The Tolerance of Gut Commensal Faecalibacterium to Oxidative Stress Is Strain Dependent and Relies on Detoxifying Enzymes.

Obligate anaerobic bacteria in genus Faecalibacterium are among the most dominant taxa in the colon of healthy individuals and contribute to intestinal homeostasis. A decline in the abundance of this genus is associated with the occurrence of various gastrointestinal disorders, including inflammatory bowel diseases. In the colon, these diseases are accompanied by an imbalance between the generation and elimination of reactive oxygen species (ROS), and oxidative stress is closely linked to disruptions in anaerobiosis. In this work, we explored the impact of oxidative stress on several strains of faecalibacteria. An in silico analysis of complete genomes of faecalibacteria revealed the presence of genes encoding O2- and/or ROS-detoxifying enzymes, including flavodiiron proteins, rubrerythrins, reverse rubrerythrins, superoxide reductases, and alkyl peroxidase. However, the presence and the number of these detoxification systems varied greatly among faecalibacteria. These results were confirmed by O2 stress survival tests, in which we found that strains differed widely in their sensitivity. We showed the protective role of cysteine, which limited the production of extracellular O2•- and improved the survival of Faecalibacterium longum L2-6 under high O2 tension. In the strain F. longum L2-6, we observed that the expression of genes encoding detoxifying enzymes was upregulated in the response to O2 or H2O2 stress but with different patterns of regulation. Based on these results, we propose a first model of the gene regulatory network involved in the response to oxidative stress in F. longum L2-6. IMPORTANCE Commensal bacteria in the genus Faecalibacterium have been proposed for use as next-generation probiotics, but efforts to cultivate and exploit the potential of these strains have been limited by their sensitivity to O2. More broadly, little is known about how commensal and health-associated bacterial species in the human microbiome respond to the oxidative stress that occurs as a result of inflammation in the colon. In this work, we provide insights regarding the genes that encode potential mechanisms of protection against O2 or ROS stress in faecalibacteria, which may facilitate future advances in work with these important bacteria.

Intraspecific Diversity of Microbial Anti-Inflammatory Molecule (MAM) from Faecalibacterium prausnitzii.

The commensal bacterium Faecalibacterium prausnitzii has unique anti-inflammatory properties, at least some of which have been attributed to its production of MAM, the Microbial Anti-inflammatory Molecule. Previous phylogenetic studies of F. prausnitzii strains have revealed the existence of various phylogroups. In this work, we address the question of whether MAMs from different phylogroups display distinct anti-inflammatory properties. We first performed wide-scale identification, classification, and phylogenetic analysis of MAM-like proteins encoded in different genomes of F. prausnitzii. When combined with a gene context analysis, this approach distinguished at least 10 distinct clusters of MAMs, providing evidence for functional diversity within this protein. We then selected 11 MAMs from various clusters and evaluated their anti-inflammatory capacities in vitro. A wide range of anti-inflammatory activity was detected. MAM from the M21/2 strain had the highest inhibitory effect (96% inhibition), while MAM from reference strain A2-165 demonstrated only 56% inhibition, and MAM from strain CNCM4541 was almost inactive. These results were confirmed in vivo in murine models of acute and chronic colitis. This study provides insights into the family of MAM proteins and generates clues regarding the choice of F. prausnitzii strains as probiotics for use in targeting chronic inflammatory diseases.

The regenerating family member 3 ß instigates IL-17A-mediated neutrophil recruitment downstream of NOD1/2 signalling for controlling colonisation resistance independently of microbiota community structure.

