Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

<p>Abstract</p> <p>Background</p> <p>Modulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the <it>Lactobacillus plantarum </it>species were investigated to identify genes of <it>L. plantarum </it>with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs).</p> <p>Results</p> <p>A total of 42 <it>Lactobacillus plantarum </it>strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The <it>L. plantarum </it>strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with <it>L. plantarum </it>WCFS1 DNA microarrays (also termed gene-trait matching) resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an <it>N</it>-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Deletion of these genes in <it>L. plantarum </it>WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells.</p> <p>Conclusions</p> <p>The altered PBMC cytokine profiles obtained with the <it>L. plantarum </it>WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 <it>L. plantarum </it>strains. This study therefore resulted in the identification of genes present in certain strains of <it>L. plantarum </it>which might be responsible for the stimulation of anti- or pro-inflammatory immune responses in the gut.</p

The <i>agr</i> inhibitors solonamide B and analogues alter immune responses to <i>Staphylococccus aureus</i> but do not exhibit adverse effects on immune cell functions

Staphylococcus aureus infections are becoming increasingly difficult to treat due to antibiotic resistance with the community-associated methicillin-resistant S. aureus (CA-MRSA) strains such as USA300 being of particular concern. The inhibition of bacterial virulence has been proposed as an alternative approach to treat multi-drug resistant pathogens. One interesting anti-virulence target is the agr quorum-sensing system, which regulates virulence of CA-MRSA in response to agr-encoded autoinducing peptides. Agr regulation confines exotoxin production to the stationary growth phase with concomitant repression of surface-expressed adhesins. Solonamide B, a non-ribosomal depsipeptide of marine bacterial origin, was recently identified as a putative anti-virulence compound that markedly reduced expression of α-hemolysin and phenol-soluble modulins. To further strengthen solonamide anti-virulence candidacy, we report the chemical synthesis of solonamide analogues, investigation of structure-function relationships, and assessment of their potential to modulate immune cell functions. We found that structural differences between solonamide analogues confer significant differences in interference with agr, while immune cell activity and integrity is generally not affected. Furthermore, treatment of S. aureus with selected solonamides was found to only marginally influence the interaction with fibronectin and biofilm formation, thus addressing the concern that application of compounds inducing an agr-negative state may have adverse interactions with host factors in favor of host colonization

Alterations in early cytokine-mediated immune responses to Plasmodium falciparum infection in Tanzanian children with mineral element deficiencies: a cross-sectional survey

BACKGROUND: Deficiencies in vitamins and mineral elements are important causes of morbidity in developing countries, possibly because they lead to defective immune responses to infection. The aim of the study was to assess the effects of mineral element deficiencies on early innate cytokine responses to Plasmodium falciparum malaria. METHODS: Peripheral blood mononuclear cells from 304 Tanzanian children aged 6-72 months were stimulated with P. falciparum-parasitized erythrocytes obtained from in vitro cultures. RESULTS: The results showed a significant increase by 74% in geometric mean of TNF production in malaria-infected individuals with zinc deficiency (11% to 240%; 95% CI). Iron deficiency anaemia was associated with increased TNF production in infected individuals and overall with increased IL-10 production, while magnesium deficiency induced increased production of IL-10 by 46% (13% to 144%) in uninfected donors. All donors showed a response towards IL-1beta production, drawing special attention for its possible protective role in early innate immune responses to malaria. CONCLUSIONS: In view of these results, the findings show plasticity in cytokine profiles of mononuclear cells reacting to malaria infection under conditions of different micronutrient deficiencies. These findings lay the foundations for future inclusion of a combination of precisely selected set of micronutrients rather than single nutrients as part of malaria vaccine intervention programmes in endemic countries

Structure Dependent-Immunomodulation by Sugar Beet Arabinans via a SYK Tyrosine Kinase-Dependent Signaling Pathway

There is much interest in the immunomodulatory properties of dietary fibers but their activity may be influenced by contamination with microbial-associated molecular patterns (MAMPs) such as lipopolysaccharide (LPS) and lipoteichoic acids, which are difficult to remove completely from biological samples. Bone marrow-derived dendritic cells (BMDCs) from TLR2x4 double-KO mice were shown to be a reliable approach to analyse the immunomodulatory properties of a diverse range of dietary fibers, by avoiding immune cell activation due to contaminating MAMPs. Several of the 44 tested dietary fiber preparations induced cytokine responses in BMDCs from TLR2x4 double-KO mice. The particulate fractions of linear arabinan (LA) and branched arabinan (BA) from sugar beet pectin were shown to be strongly immune stimulatory with LA being more immune stimulatory than BA. Enzymatic debranching of BA increased its immune stimulatory activity, possibly due to increased particle formation by the alignment of debranched linear arabinan. Mechanistic studies showed that the immunostimulatory activity of LA and BA was independent of the Dectin-1 recognition but Syk kinase-dependent

Effect of nutrient deficiencies on in vitro Th1 and Th2 cytokine response of peripheral blood mononuclear cells to Plasmodium falciparum infection

