PATHOGEN AND PROBIOTIC BACTERIA DIFFERENTIALLY STIMULATE NITRIC OXIDE PRODUCTION AND S100B PROTEIN EXPRESSION IN HUMAN ENTEROGLIAL CELLS

Background and aim: Enteric glial cells (EGC) are involved in intestinal homeostasis and may contribute to regulate host-bacteria interaction. Astrocytes, the equivalent of enteroglial cells (EGC) in Central Nervous System respond to bacteria releasing nitric oxide (NO), whether this does occur in bacterial-EGC interaction is not known. We aimed to investigate whether human EGC generate NO in response to pathogen and probiotic bacteria and whether this is associated with S100B overexpression.Material and methods: Human EGC were obtained according to a method previously described by our group. Briefly, myenteric plexus preparations were isolated from ileum of patients undergoing surgery and enzimatically dissociated. Ganglia were plated and cell cultures were grown to subconflu- ence. After 21 days, EGC were purified by incubation with the anti-Thy-1.1 ab-coated magnetic beads and separated using a Dynal Magnet ® . EGC were incubated for 24 hours with the probiotic Lactobacillus Paracasei F19 (LP F19) and the pathogen Enteroinvasive Escherichia Coli (EIEC). 2 different bacteria/cells ratios were used (0.1/1 and 10/1, respectively). Nitrite assay and Western Blot analysis were respectively used to evaluate NO release and S100B expression in stimulated cells compared to unstimulated cells that served as controls. Data are expressed as mean ± SD of 3 independent experiments. Results: Glial derived S100B protein expression was significantly higher in response to EIEC than to LP F19 (+2.9 ± 0.2 and +0.9 ± 0.3 fold increase vs control; p < 0.05). EIEC induced a significantly higher NO release than LP F19 both at a 0.1/1 (17.7 ± 0.7 vs 4.0 ± 0.1 nmol x 10ˆ6 cells; p < 0.001) and at 10/1 ratio (20.7 ± 2.1 vs 9.0 ± 0.1 nmol x 10ˆ6 cells; p < 0.001). Compared to control conditions (3.7 ± 0.1 nmol x 10ˆ6 cells), EIEC and high concentration of LP F19 induced a significant increase of NO release (all p < 0.001). Conclusions: We show that EGC are able to release nitric oxide when challenged with bacteria and that this is likely dependent on the different expression of S100B protein. As EGC-released NO was different between pathogen and probiotic bacteria we suggest that human EGCs likely participate to host-bacteria interaction via a different NO release