplasm via a channel exclusively present in the AcrB L protomer. Thus, our work unveils a class of allosteric efflux-pump inhibitors that likely act by preventing the functional catalytic cycle of the RND pump. Efflux transporters of the RND family confer resistance to multiple antibiotics in Gram-negative bacteria. Here, the authors identify pyridylpiperazine-based compounds that potentiate antibiotic activity in E. coli through allosteric inhibition of its primary RND transporter.

Pollution, including polycyclic aromatic hydrocarbons (PAH), may contribute to increased prevalence of asthma. PAH can bind to the Aryl hydrocarbon Receptor (AhR), a transcription factor involved in Th17/Th22 type polarization. These cells produce IL17A and IL-22, which allow neutrophil recruitment, airway smooth muscle proliferation and tissue repair and remodeling. Increased IL-17 and IL-22 productions have been associated with asthma. We hypothesized that PAH might affect, through their effects on AhR, IL-17 and IL-22 production in allergic asthmatics. Activated peripheral blood mononuclear cells (PBMCs) from 16 nonallergic nonasthmatic (NA) and 16 intermittent allergic asthmatic (AA) subjects were incubated with PAH, and IL-17 and IL-22 productions were assessed. At baseline, activated PBMCs from AA exhibited an increased IL-17/IL-22 profile compared with NA subjects. Diesel exhaust particle (DEP)-PAH and Benzo[a]Pyrene (B[a]P) stimulation further increased IL-22 but decreased IL-17A production in both groups. The PAH-induced IL-22 levels in asthmatic patients were significantly higher than in healthy subjects. Among PBMCs, PAH-induced IL-22 expression originated principally from single IL-22- but not from IL-17- expressing CD4 T cells. The Th17 transcription factors RORA and RORC were down regulated, whereas AhR target gene CYP1A1 was upregulated. IL-22 induction by DEP-PAH was mainly dependent upon AhR whereas IL-22 induction by B[a]P was dependent upon activation of PI3K and JNK. Altogether, these data suggest that DEP-PAH and B[a]P may contribute to increased IL22 production in both healthy and asthmatic subjects through mechanisms involving both AhR -dependent and -independent pathways.

Although the heavy metals cadmium (Cd) and lead (Pb) are known environmental health concerns, their long-term impacts on gut ecology and susceptibility to gastrointestinal autoimmune diseases have not been extensively investigated. We sought to determine whether subchronic oral exposure to Cd or Pb is a risk factor for the development and progression of inflammatory bowel disease (IBD). Mice were exposed to various doses of CdCl 2 or PbCl 2 in drinking water for 1, 4 or 6 weeks prior to infection with Salmonella , the induction of colitis with dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS). In human cell-based models, exposure to Cd and Pb is associated with reduced transepithelial electric resistance and changes in bacteria-induced cytokine responses. Although 1- and 6-week exposures did not have clear effects on the response to Salmonella infectious challenges, 1-week short-term treatments with CdCl 2 tended to enhance intestinal inflammation in mice. Unexpectedly, subchronic exposure to Cd and (to a lesser extent) Pb significantly mitigated some of the symptoms of DSS-induced colitis and reduced the severity of TNBS colitis in a dose-dependent manner. The possible adaptive and immunosuppressive mechanisms by which heavy metals might reduce intestinal inflammation are explored and discussed.

The beneficial effects of carbohydrate-derived fibers are mainly attributed to modulation of the microbiota, increased colonic fermentation, and the production of short-chain fatty acids. We studied the direct immune responses to alimentary fibers in in vitro and in vivo models. Firstly, we evaluated the immunomodulation induced by nine different types of low-digestible fibers on human peripheral blood mononuclear cells. None of the fibers tested induced cytokine production in baseline conditions. However, only one from all fibers almost completely inhibited the production of anti- and proinflammatory cytokines induced by bacteria. Secondly, the impact of short- (five days) and long-term (three weeks) oral treatments with selected fibers was assessed in the trinitrobenzene-sulfonic acid colitis model in mice. The immunosuppressive fiber significantly reduced levels of inflammatory markers over both treatment periods, whereas a nonimmunomodulatory fiber had no effect. The two fibers did not differ in terms of the observed fermentation products and colonic microbiota after three weeks of treatment, suggesting that the anti-inflammatory action was not related to prebiotic properties. Hence, we observed a direct effect of a specific fiber on the murine immune system. This intrinsic, fiber-dependent immunomodulatory potential may extend prebiotic-mediated protection in inflammatory bowel disease.

