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Metabolic disorders are an increasing concern in the industrialized world. Current research has shown a direct link between the composition of the gut microbiota and the pathogenesis of obesity and diabetes. In only a few weeks, an obesity-inducing diet can lead to increased gut permeability and microbial dysbiosis, which contributes to chronic inflammation in the gut and adipose tissues, and to the development of insulin resistance. In this review, we examine the interplay between gut inflammation, insulin resistance, and the gut microbiota, and discuss how some probiotic species can be used to modulate gut homeostasis. We focus primarily on Faecalibacterium prausnitzii , a highly abundant butyrate-producing bacterium that has been proposed both as a biomarker for the development of different gut pathologies and as a potential treatment due to its production of anti-inflammatory metabolites.

Food-grade Lactic Acid Bacteria (LAB) have been safely consumed for centuries by humans in fermented foods. Thus, they are good candidates to develop novel oral vectors, constituting attractive alternatives to attenuated pathogens, for mucosal delivery strategies. Herein, this review summarizes our research, up until now, on the use of LAB as mucosal delivery vectors for therapeutic proteins and DNA vaccines. Most of our work has been based on the model LAB Lactococcus lactis , for which we have developed efficient genetic tools, including expression signals and host strains, for the heterologous expression of therapeutic proteins such as antigens, cytokines and enzymes. Resulting recombinant lactococci strains have been tested successfully for their prophylactic and therapeutic effects in different animal models: i) against human papillomavirus type 16 (HPV-16)-induced tumors in mice, ii) to partially prevent a bovine β-lactoglobulin (BLG)-allergic reaction in mice and iii) to regulate body weight and food consumption in obese mice. Strikingly, all of these tools have been successfully transposed to the Lactobacillus genus, in recent years, within our laboratory. Notably, anti-oxidative Lactobacillus casei strains were constructed and tested in two chemically-induced colitis models. In parallel, we also developed a strategy based on the use of L. lactis to deliver DNA at the mucosal level, and were able to show that L. lactis is able to modulate the host response through DNA delivery. Today, we consider that all of our consistent data, together with those obtained by other groups, demonstrate and reinforce the interest of using LAB, particularly lactococci and lactobacilli strains, to develop novel therapeutic protein mucosal delivery vectors which should be tested now in human clinical trials.

IL22 is a cytokine with many immunoregulatory effects. It also promotes mucus production, cell proliferation and wound healing, making it a key component of intestinal health. An increasing body of studies highlight the potential of IL22 as a therapeutic drug against NAFLD through the induction of antimicrobial peptides, especially of the Reg3 lectins family, and the regulation of gut microbiota. However, adverse reactions to this molecule in long term administration are little-studied. In this study, we gave daily doses of recombinant L. lactis delivering DNA vaccine for REG3A and IL-22 production to mice fed with a butter-based high fat diet (milk-fat diet or MFD). We aim to compare the effects of REG3A and IL-22 expression by the epithelial cells on the development of obesity-related conditions and the composition of the microbiota. We show that they have opposite effects on fat accumulation in the liver and insulin resistance development despite inducing a similar shift in fecal microbiota composition. Here, we provide evidence that daily long term administration of IL22 can aggravate NAFLD by mechanisms independent from gut microbiota.

Background Microbial anti-inflammatory molecule (MAM) is a key effector of the next-generation probiotic Faecalibacterium duncaniae A2-165, a species whose depletion in the gut microbiota is strongly linked to inflammatory bowel disease (IBD) and other conditions. Despite its importance, the direct anti-inflammatory effects of purified MAM have never been evaluated in vitro or in intestinal inflammation models. Prior studies have relied on bacterial supernatants, synthetic peptides, or DNA delivery systems, each with inherent limitations. Results In this study, we produced and purified recombinant MAM (R-MAM) under denaturing conditions and, for the first time, demonstrated its direct anti-inflammatory activity in vitro and its protective effects in a colitis murine model. Despite numerous attempts, we were not able to obtain a non-aggregated R-MAM. Therefore, we can assume that the R-MAM used here is partly or totally denatured. Nevertheless, in vitro assays with human intestinal epithelial cells (HT-29) and peripheral blood mononuclear cells (PBMCs) confirmed the ability of MAM to induce an anti-inflammatory cytokine profile. In addition, in a DNBS-induced colitis model, oral administration of R-MAM significantly prevented weight loss and reduced colon weight and thickness, key macroscopic indicators of inflammation. Conclusions These findings provide a critical validation step for the therapeutic potential of MAM in intestinal inflammation, despite its purification under denaturing conditions. Future studies should focus on optimizing protein stability and conformational integrity to increase its therapeutic potential as a biotherapeutic agent. Graphical Abstract