OBJECTIVE: Loss of the Crohn's disease predisposing NOD2 gene results in an intestinal microenvironment conducive for colonisation by attaching-and-effacing enteropathogens. However, it remains elusive whether it relies on the intracellular recruitment of the serine-threonine kinase RIPK2 by NOD2, a step that is required for its activation of the transcription factor NF-κB. DESIGN: Colonisation resistance was evaluated in wild type and mutant mice, as well as in ex-germ-free (ex-GF) mice which were colonised either with faeces from Ripk2-deficient mice or with bacteria with similar preferences for carbohydrates to those acquired by the pathogen. The severity of the mucosal pathology was quantified at several time points postinfection by using a previously established scoring. The community resilience in response to infection was evaluated by 16S ribosomal RNA gene sequence analysis. The control of pathogen virulence was evaluated by monitoring the secretion of Citrobacter-specific antibody response in the faeces. RESULTS: Primary infection was similarly outcompeted in ex-GF Ripk2-deficient and control mice, demonstrating that the susceptibility to infection resulting from RIPK2 deficiency cannot be solely attributed to specific microbiota community structures. In contrast, delayed clearance of Citrobacter rodentium and exacerbated histopathology were preceded by a weakened propensity of intestinal macrophages to afford innate lymphoid cell activation. This tissue protection unexpectedly required the regenerating family member 3ß by instigating interleukin (IL) 17A-mediated neutrophil recruitment to the intestine and subsequent phosphorylation of signal transducer and activator of transcription 3. CONCLUSIONS: These results unveil a previously unrecognised mechanism that efficiently protects from colonisation by diarrhoeagenic bacteria early in infection.

Oral delivery of pancreatitis-associated protein by Lactococcus lactis displays protective effects in dinitro-benzenesulfonic-acid-induced colitis model and is able to modulate the composition of the microbiota.

Antimicrobial peptides secreted by intestinal immune and epithelial cells are important effectors of innate immunity. They play an essential role in the maintenance of intestinal homeostasis by limiting microbial epithelium interactions and preventing unnecessary microbe-driven inflammation. Pancreatitis-associated protein (PAP) belongs to Regenerating islet-derived III proteins family and is a C-type (Ca+2 dependent) lectin. PAP protein plays a protective effect presenting anti-inflammatory properties able to reduce the severity of colitis, preserving gut barrier and epithelial inflammation. Here, we sought to determine whether PAP delivered at intestinal lumen by recombinant Lactococcus lactis strain (LL-PAP) before and after chemically induced colitis is able to reduce the severity in two models of colitis. After construction and characterization of our recombinant strains, we tested their effects in dinitro-benzenesulfonic-acid (DNBS) and Dextran sulfate sodium (DSS) colitis model. After the DNBS challenge, mice treated with LL-PAP presented less severe colitis compared with PBS and LL-empty-treated mice groups. After the DSS challenge, no protective effects of LL-PAP could be detected. We determined that after 5 days administration, LL-PAP increase butyrate producer's bacteria, especially Eubacterium plexicaudatum. Based on our findings, we hypothesize that a treatment with LL-PAP shifts the microbiota preventing the severity of colon inflammation in DNBS colitis model. These protective roles of LL-PAP in DNBS colitis model might be through intestinal microbiota modulation.

Secretion of biologically active pancreatitis-associated protein I (PAP) by genetically modified dairy Lactococcus lactis NZ9000 in the prevention of intestinal mucositis.

BACKGROUND: Mucositis is one of the most relevant gastrointestinal inflammatory conditions in humans, generated by the use of chemotherapy drugs, such as 5-fluoracil (5-FU). 5-FU-induced mucositis affects 80% of patients undergoing oncological treatment causing mucosal gut dysfunctions and great discomfort. As current therapy drugs presents limitations in alleviating mucositis symptoms, alternative strategies are being pursued. Recent studies have shown that the antimicrobial pancreatitis-associated protein (PAP) has a protective role in intestinal inflammatory processes. Indeed, it was demonstrated that a recombinant strain of Lactococcus lactis expressing human PAP (LL-PAP) could prevent and improve murine DNBS-induced colitis, an inflammatory bowel disease (IBD) that causes severe inflammation of the colon. Hence, in this study we sought to evaluate the protective effects of LL-PAP on 5-FU-induced experimental mucositis in BALB/c mice as a novel approach to treat the disease. RESULTS: Our results show that non-recombinant L. lactis NZ9000 have antagonistic activity, in vitro, against the enteroinvasive gastrointestinal pathogen L. monocytogenes and confirmed PAP inhibitory effect against Opportunistic E. faecalis. Moreover, L. lactis was able to prevent histological damage, reduce neutrophil and eosinophil infiltration and secretory Immunoglobulin-A in mice injected with 5-FU. Recombinant lactococci carrying antimicrobial PAP did not improve those markers of inflammation, although its expression was associated with villous architecture preservation and increased secretory granules density inside Paneth cells in response to 5-FU inflammation. CONCLUSIONS: We have demonstrated for the first time that L. lactis NZ9000 by itself, is able to prevent 5-FU-induced intestinal inflammation in BALB/c mice. Moreover, PAP delivered by recombinant L. lactis strain showed additional protective effects in mice epithelium, revealing to be a promising strategy to treat intestinal mucositis.