An original immuno-regulatory strategy against inflammatory bowel diseases based on the use of 28 kDa glutathione S-transferase (P28GST), a unique schistosome protein, was recently proposed. Improvement of intestinal inflammation occurs through restoration of the immunological balance between pro-inflammatory T-helper 1 (Th1) responses and both T-helper 2 (Th2) and regulatory responses. However, detailed mechanisms explaining how P28GST prevents colitis and promotes gut homeostasis remain unknown. Considering the complex interplay between the adaptive and innate immune system and the intestinal microbiota, we raised the question of the possible role of the microbial ecosystem in the anti-inflammatory effects mediated by the helminth-derived P28GST protein. We first analyzed, by 16S rRNA sequencing, the bacterial profiles of mice fecal microbiota at several time points of the P28GST-immunomodulation period prior to trinitrobenzene sulfonic acid (TNBS)-colitis. The influence of gut microbiota in the P28GST-mediated anti-inflammatory effects was then assessed by fecal microbiota transplantation experiments from P28GST-immunized mice to either conventional or microbiota depleted naïve recipient mice. Finally, the experimental data were supplemented by the temporal fecal microbiota compositions of P28GST-treated Crohn’s disease patients from a pilot clinical study (NCT02281916). The P28GST administration slightly modulated the diversity and composition of mouse fecal microbiota while it significantly reduced experimental colitis in mice. Fecal microbiota transplantation experiments failed to restore the P28GST-induced anti-inflammatory effects. In Crohn’s disease patients, P28GST also induced slight changes in their overall fecal bacterial composition. Collectively, these results provide key elements in both the anti-inflammatory mechanisms and the safe therapeutic use of immunomodulation with such promising helminth-derived molecules.

Asthma is a chronic lung disease with persistent airway inflammation, bronchial hyper-reactivity, mucus overproduction, and airway remodeling. Antagonizing T2 responses by triggering the immune system with microbial components such as Toll-like receptors (TLRs) has been suggested as a therapeutic concept for allergic asthma. The aim of this study was to evaluate the effect of a TLR2/6 agonist, FSL-1 (Pam2CGDPKHPKSF), administered by intranasal instillation after an allergic airway reaction was established in the ovalbumin (OVA) mouse model and to analyze the role of natural killer (NK) cells in this effect. We showed that FSL-1 decreased established OVA-induced airway hyper-responsiveness and eosinophilic inflammation but did not reduce the T2 or T17 response. FSL-1 increased the recruitment and activation of NK cells in the lung parenchyma and modified the repartition of NK cell subsets in lung compartments. Finally, the transfer or depletion of NK cells did not modify airway hyper-responsiveness and eosinophilia after OVA and/or FSL-1 treatment. Thus, the administration of FSL-1 reduces airway hyper-responsiveness and bronchoalveolar lavage eosinophilia. However, despite modifications of their functions following OVA sensitization, NK cells play no role in OVA-induced asthma and its inhibition by FSL-1. Therefore, the significance of NK cell functions and localization in the airways remains to be unraveled in asthma.

Gut microbiota plays a key role in the maintenance of homeostasis and host physiology, comprising development, metabolism, and immunity. Profiling the composition and the gastrointestinal microbiome with a reliable methodology is of substantial interest to yield new insights into the pathogenesis of many diseases as well as defining new prophylactic and therapeutic interventions. Here, we briefly present our methodology applied to fecal samples from mice and then further extended to the samples from a cat and a single human subject at 4 different time points as examples to illustrate the methodological strengths. Both interindividual and time-related variations are demonstrated and discussed.

In the article titled “Snapshot on a Pilot Metagenomic Study for the Appraisal of Gut Microbial Diversity in Mice, Cat, and Man”, [1] the last author’s first and last names were reversed. The author’s name should have been written Benoît Foligné. The revised author list is shown above.

Natural killer (NK) cells were originally described as cytolytic effector cells, but since then have been recognized to possess regulatory functions on immune responses. Chemokines locate NK cells throughout the body in homeostatic and pathological conditions. They may also directly stimulate immune cells. CCL18 is a constitutive and inducible chemokine involved in allergic diseases. The aim of this study was to evaluate CCL18’s effect on NK cells from allergic and nonallergic donors in terms of both chemotactic and immune effects. Results showed that CCL18 was able to induce migration of NK cells from nonallergic donors in a G-protein-dependent manner, suggesting the involvement of a classical chemokine receptor from the family of seven-transmembrane domain G-protein-coupled receptors. In contrast, NK cells from allergic patients were unresponsive. Similarly, CCL18 was able to induce NK cell cytotoxicity only in nonallergic subjects. Purified NK cells did not express CCR8, one of the receptors described to be involved in CCL18 functions. Finally, the defect in CCL18 response by NK cells from allergic patients was unrelated to a defect in CCL18 binding to NK cells. Overall, our results suggest that some NK cell functions may be defective in allergic diseases.

Natural killer (NK) cells were originally described as cytolytic effector cells, but since then have been recognized to possess regulatory functions on immune responses. Chemokines locate NK cells throughout the body in homeostatic and pathological conditions. They may also directly stimulate immune cells. CCL18 is a constitutive and inducible chemokine involved in allergic diseases. The aim of this study was to evaluate CCL18’s effect on NK cells from allergic and nonallergic donors in terms of both chemotactic and immune effects. Results showed that CCL18 was able to induce migration of NK cells from nonallergic donors in a G-protein-dependent manner, suggesting the involvement of a classical chemokine receptor from the family of seven-transmembrane domain G-protein-coupled receptors. In contrast, NK cells from allergic patients were unresponsive. Similarly, CCL18 was able to induce NK cell cytotoxicity only in nonallergic subjects. Purified NK cells did not express CCR8, one of the receptors described to be involved in CCL18 functions. Finally, the defect in CCL18 response by NK cells from allergic patients was unrelated to a defect in CCL18 binding to NK cells. Overall, our results suggest that some NK cell functions may be defective in allergic diseases.