Two recent papers show that probiotics colonize the gut in permissive volunteers only and delay the reconstitution of the microbiome after antibiotics treatment. In the absence of any clinical readouts, it is still difficult to extrapolate these observations in terms of short-term or long-term health consequences for patients.

Abundance of Faecalibacterium prausnitzii, a dominant bacterium of the human microbiota that exhibits antiinflammatory effects, is decreased in patients with inflammatory bowel diseases (IBD). In humans, colonic lamina propria contains IL-10–secreting, Foxp3– Tregs characterized by a double expression of CD4 and CD8α (DP8α) and a specificity for F. prausnitzii. This Treg subset is decreased in IBD. The in vivo effect of DP8α cells has not been evaluated yet to our knowledge. Here, using a humanized model of a NSG immunodeficient mouse strain that expresses the HLA D–related allele HLA-DR*0401 but not murine class II (NSG-Ab° DR4) molecules, we demonstrated a protective effect of a HLA-DR*0401–restricted DP8α Treg clone combined with F. prausnitzii administration in a colitis model. In a cohort of patients with IBD, we showed an independent association between the frequency of circulating DP8α cells and disease activity. Finally, we pointed out a positive correlation between F. prausnitzii–specific DP8α Tregs and the amount of F. prausnitzii in fecal microbiota in healthy individuals and patients with ileal Crohn’s disease.

With its antimicrobial and immunomodulating properties, the cathelicidin (LL37) plays an important role in innate immune system. Here, we attempted to alleviate chemically induced colitis using a lactococci strain that either directly expressed the precursor to LL37, hCAP18 (LL-pSEC:hCAP18), or delivered hCAP18 cDNA to host cells under the control of the cytomegalovirus promoter (LL-Probi-H1:hCAP18). We also investigated whether the alleviation of symptoms could be explained through modification of the gut microbiota by hCAP18. Mice were administered daily doses of LL-pSEC:hCAP18 or LL-Probi-H1:hCAP18. On day 7, colitis was induced by DNBS. During autopsy, we assessed macroscopic tissue damage in the colon and collected tissue samples for the characterization of inflammation markers and histological analysis. Feces were collected at day 7 for 16S DNA sequencing. We also performed a fecal transplant experiment in which mice underwent colon washing and received feces from Lactococcus lactis -treated mice before DNBS-colitis induction. Treatment with LL-Probi-H1:hCAP18 reduced the severity of colitis symptoms. The protective effects were accompanied by increased levels of IL17A and IL10 in mesenteric lymph node cells. L. lactis administration altered the abundance of Lachnospiraceae and Muribaculaceae . However, fecal transplant from L. lactis -treated mice did not improve DNBS-induced symptoms in recipient mice.

•Validation of Lactococcus lactis as a DNA delivery vehicle for major antigen-presenting cell, dendritic cells.•Recombinant L. lactis expressing Listeria monocytogenes mutated internalin A (mInlA) is a better vector than wild type strain. Lactococcus lactis (L. lactis), a generally regarded as safe (GRAS) bacterium has recently been investigated as a mucosal delivery vehicle for DNA vaccines. Because of its GRAS status, L. lactis represents an attractive alternative to attenuated pathogens. Previous studies showed that eukaryotic expression plasmids could be delivered into intestinal epithelial cells (IECs) by L. lactis, or recombinant invasive strains of L. lactis, leading to heterologous protein expression. Although expression of antigens in IECs might lead to vaccine responses, it would be of interest to know whether uptake of L. lactis DNA vaccines by dendritic cells (DCs) could lead to antigen expression as they are unique in their ability to induce antigen-specific T cell responses. To test this, we incubated mouse bone marrow-derived DCs (BMDCs) with invasive L. lactis strains expressing either Staphylococcus aureus Fibronectin Binding Protein A (LL-FnBPA+), or Listeria monocytogenes mutated Internalin A (LL-mInlA+), both strains carrying a plasmid DNA vaccine (pValac) encoding for the cow milk allergen β-lactoglobulin (BLG). We demonstrated that they can transfect BMDCs, inducing the secretion of the pro-inflammatory cytokine IL-12. We also measured the capacity of strains to invade a polarized monolayer of IECs, mimicking the situation encountered in the gastrointestinal tract. Gentamycin survival assay in these cells showed that LL-mInlA+ is 100 times more invasive than L. lactis. The cross-talk between differentiated IECs, BMDCs and bacteria was also evaluated using an in vitro transwell co-culture model. Co-incubation of strains in this model showed that DCs incubated with LL-mInlA+ containing pValac:BLG could express significant levels of BLG. These results suggest that DCs could sample bacteria containing the DNA vaccine across the epithelial barrier and express the antigen.

