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Genome-wide Immune Modulation of TLR3-mediated Inflammation in Intestinal Epithelial Cells Differ between Single versus Multi-Strain Probiotic Combination

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE71515 | GEO | 2015/08/25

SECONDARY ACCESSION(S): PRJNA293740

REPOSITORIES: GEO

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Project description:One of the key impacts of probiotic bacteria is the ability to beneficially modulate innate and adaptive immune responses of various cell types of the gut-associated lymphoid tissue. In this study we used an in vitro intestinal epithelial cell model to investigate the impact of three probiotic bacteria Lactobacillus helveticus R0052, Bifidobacterium longum subsp. infantis R0033 and Bifidobacterium bifidum R0071 individually, in combination (R0033, R0052 and R0071) and of a specific surface-layer protein partially purified from L. helveticus R0052 (R0052-SLP) in response to a known dsRNA pro-inflammatory stimulus, poly (I:C). Genome-wide human expression microarray chips were used to evaluate other cellular pathways and gain a deeper insight into global gene and pathway modulation. Here we report that the challenge by poly (I:C)-only resulted in the differential expression of 863 genes; whereas, the co-challenge with either R0033, R0052, R0071, R0052-SLP and the multi-strain combination (R0052, R0033 and R0071) resulted in differential expression of 467 genes, 369 genes, 293 genes, 219 genes and 132 genes; respectively. The multi-strain combination had a greater impact than each single component strain at reducing the number of genes modulated. Among the major human pathways modulated by probiotics or R0052-SLP in response to poly (I:C) include: immune/virus, cellular/signaling, nervous/endocrine and autoimmune disorder related pathways. Cytokine and chemokine profiling was performed at the protein level based on specific markers from the TNF-α and NF-KB signaling pathways. The results revealed single strain, multi-strain and R0052-SLP specific attenuation for the majority of proteins measured (TNF-α, IL-8, CXCL1, CXCL2, CXCL10 and IL-10) indicating potentially different mechanisms for each strain, multi-strain and protein component and reinforcing the rationale for multi-strain combinations. The overall study design consisted of 8 different treatments/challenges of HT-29. Briefly, HT-29 cells were co-challenge for 3 h with either the single strains (R0033, R0052 or R0071) or probiotic combination (PC) alone or in combination with poly (I:C) at 10 µg/mL. Also, a specific surface layer protein purified from R0052 (R0052-SLP) was also co-challenged with poly (I:C) in HT-29 cells. Treatment/challenges of poly (I:C)-only, surface layer protein-only (R0052-SLP-only) and probiotic combination-only or PC-only were also evaluated. All challenges and co-challenges were compared to unchallenged control HT-29 cells. 4 biological reps were performed for each of the 8 samples/treatments.

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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Genome-wide Immune Modulation of TLR3-mediated Inflammation in Intestinal Epithelial Cells Differ between Single versus Multi-Strain Probiotic Combination

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