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

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. 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.