Lactococcus lactis carrying the pValac eukaryotic expression vector coding for IL-4 reduces chemically-induced intestinal inflammation by increasing the levels of IL-10-producing regulatory cells.

BACKGROUND: Inflammatory bowel diseases are characterized by chronic intestinal inflammation that leads to severe destruction of the intestinal mucosa. Therefore, the understanding of their aetiology as well as the development of new medicines is an important step for the treatment of such diseases. Consequently, the development of Lactococcus lactis strains capable of delivering a eukaryotic expression vector encoding the interleukin 4 (IL-4) of Mus musculus would represent a new strategy for the elaboration of a more effective alternative therapy against Crohn's disease. RESULTS: The murine IL-4 ORF was cloned into the eukaryotic expression vector pValac::dts. The resulting plasmid-pValac::dts::IL-4-was transfected into CHO cells so that its functionality could be evaluated in vitro. With fluorescent confocal microscopy, flow cytometry and ELISA, it was observed that pValac::dts::IL-4-transfected cells produced IL-4, while non-transfected cells and cells transfected with the empty vector did not. Then, pValac::dts::IL-4 was inserted into L. lactis MG1363 FnBPA(+) in order to evaluate the therapeutic potential of the recombinant strain against TNBS-induced colitis. Intragastric administration of L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) was able to decrease the severity of colitis, with animals showing decreased levels of IL-12, IL-6 and MPO activity; and increased levels of IL-4 and IL-10. Finally, LP-isolated cells from mice administered TNBS were immunophenotyped so that the main IL-4 and IL-10 producers were identified. Mice administered the recombinant strain presented significantly higher percentages of F4/80(+)MHCII(+)Ly6C(-)IL-4(+), F4/80(+)MHCII(+)Ly6C(-)IL-10(+), F4/80(+)MHCII(+)Ly6C(-)CD206(+)CD124(+)IL-10(+) and CD4(+)Foxp3(+)IL10(+) cells compared to the other groups. CONCLUSIONS: This study shows that L. lactis MG1363 FnBPA(+) (pValac::dts::IL-4) is a good candidate to maintain the anti-inflammatory and proinflammatory balance in the gastrointestinal tract, increasing the levels of IL-10-secreting regulatory cells and, thus, demonstrating the effectiveness of this novel DNA delivery-based strategy.

Surface display of an anti-DEC-205 single chain Fv fragment in Lactobacillus plantarum increases internalization and plasmid transfer to dendritic cells in vitro and in vivo.

BACKGROUND: Lactic acid bacteria (LAB) are promising vehicles for delivery of a variety of medicinal compounds, including antigens and cytokines. It has also been established that LAB are able to deliver cDNA to host cells. To increase the efficiency of LAB-driven DNA delivery we have constructed Lactobacillus plantarum strains targeting DEC-205, which is a receptor located at the surface of dendritic cells (DCs). The purpose was to increase uptake of bacterial cells, which could lead to improved cDNA delivery to immune cells. RESULTS: Anti-DEC-205 antibody (aDec) was displayed at the surface of L. plantarum using three different anchoring strategies: (1) covalent anchoring of aDec to the cell membrane (Lipobox domain, Lip); (2) covalent anchoring to the cell wall (LPXTG domain, CWA); (3) non-covalent anchoring to the cell wall (LysM domain, LysM). aDec was successfully expressed in all three strains, but surface location of the antibody could only be demonstrated for the two strains with cell wall anchors (CWA and LysM). Co-incubation of the engineered strains and DCs showed increased uptake when anchoring aDec using the CWA or LysM anchors. In a competition assay, free anti-DEC abolished the increased uptake, showing that the internalization is due to specific interactions between the DEC-205 receptor and aDec. To test plasmid transfer, a plasmid for expression of GFP under control of an eukaryotic promoter was transformed into the aDec expressing strains and GFP expression in DCs was indeed increased when using the strains producing cell-wall anchored aDec. Plasmid transfer to DCs in the gastro intestinal tract was also detected using a mouse model. Surprisingly, in mice the highest expression of GFP was observed for the strain in which aDec was coupled to the cell membrane. CONCLUSION: The results show that surface expression of aDec leads to increased internalization of L. plantarum and plasmid transfer in DCs and that efficiency depends on the type of anchor used. Interestingly, in vitro data indicates that cell wall anchoring is more effective, whereas in vivo data seem to indicate that anchoring to the cell membrane is preferable. It is likely that the more embedded localization of aDec in the latter case is favorable when cells are exposed to the harsh conditions of the gastro-intestinal tract.