BACKGROUND: An appropriate balance between pro-inflammatory and anti-inflammatory cytokines that mediate innate and adaptive immune responses is required for effective protection against human malaria and to avoid immunopathology. In malaria endemic countries, this immunological balance may be influenced by micronutrient deficiencies. METHODS: Peripheral blood mononuclear cells from Tanzanian preschool children were stimulated in vitro with Plasmodium falciparum-parasitized red blood cells to determine T-cell responses to malaria under different conditions of nutrient deficiencies and malaria status. RESULTS: The data obtained indicate that zinc deficiency is associated with an increase in TNF response by 37%; 95% CI: 14% to 118% and IFN-gamma response by 74%; 95% CI: 24% to 297%. Magnesium deficiency, on the other hand, was associated with an increase in production of IL-13 by 80%; 95% CI: 31% to 371% and a reduction in IFN-gamma production. These results reflect a shift in cytokine profile to a more type I cytokine profile and cell-cell mediated responses in zinc deficiency and a type II response in magnesium deficiency. The data also reveal a non-specific decrease in cytokine production in children due to iron deficiency anaemia that is largely associated with malaria infection status. CONCLUSIONS: The pathological sequels of malaria potentially depend more on the balance between type I and type II cytokine responses than on absolute suppression of these cytokines and this balance may be influenced by a combination of micronutrient deficiencies and malaria status

Identification of Genetic Loci in Lactobacillus plantarum That Modulate the Immune Response of Dendritic Cells Using Comparative Genome Hybridization

Contains fulltext : 88219.pdf (publisher's version ) (Open Access)BACKGROUND: Probiotics can be used to stimulate or regulate epithelial and immune cells of the intestinal mucosa and generate beneficial mucosal immunomodulatory effects. Beneficial effects of specific strains of probiotics have been established in the treatment and prevention of various intestinal disorders, including allergic diseases and diarrhea. However, the precise molecular mechanisms and the strain-dependent factors involved are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we aimed to identify gene loci in the model probiotic organism Lactobacillus plantarum WCFS1 that modulate the immune response of host dendritic cells. The amounts of IL-10 and IL-12 secreted by dendritic cells (DCs) after stimulation with 42 individual L. plantarum strains were measured and correlated with the strain-specific genomic composition using comparative genome hybridisation and the Random Forest algorithm. This in silico "gene-trait matching" approach led to the identification of eight candidate genes in the L. plantarum genome that might modulate the DC cytokine response to L. plantarum. Six of these genes were involved in bacteriocin production or secretion, one encoded a bile salt hydrolase and one encoded a transcription regulator of which the exact function is unknown. Subsequently, gene deletions mutants were constructed in L. plantarum WCFS1 and compared to the wild-type strain in DC stimulation assays. All three bacteriocin mutants as well as the transcription regulator (lp_2991) had the predicted effect on cytokine production confirming their immunomodulatory effect on the DC response to L. plantarum. Transcriptome analysis and qPCR data showed that transcript level of gtcA3, which is predicted to be involved in glycosylation of cell wall teichoic acids, was substantially increased in the lp_2991 deletion mutant (44 and 29 fold respectively). CONCLUSION: Comparative genome hybridization led to the identification of gene loci in L. plantarum WCFS1 that modulate the immune response of DCs

Pili-like proteins of Akkermansia muciniphila modulate host immune responses and gut barrier function

Gut barrier function is key in maintaining a balanced response between the host and its microbiome. The microbiota can modulate changes in gut barrier as well as metabolic and inflammatory responses. This highly complex system involves numerous microbiota-derived factors. The gut symbiont Akkermansia muciniphila is positively correlated with a lean phenotype, reduced body weight gain, amelioration of metabolic responses and restoration of gut barrier function by modulation of mucus layer thickness. However, the molecular mechanisms behind its metabolic and immunological regulatory properties are unexplored. Herein, we identify a highly abundant outer membrane pili-like protein of A. muciniphila MucT that is directly involved in immune regulation and enhancement of trans-epithelial resistance. The purified Amuc_1100 protein and enrichments containing all its associated proteins induced production of specific cytokines through activation of Toll-like receptor (TLR) 2 and TLR4. This mainly leads to high levels of IL-10 similar to those induced by the other beneficial immune suppressive microorganisms such as Faecalibacterium prausnitzii A2-165 and Lactobacillus plantarum WCFS1. Together these results indicate that outer membrane protein composition and particularly the newly identified highly abundant pili-like protein Amuc_1100 of A. muciniphila are involved in host immunological homeostasis at the gut mucosa, and improvement of gut barrier function.Peer reviewe

Prebiotic potential of pectin and pectic oligosaccharides to promote anti-inflammatory commensal bacteria in the human colon

