Project description:Background: In the last decade, much attention has been drawn to probiotic bacteria in the context of inflammatory bowel disease (IBD), since the potential of certain strains to attenuate inflammation was demonstrated in several animal experiments and clinical studies. Data in humans elucidating the molecular mechanism of probiotic action are still scarce. To this end, we used an organ culture system of human colon mucosa and investigated the gene expression profiles after treatment with different probiotic bacteria in phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO)) stimulated samples using whole genome microarrays. Moreover, we analyzed changes occurring in the intestinal explants cultured for 8 hours when compared to fresh, directly frozen mucosa, in order to infer the suitability of the system to study an inflammatory stimulus and likely antiinflammatory responses. Results: Culturing intestinal colon fragments during 8 hours elicited differential gene expression in 283 genes, 229 upregulated and 54 downregulated. Upregulated genes were predominantly related to apoptosis, whereas downregulated genes encoded mitochondrial proteins. No specific enrichment of genes related to inflammation or immune response could be detected, confirming the suitability of the system to further study the inmunomodulatory/anti-inflammatory properties of Lactobacillus casei BL23 (BL23), L.plantarum 299v (LP299v) and L.plantarum 299v (A-) (LP299v (A-)), a mutant strain with reduced adhesive properties to enterocytes. Intestinal explants were stimulated with PMA/IO for 3 hours and subsequently incubated with probiotic bacteria for 4 h. ANOVA analysis (p ? 0,01) revealed 205 differentially expressed genes between Control, PMA/IO (Inflamed), and the 3 bacterial treatments. Most importantly, a number of PMA/IO induced genes related to immune response and immune system process such as IL-2, IFN-?, IL17A and pro-inflammatory cytokines CXCL9 and CXCL11 were downregulated by BL23, LP299v and LP299v (A-). The behaviour of the three Lactobacillus strains was quite similar, although their presence induced differential expression of a small number of genes in a strain dependent manner. Conclusion: The human colon organ culture was found to be a suitable model for the study of inflammatory/anti-inflammatory stimuli, and therefore it constitutes a valuable tool to determine the inmunomodulatory effect of probiotic bacteria. The global transcriptional profile evoked by strains BL23, LP299v and LP299v (A-) in artificially inflamed tissue indicated a clear homeostasis restoring effect, including a decrease of the signals produced by activated T cells. Macroscopically healthy colonic intestinal tissue was obtained at surgery from 3 patients. Intestinal explants were treated with PMA and ionomycin for 3 h to induce pro-inflammatory conditions. Then, culture medium was changed and replaced with either medium or medium containing either Lactobacillus casei BL23, Lactobacillus plantarum 299v, or a nonadherent mutant of L. plantarum 299v (A-) and incubated for further 4 hours. In parallel, control intestinal explants were cultured without any treatment of PMA/ionomycin or probiotic bacteria and compared to directly frozen tissue in order to evaluate changes in gene expression which are due solely to the culture conditions.

Project description:Background: In the last decade, much attention has been drawn to probiotic bacteria in the context of inflammatory bowel disease (IBD), since the potential of certain strains to attenuate inflammation was demonstrated in several animal experiments and clinical studies. Data in humans elucidating the molecular mechanism of probiotic action are still scarce. To this end, we used an organ culture system of human colon mucosa and investigated the gene expression profiles after treatment with different probiotic bacteria in phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO)) stimulated samples using whole genome microarrays. Moreover, we analyzed changes occurring in the intestinal explants cultured for 8 hours when compared to fresh, directly frozen mucosa, in order to infer the suitability of the system to study an inflammatory stimulus and likely antiinflammatory responses. Results: Culturing intestinal colon fragments during 8 hours elicited differential gene expression in 283 genes, 229 upregulated and 54 downregulated. Upregulated genes were predominantly related to apoptosis, whereas downregulated genes encoded mitochondrial proteins. No specific enrichment of genes related to inflammation or immune response could be detected, confirming the suitability of the system to further study the inmunomodulatory/anti-inflammatory properties of Lactobacillus casei BL23 (BL23), L.plantarum 299v (LP299v) and L.plantarum 299v (A-) (LP299v (A-)), a mutant strain with reduced adhesive properties to enterocytes. Intestinal explants were stimulated with PMA/IO for 3 hours and subsequently incubated with probiotic bacteria for 4 h. ANOVA analysis (p ≤ 0,01) revealed 205 differentially expressed genes between Control, PMA/IO (Inflamed), and the 3 bacterial treatments. Most importantly, a number of PMA/IO induced genes related to immune response and immune system process such as IL-2, IFN-γ, IL17A and pro-inflammatory cytokines CXCL9 and CXCL11 were downregulated by BL23, LP299v and LP299v (A-). The behaviour of the three Lactobacillus strains was quite similar, although their presence induced differential expression of a small number of genes in a strain dependent manner. Conclusion: The human colon organ culture was found to be a suitable model for the study of inflammatory/anti-inflammatory stimuli, and therefore it constitutes a valuable tool to determine the inmunomodulatory effect of probiotic bacteria. The global transcriptional profile evoked by strains BL23, LP299v and LP299v (A-) in artificially inflamed tissue indicated a clear homeostasis restoring effect, including a decrease of the signals produced by activated T cells.