Protective effect of TSLP delivered at the gut mucosa level by recombinant lactic acid bacteria in DSS-induced colitis mouse model.

BACKGROUND: Thymic stromal lymphopoietin (TSLP) is a cytokine known to mature dendritics cells, lower pro-inflammatory IL-12 secretion, induce differentiation of anti-inflammatory FoxP3+ regulatory T cells (Treg). Moreover, Crohn's disease patients have shown a reduction of intestinal TSLP expression. To understand the role of TSLP in inflammation, we constructed Lactococcus lactis strain producing TSLP (LL-TSLP) and investigated the effect of its administration on dextran sulfate sodium (DSS)-induced colitis model in mice. RESULTS: LL-TSLP secrete an active molecule which lowers secretion of IL-12 by dendritic cells. Treatment with LL-TSLP, increases the amount of TGF-ß secreted by T cells in Mesenteric Lymph Node in healthy mice. In acute DSS-induced colitis, LL-TSLP delayed the Disease Activity Index and lowered histological score and colonic INF-γ production. In a DSS-recovery model, LL-TSLP induced a better protective effect if the strain was administered at the beginning of the colitis. At Day 4 of colitis we observed an induction of Treg by LL-TSLP. CONCLUSIONS: TSLP showed an anti-inflammatory protective role in DSS-induced colitis. We have demonstrated that a short and early administration of LL-TSLP is more efficient than a long lasting treatment.

Lactococcus lactis carrying the pValac DNA expression vector coding for IL-10 reduces inflammation in a murine model of experimental colitis.

BACKGROUND: Inflammatory bowel diseases (IBD) are intestinal disorders characterized by inflammation in the gastrointestinal tract. Interleukin-10 is one of the most important anti-inflammatory cytokines involved in the intestinal immune system and because of its role in downregulating inflammatory cascades, its potential for IBD therapy is under study. We previously presented the development of an invasive strain of Lactococcus lactis (L. lactis) producing Fibronectin Binding Protein A (FnBPA) which was capable of delivering, directly to host cells, a eukaryotic DNA expression vector coding for IL-10 of Mus musculus (pValac:il-10) and diminish inflammation in a trinitrobenzene sulfonic acid (TNBS)-induced mouse model of intestinal inflammation. As a new therapeutic strategy against IBD, the aim of this work was to evaluate the therapeutic effect of two L. lactis strains (the same invasive strain evaluated previously and the wild-type strain) carrying the therapeutic pValac:il-10 plasmid in the prevention of inflammation in a dextran sodium sulphate (DSS)-induced mouse model. RESULTS: Results obtained showed that not only delivery of the pValac:il-10 plasmid by the invasive strain L. lactis MG1363 FnBPA+, but also by the wild-type strain L. lactis MG1363, was effective at diminishing intestinal inflammation (lower inflammation scores and higher IL-10 levels in the intestinal tissues, accompanied by decrease of IL-6) in the DSS-induced IBD mouse model. CONCLUSIONS: Administration of both L. lactis strains carrying the pValac:il-10 plasmid was effective at diminishing inflammation in this murine model of experimental colitis, showing their potential for therapeutic intervention of IBD.

Correlation between fibronectin binding protein A expression level at the surface of recombinant lactococcus lactis and plasmid transfer in vitro and in vivo.