Dietary plant cell wall carbohydrates are important in modulating the composition and metabolism of the complex gut microbiota, which can impact on health. Pectin is a major component of plant cell walls. Based on studies in model systems and available bacterial isolates and genomes, the capacity to utilize pectins for growth is widespread among colonic Bacteroidetes but relatively uncommon among Firmicutes. One Firmicutes species promoted by pectin is Eubacterium eligens. E. eligens DSM3376 utilizes apple pectin and encodes a broad repertoire of pectinolytic enzymes, including a highly abundant pectate lyase of around 200 kDa that is expressed constitutively. We confirmed that certain Faecalibacterium prausnitzii strains possess some ability to utilize apple pectin and report here that F. prausnitzii strains in common with E. eligens, can utilize the galacturonide oligosaccharides DP4 and DP5 derived from sugar beet pectin. F. prausnitzii strains have been shown previously to exert anti-inflammatory effects on host cells, but we show here for the first time that E. eligens strongly promotes the production of the anti-inflammatory cytokine IL-10 in in vitro cell-based assays. These findings suggest the potential to explore further the prebiotic potential of pectin and its derivatives to re-balance the microbiota towards an anti-inflammatory profile

Lactobacillus plantarum Strains Can Enhance Human Mucosal and Systemic Immunity and Prevent Non-steroidal Anti-inflammatory Drug Induced Reduction in T Regulatory Cells

Orally ingested bacteria interact with intestinal mucosa and may impact immunity. However, insights in mechanisms involved are limited. In this randomized placebo-controlled cross-over trial, healthy human subjects were given Lactobacillus plantarum supplementation (strain TIFN101, CIP104448, or WCFS1) or placebo for 7 days. To determine whether L. plantarum can enhance immune response, we compared the effects of three stains on systemic and gut mucosal immunity, by among others assessing memory responses against tetanus toxoid (TT)-antigen, and mucosal gene transcription, in human volunteers during induction of mild immune stressor in the intestine, by giving a commonly used enteropathic drug, indomethacin [non-steroidal anti-inflammatory drug (NSAID)]. Systemic effects of the interventions were studies in peripheral blood samples. NSAID was found to induce a reduction in serum CD4+/Foxp3 regulatory cells, which was prevented by L. plantarum TIFN101. T-cell polarization experiments showed L. plantarum TIFN101 to enhance responses against TT-antigen, which indicates stimulation of memory responses by this strain. Cell extracts of the specific L. plantarum strains provoked responses after WCFS1 and TIFN101 consumption, indicating stimulation of immune responses against the specific bacteria. Mucosal immunomodulatory effects were studied in duodenal biopsies. In small intestinal mucosa, TIFN101 upregulated genes associated with maintenance of T- and B-cell function and antigen presentation. Furthermore, L. plantarum TIFN101 and WCFS1 downregulated immunological pathways involved in antigen presentation and shared downregulation of snoRNAs, which may suggest cellular destabilization, but may also be an indicator of tissue repair. Full sequencing of the L. plantarum strains revealed possible gene clusters that might be responsible for the differential biological effects of the bacteria on host immunity. In conclusion, the impact of oral consumption L. plantarum on host immunity is strain dependent and involves responses against bacterial cell components. Some strains may enhance specific responses against pathogens by enhancing antigen presentation and leukocyte maintenance in mucosa. In future studies and clinical settings, caution should be taken in selecting beneficial bacteria as closely related strains can have different effects. Our data show that specific bacterial strains can prevent immune stress induced by commonly consumed painkillers such as NSAID and can have enhancing beneficial effects on immunity of consumers by stimulating antigen presentation and memory responses

The Impact of Immune Interventions: A Systems Biology Strategy for Predicting Adverse and Beneficial Immune Effects

Despite scientific advances it remains difficult to predict the risk and benefit balance of immune interventions. Since a few years, network models have been built based on comprehensive datasets at multiple molecular/cellular levels (genes, gene products, metabolic intermediates, macromolecules, cells) to illuminate functional and structural relationships. Here we used a systems biology approach to identify key immune pathways involved in immune health endpoints and rank crucial candidate biomarkers to predict adverse and beneficial effects of nutritional immune interventions. First, a literature search was performed to select the molecular and cellular dynamics involved in hypersensitivity, autoimmunity and resistance to infection and cancer. Thereafter, molecular interaction between molecules and immune health endpoints was defined by connecting their relations by using database information. MeSH terms related to the immune health endpoints were selected resulting in the following selection: hypersensitivity (D006967: 184 genes), autoimmunity (D001327: 564 genes), infection (parasitic, bacterial, fungal and viral: 357 genes), and cancer (D009369: 3173 genes). In addition, a sequence of key processes was determined using Gene Ontology which drives the development of immune health disturbances resulting in the following selection: hypersensitivity (164 processes), autoimmunity (203 processes), infection (187 processes), and cancer (309 processes). Finally, an evaluation of the genes for each of the immune health endpoints was performed, which indicated that many genes played a role in multiple immune health endpoints, but also unique genes were observed for each immune health endpoint. This approach helps to build a screening/prediction tool which indicates the interaction of chemicals or food substances with immune health endpoint-related genes and suggests candidate biomarkers to evaluate risks and benefits. Several anti-cancer drugs and omega 3 fatty acids were evaluated as in silico test cases. To conclude, here we provide a systems biology approach to identify genes/molecules and their interaction with immune related disorders. Our examples illustrate that the prediction with our systems biology approach is promising and can be used to find both negatively and positively correlated interactions. This enables identification of candidate biomarkers to monitor safety and efficacy of therapeutic immune interventions