Differential nitric oxide production by probiotic bacteria conditioned medium in J774 cell

Differential Nitric Oxide Production By Probiotic Bacteria Conditioned Medium In J774 Cells
Mosharraf H. Sarker1, Nasima S. Chowdhury1, and Anwar R. Baydoun2,
1.Centre for Applied Science, School of Science & Engineering, Teesside University
2.School of Life Sciences, University of Hertfordshire
Introduction: Probiotic bacteria are known to stimulate the body’s immune system and increase the capacity of the host to fight against infection. They have been shown to increase nitric oxide (NO) and prostaglandin E2 (PGE2) production and play important roles in regulating cellular reactions during inflammatory response. There are evidences that health beneficial effects are mediated by unknown active metabolites produced by probiotics1. In the present study we have elucidated further the effect of probiotic conditioned cell culture medium on the modulation of NO production and iNOS and COX-2 expression in J774 Murine macrophages cells.
Methods: Probiotic conditioned medium of single probiotic strain Lactobacillus rhamnosus GG (LGG) and multi-strain preparation of 8 probiotic bacteria VSL#3 were obtained after 24 hrs of incubation of bacteria in the cell culture medium. Confluent J774 cells monolayer were incubated with probiotic conditioned medium for 24 hours and NO production was measured from nitrite in the cell culture medium by Griess assay and iNOS and COX-2 expression were measured using Western blot assay. J774 cell were loaded with Fura-2AM and ratio (340/380) of [(Ca2+)i] images were captured before and after adding VSL#3 conditioned cell culture medium.
Results: LGG conditioned medium demonstrated a low but concentration dependant increase in basal NO production. However, when LGG was applied along with LPS, NO production was reduced. Similar concentration dependant NO production was also observed with VSL#3 conditioned medium but it produced variable NO production when co-applied with LPS. Expression of iNOS protein showed the same pattern of NO production. A dose dependent basal COX-2 expression was also observed when VSL#3 was applied alone, however, no reduction of COX-2 was observed when co-applied with LPS. VSL#3 found to rise in intracellular calcium in J774 cells.
Discussion: It was apparent that the low basal increase in NO production, iNOS and COX-2 expression were not induced by bacterial endotoxin. A balanced small expression of iNOS and COX-2 is necessary for the regulation of normal cellular function. Low physiological levels of NO preserves cells integrity but excess production of it produces pro-inflammatory responses which lead to tissue injury (Korhonen et. al, 2001, Inflammation, 25(4):223-32 ). Therefore, the ability of these microfloras to modulate the basal NO, iNOS and COX-2 can be a novel approach in improving the intestinal homeostasis and immunity. Low increase in NO production due to probiotic conditioned medium probably mediated via raising intracellular calcium in J774 cells.