BACKGROUND: Fibronectin Binding Protein A (FnBPA) is an invasin from Staphylococcus aureus that allows this pathogen to internalize into eukaryote cells. It was previously demonstrated that recombinant Lactococcus lactis expressing FnBPA were invasive and able to transfer a plasmid to eukaryotic cells in vitro and in vivo. In this study, the invasivity of recombinant strains of Lactococcus lactis that express FnBPA under the control of its constitutive promoter or driven by the strong nisin inducible expression system (NICE) were studied. RESULTS: It was demonstrated that the nisA promoter allows an increase of FnBPA expression on the surface of Lactococcus lactis surface, as shown by flow cytometry, which subsequently enhanced internalization and plasmid transfer properties in vitro in Caco2 cells and Bone Marrow Dendritic Cells. In vivo, the use of nisA promoter increase the plasmid transfer in cells of both the small and large intestine of mice. CONCLUSION: FnBPA expression at the surface of recombinant L. lactis is positively correlated to internalization and DNA transfer properties. The recombinant strains of L. lactis that expresses FnBPA under the control of the nisin inducible expression system could thus be considered as an improved tool in the field of DNA transfer.

Faecalibacterium duncaniae as a novel next generation probiotic against influenza.

The gut-lung axis is critical during viral respiratory infections such as influenza. Gut dysbiosis during infection translates into a massive drop of microbially produced short-chain fatty acids (SCFAs). Among them, butyrate is important during influenza suggesting that microbiome-based therapeutics targeting butyrate might hold promises. The butyrate-producing bacterium Faecalibacterium duncaniae (formerly referred to as F. prausnitzii) is an emerging probiotic with several health-promoting characteristics. To investigate the potential effects of F. duncaniae on influenza outcomes, mice were gavaged with live F. duncaniae (A2-165 or I-4574 strains) five days before infection. Supplementation of F. duncaniae was associated with less severe disease, a lower pulmonary viral load, and lower levels of lung inflammation. F. duncaniae supplementation impacted on gut dysbiosis induced by infection, as assessed by 16S rRNA sequencing. Interestingly, F. duncaniae administration was associated with a recovery in levels of SCFAs (including butyrate) in infected animals. The live form of F. duncaniae was more potent that the pasteurized form in improving influenza outcomes. Lastly, F. duncaniae partially protected against secondary (systemic) bacterial infection. We conclude that F. duncaniae might serve as a novel next generation probiotic against acute viral respiratory diseases.

Identification of one novel candidate probiotic Lactobacillus plantarum strain active against influenza virus infection in mice by a large-scale screening.

In this study, we developed a large-scale screening of bacterial strains in order to identify novel candidate probiotics with immunomodulatory properties. For this, 158 strains, including a majority of lactic acid bacteria (LAB), were screened by two different cellular models: tumor necrosis factor alpha (TNF-α)-activated HT-29 cells and peripheral blood mononuclear cells (PBMCs). Different strains responsive to both models (pro- and anti-inflammatory strains) were selected, and their protective effects were tested in vivo in a murine model of influenza virus infection. Daily intragastric administrations during 10 days before and 10 days after viral challenge (100 PFU of influenza virus H1N1 strain A Puerto Rico/8/1934 [A/PR8/34]/mouse) of Lactobacillus plantarum CNRZ1997, one potentially proinflammatory probiotic strain, led to a significant improvement in mouse health by reducing weight loss, alleviating clinical symptoms, and inhibiting significantly virus proliferation in lungs. In conclusion, in this study, we have combined two cellular models to allow the screening of a large number of LAB for their immunomodulatory properties. Moreover, we identified a novel candidate probiotic strain, L. plantarum CNRZ1997, active against influenza virus infection in mice.

Faecalibacterium: a bacterial genus with promising human health applications.

In humans, many diseases are associated with alterations in gut microbiota, namely increases or decreases in the abundance of specific bacterial groups. One example is the genus Faecalibacterium. Numerous studies have underscored that low levels of Faecalibacterium are correlated with inflammatory conditions, with inflammatory bowel disease (IBD) in the forefront. Its representation is also diminished in the case of several diseases, including colorectal cancer (CRC), dermatitis, and depression. Additionally, the relative presence of this genus is considered to reflect, at least in part, intestinal health status because Faecalibacterium is frequently present at reduced levels in individuals with gastrointestinal diseases or disorders. In this review, we first thoroughly describe updates to the taxonomy of Faecalibacterium, which has transformed a single-species taxon to a multispecies taxon over the last decade. We then explore the links discovered between Faecalibacterium abundance and various diseases since the first IBD-focused studies were published. Next, we examine current available strategies for modulating Faecalibacterium levels in the gut. Finally, we summarize the mechanisms underlying the beneficial effects that have been attributed to this genus. Together, epidemiological and experimental data strongly support the use of Faecalibacterium as a next-generation probiotic (NGP) or live biotherapeutic product (LBP).

