Suppression of Intestinal Epithelial Cell Chemokine Production by Lactobacillus rhamnosus R0011 and Lactobacillus helveticus R0389 Is Mediated by Secreted Bioactive Molecules

Abstract

Host intestinal epithelial cells (IEC) present at the gastrointestinal interface are exposed to pathogenic and non-pathogenic bacteria and their products. Certain probiotic lactic acid bacteria (LAB) have been associated with a range of host-immune modulatory activities including down-regulation of pro-inflammatory gene expression and cytokine production by IEC, with growing evidence suggesting that these bacteria secrete bioactive molecules with immunomodulatory activity. The aim of this study was to determine whether two lactobacilli with immunomodulatory activity [Lactobacillus rhamnosus R0011 (Lr) and Lactobacillus helveticus R0389 (Lh)], produce soluble mediators able to influence IEC responses to Pattern Recognition Receptor (PRR) ligands and pro-inflammatory cytokines [Tumor Necrosis Factor α (TNFα), Interleukin-1β (IL-1β)], signals inducing IEC chemokine production during infection. To this end, the effects of cell-free supernatants (CFS) from Lr and Lh on IEC production of the pro-inflammatory chemokines interleukin (IL)-8 and cytokine-induced neutrophil chemoattractant 1 (CINC-1) induced by a range of host- or pathogen-derived pro-inflammatory stimuli were determined, and the impact on human HT-29 IEC and a primary IEC line (rat IEC-6) was compared. The Lr-CFS and Lh-CFS did not significantly modulate basal IL-8 production from HT-29 IECs or CINC-1 production from IEC-6 cells. However, both Lr-CFS and Lh-CFS significantly down-regulated IL-8 production from HT-29 IECs challenged with varied PRR ligands. Lr-CFS and Lh-CFS had differential effects on PRR-induced CINC-1 production by rat IEC-6 IECs, with no significant down-regulation of CINC-1 observed from IEC-6 IECs cultured with Lh-CFS. Further analysis of the Lr-CFS revealed down-regulation of IL-8 production induced by the pro-inflammatory cytokines IL-1β and TNFα Preliminary characterization of the bioactive constituent(s) of the Lr-CFS indicates that it is resistant to treatment with DNase, RNase, and an acidic protease, but is sensitive to alterations in pH. Taken together, these results indicate that these lactobacilli secrete bioactive molecules of low molecular weight that may modulate host innate immune activity through interactions with IEC.

Extracellular Vesicles with Possible Roles in Gut Intestinal Tract Homeostasis and IBD

“…Cells. IECs promote the development of dendritic cells (DCs) and macrophages with tolerogenic properties by producing numerous immunoregulatory signals, including TGF-β, thymic stromal lymphopoietin (TSLP), and retinoic acid [63][64][65]. Professional antigen-presenting cells (APC) have been verified to secrete major histocompatibility complex-(MHC-) bearing vesicles called exosomes, which are a subset of EVs [66].…”

Secretome-Mediated Interactions with Intestinal Epithelial Cells: A Role for Secretome Components from Lactobacillus rhamnosus R0011 in the Attenuation of Salmonella enterica Serovar Typhimurium Secretome and TNF-α–Induced Proinflammatory Responses

“…Lyophilized L. rhamnosus R0011 was obtained from Rosell Institute for Microbiome and Probiotics (Montreal, QC). The LrS was prepared as previously described (21). Briefly, bacteria were grown in de Man, Rogosa, and Sharpe medium (Difco) at 37˚C for 17 h in a shaking incubator and then diluted in nonsupplemented RPMI 1640 medium and allowed to further propagate for an additional 23 h under the same conditions.…”

“…The HT-29 human colorectal adenocarcinoma cell line was obtained from the American Type Culture Collection (no. HTB-38) and was maintained in RPMI 1640 medium supplemented with 10% bovine calf serum and 0.05 mg/ml gentamicin (Sigma-Aldrich) and were grown in 75-cm 2 tissue culture flasks (Greiner Bio-One) at 37˚C, 5% CO 2 in a humidified incubator (Thermo Fisher Scientific), as described previously (21). HT-29 IECs were enumerated and viability determined using trypan blue following subculturing.…”

“…We previously reported that the L. rhamnosus R0011 secretome (LrS) downregulates the production of IL-8 from IECs challenged with a wide array of innate immune stimulants including TNF-a, TLR, and NOD1 agonists (21). In the current study, we aimed to further interrogate the signaling pathways modulated by the LrS in HT-29 IECs exposed to TNF-a to better understand the mechanism(s) of action behind the observed bioactivity, as TNF-a induction is a common feature of many pattern recognition receptor (PRR)-mediated pathways.…”

Milk fermented with Lactobacillus rhamnosus R0011 induces a regulatory cytokine profile in LPS-challenged U937 and THP-1 macrophages

“…In this study, the impact of dairy milk-cultured L. rhamnosus R0011 on macrophage cytokine production profiles was examined. Secretome derived from L. rhamnosus R0011 down-regulates inflammatory mediator production from challenged intestinal epithelial cells, suggesting a role for bioactive molecule production by this lactic acid bacterium ( Jeffrey et al., 2018 ). When cultured in milk, L. rhamnosus R0011 produces soluble mediators that down-regulate interleukin (IL)-1β-induced prostaglandin production, a key lipid mediator in the inflammatory response, from human intestinal epithelial cells (IEC) ( Fiander et al, 2005 ).…”