Background The use of food-grade Lactic Acid Bacteria (LAB) as DNA delivery vehicles represents an attractive strategy to deliver DNA vaccines at the mucosal surfaces as they are generally regarded as safe (GRAS). We previously showed that either native Lactococcus lactis (LL) or recombinant invasive LL expressing Fibronectin Binding Protein A of Staphylococcus aureus (LL-FnBPA+) or Internalin A of Listeria monocytogenes (LL-InlA+), were able to deliver and trigger DNA expression by epithelial cells, either in vitro or in vivo . InlA does not bind to its receptor, the murine E-cadherin, thus limiting the use of LL-InlA+ in in vivo murine models. Moreover, FnBPA binds to its receptors, integrins, via fibronectin introducing another limiting factor. In order to avoid the limitations of LL-InlA+ and LL-FnBPA+, a new L . lactis strain was engineered to produce a previously described mutated form of InlA (LL-mInlA+) allowing the binding of mInlA on murine E-cadherin. Results After showing the expression of mInLA at the surface of LL-mInlA+ strain, in vitro gentamycin survival assay in Caco-2 cells showed that LL-mInlA+ is 1000 times more invasive than LL. LL-mInlA+ invasivity was also validated by fluorescence microscopy. LL and LL-mInlA+ were transformed with pValacBLG, a plasmid containing the cDNA of bovine β-Lactoglobulin (BLG), resulting in strains LL-BLG and LL-mInlA+BLG. The plasmid transfer in vitro using LL-mInlA+BLG was increased 10 times compared to LL-BLG. Moreover, the number of mice producing BLG in isolated enterocytes after oral administration of LL-mInlA+BLG in vivo was slightly higher than after oral administration of LL-BLG. Conclusions We confirmed in this study that the production of mInlA at the surface of L . lactis is a promising strategy for plasmid transfer in vitro and in vivo .

Lactococci are noninvasive lactic acid bacteria frequently used as protein delivery vectors and, more recently, as DNA delivery vehicles. We previously showed that Lactococcus lactis (LL) expressing the Fibronectin-Binding Protein A of Staphylococcus aureus (LL-FnBPA+) showed higher internalization rates in vitro in Caco-2 cells than the native (wt) lactococci and were able to deliver a eukaryotic Green Fluorescent Protein (GFP) expression plasmid in 1% of human Caco-2 cells. Here, using the bovine beta-lactoglobulin (BLG), one of the major cow's milk allergen, and GFP we characterized the potential of LL-FnBPA+ as an in vivo DNA vaccine delivery vehicle. We first showed that the invasive strain LL-FnBPA+ carrying the plasmid pValac:BLG (LL-FnBPA+ BLG) was more invasive than LL-BLG and showed the same invasivity as LL-FnBPA+. Then we demonstrated that the Caco-2 cells, co-incubated with LL-FnBPA+ BLG produced up to 30 times more BLG than the Caco-2 cells co-incubated with the non invasive LL-BLG. Using two different gene reporters, BLG and GFP, and two different methods of detection, EIA and fluorescence microscopy, we showed in vivo that: i) in order to be effective, LL-FnBPA+ required a pre-coating with Fetal Calf Serum before oral administration; ii) plasmid transfer occurred in enterocytes without regard to the strains used (invasive or not); iii) the use of LL-FnBPA+ increased the number of mice producing BLG, but not the level of BLG produced. We thus confirmed the good potential of invasive recombinant lactic acid bacteria as DNA delivery vector in vivo.