Background and Aim: Enteric glial cells (EGC) are involved in intestinal inflammation and can be activated by exogenous stimuli, and foreign antigens (i.e. gliadin). Toll-Like Receptors (TLRs) are immune receptors able to recognize pathogen conserved molecular patterns. Whether EGC express TLRs or respond to bacterial stimuli is unknown. We aimed to investigate if TLRs are expressed by human EGC and if their expression is differentially modulated by pathogens or probiotics bacteria. Materials and Methods: Pure EGC were obtained according to 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 subconfluence. After 21days, EGC were purified using Dynal Magnet® and characterized by immunofluorescence to confirm the absence of contaminating cells (i.e. fibroblasts). TLRs 1, 2, 4, 7 and 9 mRNA expression were evaluated at baseline and after 24 hours (h) incubation of EGC with the probiotic strain Lactobacillus Paracasei F19 (LP F19) and the pathogen Enteroinvasive Escherichia coli (EIEC). The bacteria/cells ratio was 0.1/1 and 100/1. Quantitative Real Time PCR was performed to evaluate specific mRNAs fold increases respect to untreated controls. Data were obtained with 3 experiments and expressed as mean±SD. Results: Human purified EGC physiologically expressed TLR 1, 2, 4, 7 and 9 mRNAs, with TLR 1 and 4 transcripts being the most expressed. With a bacteria/cells ratio of 0.1/1, EIEC induced a higher significant increase than LP F19 of TLR 2 (4.1±0.1 and 2.7±0.4; p<0.05), TLR 7 (8.0±0.2 and 0.6±0.2; p<0.05) and TLR 9 (8.8±1.7 and 1.0±0.2; p<0.05). Conversely, only a trend to an increased expression of TLR 4 was observed with LP F19 respect to EIEC (2.4±0.1 and 1.3±0.4; p=NS). At 100/1 bacteria/cell ratio EIEC determined an 80% of cellular mortality, while LP F19 induced an higher expression of TLR 7 (17.8±4.1) and 9 (21.2±4.9) respect to lowest bacteria/cell ratio, while the other TLRs remained unmodified. Conclusion: We showed, for the first time, that pure cultures of human EGC express TLRs' mRNA and are able to discriminate between pathogen and probiotic bacteria. This data support the hypothesis that enteric glia is involved in the host-pathogen interaction.
Human Derived Enteroglial Cells Toll-Like Receptor smRNA Expression and Modulation by Pathogen and Probiotic Bacteria / F., Turco; C., Cirillo; Sarnelli, Giovanni; A., Mango; M., Cammarota; M., Giuliano; M., Cartenì; Cuomo, Rosario. - In: GASTROENTEROLOGY. - ISSN 0016-5085. - ELETTRONICO. - 138:(2010), pp. S37-S37. [10.1016/S0016-5085(10)60171-0]
Human Derived Enteroglial Cells Toll-Like Receptor smRNA Expression and Modulation by Pathogen and Probiotic Bacteria
SARNELLI, GIOVANNI;CUOMO, ROSARIO
2010
Abstract
Background and Aim: Enteric glial cells (EGC) are involved in intestinal inflammation and can be activated by exogenous stimuli, and foreign antigens (i.e. gliadin). Toll-Like Receptors (TLRs) are immune receptors able to recognize pathogen conserved molecular patterns. Whether EGC express TLRs or respond to bacterial stimuli is unknown. We aimed to investigate if TLRs are expressed by human EGC and if their expression is differentially modulated by pathogens or probiotics bacteria. Materials and Methods: Pure EGC were obtained according to 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 subconfluence. After 21days, EGC were purified using Dynal Magnet® and characterized by immunofluorescence to confirm the absence of contaminating cells (i.e. fibroblasts). TLRs 1, 2, 4, 7 and 9 mRNA expression were evaluated at baseline and after 24 hours (h) incubation of EGC with the probiotic strain Lactobacillus Paracasei F19 (LP F19) and the pathogen Enteroinvasive Escherichia coli (EIEC). The bacteria/cells ratio was 0.1/1 and 100/1. Quantitative Real Time PCR was performed to evaluate specific mRNAs fold increases respect to untreated controls. Data were obtained with 3 experiments and expressed as mean±SD. Results: Human purified EGC physiologically expressed TLR 1, 2, 4, 7 and 9 mRNAs, with TLR 1 and 4 transcripts being the most expressed. With a bacteria/cells ratio of 0.1/1, EIEC induced a higher significant increase than LP F19 of TLR 2 (4.1±0.1 and 2.7±0.4; p<0.05), TLR 7 (8.0±0.2 and 0.6±0.2; p<0.05) and TLR 9 (8.8±1.7 and 1.0±0.2; p<0.05). Conversely, only a trend to an increased expression of TLR 4 was observed with LP F19 respect to EIEC (2.4±0.1 and 1.3±0.4; p=NS). At 100/1 bacteria/cell ratio EIEC determined an 80% of cellular mortality, while LP F19 induced an higher expression of TLR 7 (17.8±4.1) and 9 (21.2±4.9) respect to lowest bacteria/cell ratio, while the other TLRs remained unmodified. Conclusion: We showed, for the first time, that pure cultures of human EGC express TLRs' mRNA and are able to discriminate between pathogen and probiotic bacteria. This data support the hypothesis that enteric glia is involved in the host-pathogen interaction.File | Dimensione | Formato | |
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