New gene markers for classification and quantification of Faecalibacterium spp. in the human gut.

Faecalibacterium prausnitzii is a promising biomarker of a healthy human microbiota. However, previous studies reported the heterogeneity of this species and found the presence of several distinct groups at the species level among F. prausnitzii strains. Our recent study revealed that methods previously developed for quantification of F. prausnitzii were not specific to the species level because of the heterogeneity within the F. prausnitzii species and the application of 16S rRNA gene, which is an invalid genetic marker for the species. Therefore, previously available data failed to provide information on different groups, which limits our understanding of the importance of this organism for host health. Here, we propose an alternative gene marker for quantification of F. prausnitzii-related taxa. A total of nine group-specific primer pairs were designed by targeting rpoA gene sequences. The newly developed rpoA-based qPCR successfully quantified targeted groups. Application of the developed qPCR assay in six healthy adults revealed marked differences in abundance and prevalence among the different targeted groups in stool samples. The developed assay will facilitate detailed understanding of the impact of Faecalibacterium populations at the group level on human health and to understand the links between depletion of specific groups in Faecalibacterium and different human disorders.

Staphylococcus epidermidis isolates from atopic or healthy skin have opposite effect on skin cells: potential implication of the AHR pathway modulation.

Introduction: Staphylococcus epidermidis is a commensal bacterium ubiquitously present on human skin. This species is considered as a key member of the healthy skin microbiota, involved in the defense against pathogens, modulating the immune system, and involved in wound repair. Simultaneously, S. epidermidis is the second cause of nosocomial infections and an overgrowth of S. epidermidis has been described in skin disorders such as atopic dermatitis. Diverse isolates of S. epidermidis co-exist on the skin. Elucidating the genetic and phenotypic specificities of these species in skin health and disease is key to better understand their role in various skin conditions. Additionally, the exact mechanisms by which commensals interact with host cells is partially understood. We hypothesized that S. epidermidis isolates identified from different skin origins could play distinct roles on skin differentiation and that these effects could be mediated by the aryl hydrocarbon receptor (AhR) pathway. Methods: For this purpose, a library of 12 strains originated from healthy skin (non-hyperseborrheic (NH) and hyperseborrheic (H) skin types) and disease skin (atopic (AD) skin type) was characterized at the genomic and phenotypic levels. Results and discussion: Here we showed that strains from atopic lesional skin alter the epidermis structure of a 3D reconstructed skin model whereas strains from NH healthy skin do not. All strains from NH healthy skin induced AhR/OVOL1 path and produced high quantities of indole metabolites in co-culture with NHEK; especially indole-3-aldehyde (IAld) and indole-3-lactic acid (ILA); while AD strains did not induce AhR/OVOL1 path but its inhibitor STAT6 and produced the lowest levels of indoles as compared to the other strains. As a consequence, strains from AD skin altered the differentiation markers FLG and DSG1. The results presented here, on a library of 12 strains, showed that S. epidermidis originated from NH healthy skin and atopic skin have opposite effects on the epidermal cohesion and structure and that these differences could be linked to their capacity to produce metabolites, which in turn could activate AHR pathway. Our results on a specific library of strains provide new insights into how S. epidermidis may interact with the skin to promote health or disease.

Microbiota-induced regulatory T cells associate with FUT2-dependent susceptibility to rotavirus gastroenteritis.

The FUT2 α1,2fucosyltransferase contributes to the synthesis of fucosylated glycans used as attachment factors by several pathogens, including noroviruses and rotaviruses, that can induce life-threatening gastroenteritis in young children. FUT2 genetic polymorphisms impairing fucosylation are strongly associated with resistance to dominant strains of both noroviruses and rotaviruses. Interestingly, the wild-type allele associated with viral gastroenteritis susceptibility inversely appears to be protective against several inflammatory or autoimmune diseases for yet unclear reasons, although a FUT2 influence on microbiota composition has been observed. Here, we studied a cohort of young healthy adults and showed that the wild-type FUT2 allele was associated with the presence of anti-RVA antibodies, either neutralizing antibodies or serum IgA, confirming its association with the risk of RVA gastroenteritis. Strikingly, it was also associated with the frequency of gut microbiota-induced regulatory T cells (Tregs), so-called DP8α Tregs, albeit only in individuals who had anti-RVA neutralizing antibodies or high titers of anti-RVA IgAs. DP8α Tregs specifically recognize the human symbiont Faecalibacterium prausnitzii, which strongly supports their induction by this anti-inflammatory bacterium. The proportion of F. prausnitzii in feces was also associated with the FUT2 wild-type allele. These observations link the FUT2 genotype with the risk of RVA gastroenteritis, the microbiota and microbiota-induced DP8α Treg cells, suggesting that the anti-RVA immune response might involve an induction/expansion of these T lymphocytes later providing a balanced immunological state that confers protection against inflammatory diseases.