Project description:The chronic inflammatory immune response triggered by the infection of the tooth root canal system results in the local upregulation of RANKL, resulting in periapical bone loss. While RANKL has a well-characterized role in the control of bone homeostasis/pathology, it can play important roles in the regulation of the immune system, although its possible immunoregulatory role in infectious inflammatory osteolytic conditions remains largely unknown. Here, we used a mouse model of infectious inflammatory periapical lesions subjected to continuous or transitory anti-RANKL inhibition, followed by the analysis of lesion outcome and multiple host response parameters. Anti-RANKL administration resulted in arrest of bone loss but interfered in the natural immunoregulation of the lesions observed in the untreated group. RANKL inhibition resulted in an unremitting proinflammatory response, persistent high proinflammatory and effector CD4 response, decreased regulatory T-cell (Treg) migration, and lower levels of Treg-related cytokines IL-10 and TGFb. Anti-RANKL blockade impaired the immunoregulatory process only in early disease stages, while the late administration of anti-RANKL did not interfere with the stablished immunoregulation. The impaired immunoregulation due to RANKL inhibition is characterized by increased delayed-type hypersensitivity in vivo and T-cell proliferation in vitro to the infecting bacteria, which mimic the effects of Treg inhibition, reinforcing a possible influence of RANKL on Treg-mediated suppressive response. The adoptive transfer of CD4+FOXp3+ Tregs to mice receiving anti-RANKL therapy restored the immunoregulatory capacity, attenuating the inflammatory response in the lesions, reestablishing normal T-cell response in vivo and in vitro, and preventing lesion relapse upon anti-RANKL therapy cessation. Therefore, while RANKL inhibition efficiently limited the periapical bone loss, it promoted an unremitting host inflammatory response by interfering with Treg activity, suggesting that this classic osteoclastogenic mediator plays a role in immunoregulation.

Project description:Genome-wide transcriptional analysis in intestinal epithelial cells (IEC) can aid in elucidating the impact of single versus multi-stain probiotic combinations on immunological and cellular mechanism of action. In this study we used an in vitro intestinal epithelial cell model to investigate the impact of three probiotic bacteria individually or in combination and a surface-layer protein (SLP) partially purified from one of the bacteria on HT-29 cells’ response to a known pro-inflammatory stimulus, polyinosinic:polycytidylic, poly(I:C). Human expression microarray chips were used to evaluate the effect of Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033 and Bifidobacterium bifidum R0071 individually, in combination and of a surface-layer protein (SLP) partially purified from R0052 on HT-29 cells’ transcriptional profile to poly(I:C)-induced inflammation. Hierarchical heat map clustering, Set Distiller and String analyses revealed that the effects of R0052 and R0071 diverged from that of R0033 and R0052-SLP. It was evident from the global analyses with respect to the immune, cellular and homeostasis related pathways that the co-challenge with probiotic combination (PC) vastly differed in its effect from the single strains and R0052-SLP treatments. The multi-strain PC resulted in a greater reduction of modulated genes, found through functional connections between immune and cellular pathways. Cytokine and chemokine analyses based on specific outcomes from the TNF-α and NF-κB signaling pathways revealed single, multi-strain and R0052-SLP specific attenuation of the majority of proteins measured (TNF-α, IL-8, CXCL1, CXCL2 and CXCL10), indicating potentially different mechanisms. These findings indicate a synergistic effect of the bacterial combinations relative to the single strain and R0052-SLP treatments in resolving toll-like receptor 3 (TLR3)-induced inflammation in IEC and maintaining cellular homeostasis, reinforcing the rationale for using multi-strain formulations as a probiotic.

Project description:A commercially available product containing three probiotic bacterial strains (Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033, and Bifidobacterium bifidum R0071) was previously shown in animal trials to modulate both TH1 and TH2 immune responses. Clinical studies on this combination of bacteria have also shown positive health effects against seasonal winter diseases and rotavirus infection. The goal of this study was to use a well-established in vitro intestinal epithelial (HT-29) cell model that has been shown to constitutively express double-stranded RNA (dsRNA) sensors (Toll-like receptor 3[TLR3], retinoic acid-inducible gene I, melanoma differentiation-associated gene 5, and dsRNA-activated protein kinase). By using the HT-29 cell model, we wanted to evaluate whether or not this combination of three bacteria had the capacity to immune modulate the host cell response to a dsRNA ligand, poly(IM-bM-^@M-"C). Using a custom-designed, two-color expression microarray targeting genes of the human immune system, we investigated the response of HT-29 cells challenged with poly(IM-bM-^@M-"C) both in the presence and in the absence of the three probiotic bacteria. We observed that the combination of the three bacteria had a major impact on attenuating the expression of genes connected to proinflammatory TH1 and antiviral innate immune responses compared to that obtained by the poly(IM-bM-^@M-"C)-only challenge. Major pathways through which the multistrain combination may be eliciting its immune-modulatory effect include the TLR3-TRIF, mitogen-activated protein kinase, and NF-KB signaling pathways.Such a model may be useful for selecting potential biomarkers for the design of future clinical trials The overall design consisted of 3 samples of HT-29 cells treated with poly (I:C)-only, probiotic combination (PC) and poly (I:C) + PC versus unchallenged HT-29 cells. A minimum of four dye-swap hybridizations (4 biological replicates) were performed for each of the 3 samples analyzed. Unchallenged HT-29 cells were controls and challenged HT-29 cells with poly (I:C)-only, PC-only and poly (I:C) + PC were treated samples.

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A patient-based probiotic approach identifies a Treg-centered immunoregulation via modulation of gut microbiota metabolites

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