LACTOBACILLUS SPP INHIBIT LIPOPOLYSACCHARIDE INDUCED IL-8 PRODUCTION IN MACROPHAGES

S Lebel, NL Jones

Hospital for Sick Children and Department of Paediatrics and Physiology, University of Toronto, Toronto, Ontario

Probiotics have beneficial effects in several models of inflammatory bowel disease (IBD). The intestinal flora and lipopolysaccharide (LPS), play an important role in the pathogenesis of IBD. Macrophages play a critical role in response to LPS stimulation. Therefore, we hypothesized that probiotics would prevent LPS-mediated inflammatory responses in macrophages.
AIM: The aim of this study was to determine the role of probiotics in the modulation of LPS-mediated IL-8 production in macrophages.
METHODS: THP-1 monocytic cells were infected with Lactobacillus rhamnosus R011 or L. acidophilus R052 at concentration of 10⁷ to 10⁹ CFU/ml for up to 24 hours. Uninfected cells served as controls. Cells were then stimulated with Escherichia coli 026:B6 LPS (100ng/ml) for 24 hours. IL-8 production was measured by immunoassay. The expression of the LPS receptor, Toll-like receptor 4 (TLR4), was detected by Western blotting. Cell death was assessed by fluorescence microscopy following acridine orange/ethidium bromide staining.
RESULTS: THP-1 cells produced IL-8 in response to LPS in a dose and time dependent manner with maximal IL-8 production at a dose of 100ng/ml for 24 hours (7505pg/ml±1703 p=0.0014). LPS-mediated IL-8 production was not associated with a change in TLR4 protein expression. Pretreatment with L. rhamnosus R011 at concentration of 10⁸CFU/ml inhibited LPS mediated IL-8 production (7505pg/ml ± 1703 vs 528 pg/ml±127.2 and 800.3pg/ml±156 following 4 and 24hours pretreatment respectively) (p<0.001). L. rhamnosus R011 also inhibited LPS mediated IL-8 secretion at a concentration of 10⁹CFU/ml (3533pg/ml±1198 vs 0ng/ml and 0ng/ml following 4 and 24 hours pretreatment respectively) (p>0.05). Similar results were documented with L. acidophilus R052 at concentration of 10⁸ and 10⁹CFU/ml. Both L. rhamnosus and L. acidophilus induced cell death of THP-1 cells in a time and dose dependent manner with a peak cytotoxicity at 24 hours at a concentration of 10⁹CFU/ml (control 12.5%±3.8 vs L. rhamnosus 90% vs L. acidophilus: 57.5%±37.5 [n=2]).
CONCLUSION: Taken together, these results indicate that Lactobacillus spp trigger cell death in activated macrophages resulting in a reduced production of pro-inflammatory mediators. This provides one possible explanation for their beneficial effect in intestinal inflammation.