Growth differentiation factor 11 delivered by dairy Lactococcus lactis strains modulates inflammation and prevents mucosal damage in a mice model of intestinal mucositis.

Mucositis is an inflammation of the gastrointestinal mucosa that debilitate the quality of life of patients undergoing chemotherapy treatments. In this context, antineoplastic drugs, such as 5-fluorouracil, provokes ulcerations in the intestinal mucosa that lead to the secretion of pro-inflammatory cytokines by activating the NF-κB pathway. Alternative approaches to treat the disease using probiotic strains show promising results, and thereafter, treatments that target the site of inflammation could be further explored. Recently, studies reported that the protein GDF11 has an anti-inflammatory role in several diseases, including in vitro and in vivo results in different experimental models. Hence, this study evaluated the anti-inflammatory effect of GDF11 delivered by Lactococcus lactis strains NCDO2118 and MG1363 in a murine model of intestinal mucositis induced by 5-FU. Our results showed that mice treated with the recombinant lactococci strains presented improved histopathological scores of intestinal damage and a reduction of goblet cell degeneration in the mucosa. It was also observed a significant reduction of neutrophil infiltration in the tissue in comparison to positive control group. Moreover, we observed immunomodulation of inflammatory markers Nfkb1, Nlrp3, Tnf, and upregulation of Il10 in mRNA expression levels in groups treated with recombinant strains that help to partially explain the ameliorative effect in the mucosa. Therefore, the results found in this study suggest that the use of recombinant L. lactis (pExu:gdf11) could offer a potential gene therapy for intestinal mucositis induced by 5-FU.

In vitro and in vivo characterization of DNA delivery using recombinant Lactococcus lactis expressing a mutated form of L. monocytogenes Internalin A.

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 ß-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.

Gene co-expression network analysis of the human gut commensal bacterium Faecalibacterium prausnitzii in R-Shiny.

Faecalibacterium prausnitzii is abundant in the healthy human intestinal microbiota, and the absence or scarcity of this bacterium has been linked with inflammatory diseases and metabolic disorders. F. prausnitzii thus shows promise as a next-generation probiotic for use in restoring the balance of the gut microbial flora and, due to its strong anti-inflammatory properties, for the treatment of certain pathological conditions. However, very little information is available about gene function and regulation in this species. Here, we utilized a systems biology approach-weighted gene co-expression network analysis (WGCNA)-to analyze gene expression in three publicly available RNAseq datasets from F. prausnitzii strain A2-165, all obtained in different laboratory conditions. The co-expression network was then subdivided into 24 co-expression gene modules. A subsequent enrichment analysis revealed that these modules are associated with different kinds of biological processes, such as arginine, histidine, cobalamin, or fatty acid metabolism as well as bacteriophage function, molecular chaperones, stress response, or SOS response. Some genes appeared to be associated with mechanisms of protection against oxidative stress and could be essential for F. prausnitzii's adaptation and survival under anaerobic laboratory conditions. Hub and bottleneck genes were identified by analyses of intramodular connectivity and betweenness, respectively; this highlighted the high connectivity of genes located on mobile genetic elements, which could promote the genetic evolution of F. prausnitzii within its ecological niche. This study provides the first exploration of the complex regulatory networks in F. prausnitzii, and all of the "omics" data are available online for exploration through a graphical interface at https://shiny.migale.inrae.fr/app/faeprau.

Lactococcus lactis engineered to deliver hCAP18 cDNA alleviates DNBS-induced colitis in C57BL/6 mice by promoting IL17A and IL10 cytokine expression.

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.