Mechanism Of Mast Cell In Bacterial Diarrhea

The mechanisms of diarrhea have been well discussed,but the exact mechanism needs to be further understood.In fact,intestinal epithelial cells are thought to be tolerant to commensal bacterial toxins and components;however,how these microbial products break down the established "tolerance" and induce diarrhea and other inflammatory reactions remains unclear.Mast cells are strategically localized at the host-environment interfaces such as the skin,the airway,and the intestine.Mast cells thus are regarded as a critical component of host defense against bacterial infections.However,the mechanism of diarrhea caused by microbial product-induced mast cell mediator release remains largely unknown.Staphylococcus aureus(S.aureus) is one of the pathogens associated with infectious diarrhea.Peptidoglycan(Peptidoglycan,PGN) is a cell wall component of almost all gram positive(including S.aureus) and gram-negative bacteria that has strong immune activity.It is capable of modifying the functions of some immune cells and may play a critical role in bacterial diarrhea.The objectives of this study involved testing 1)the influence to T84 monolayer's barrier function by PGN,2)whether HMC-1 dyfuction the T84 monolayer's barrier,3) the uptake pathway of PGN by HMC-1,4)whether oral PGN induces diarrhea in mice,5)whether TLR2 and nucleotide-binding oligomerization domain(NODs) mediate PGN-induced mast cell activation in the intestine,and 6)whether mast cell activation is required in PGN-induced diarrhea. Methods1.Cell culture and evaluation of the barrier function of T84 monolayerT84(T84 Monolayer) cells were employed in this study.Only passages 35-45 were used in the experiments.To evaluate the transport of PGN by T84 monolayers,purified PGN and HRP(Horseradish peroxidase,HRP) was added to the apical chambers of transwell.PGN contents and HRP in samples were determined by enzyme-linked immune assay(ELISA).HRP flux was performed to evaluate the permeability of T84 monolayers.2.Measurement of PGN uptake by HMC-1 cells(Human mast cell strain),THP-1(Human mononuclear cell strain) and HEK293(Human embryo kidney cell strain)T84 cells and HMC-1 cells(or THP-1 and HEK293) were co-cultured in transwell systems.PGN was added to the apical chambers.Cells were collected from the basal chambers 2 h after the addition of PGN.Total proteins were extracted from the cells to determine the contents of PGN in HMC-1 cells(or THP-1 and HEK293) with western blot.A portion of the cells was fixed with cold acetone,stained with anti-PGN monoclonal antibody and fluorescein-conjugated rabbit anti-mouse second antibody The stained cells wore observed under a confocal microscope.3.Histamine assayHistamine and 5-HT levels in HMC-1 cell(THP-1,HEK293),colon mucosal mast cells and P815 cells were determined by ELISA.4.Expression of TLR2 and NOD1 in P815 cellsExpression of TLR2 and NOD1 in P815 cells with immunocytochemistry and Western-Blot5.mRNA expression of TLR2 and NOD2 in knock down HMC-1 cellsRNA interference(RNAi) was employed to knock down the mRNA expression of TLR2 and NOD2 in HMC-1 cells,respectively.The efficiency of siRNA transfection was determined by real-time reverse transcription(RT)-PCR(or qPCR) and western blotting,respectively. 6.A murine model of diarrhea elicited by PGNDiarrhea was induced by oral administration with graded doses of purified PGN. Mast cell mediator antagonist pretreat mice before gavage.The percentage of water in stool was calculated for each mouse.The wet weight of the small and large intestines as well as the body weight were recorded.The ratios of the small and large intestine/body weight were calculated to assess the watery fluid accumulation in the intestinal tract.7.Expression of TLR2,NOD(1,2)in intestinal mucosal mast cell and P815 cellsExpression of TLR2,NOD(1 or 2)in intestinal mucosal mast cell and P815 cells were observed with confocal microscope.8.Mast cell reconstitutionCultured mast cells(＞95%purity) were injected to W/Wv mice via the tail vein at a dose of 30,000,40,000,and 50,000 cells/mouse in 0.25 saline;the transfer was executed once more a month later.The mice were used for oral PGN experiments 2 months after the first mast cell reconstitution.Statistical AnalysisData were expressed as means±SD.Differences between two groups were analyzed with the Student's t-test;three or more groups were analyzed with the analysis of variance.Differences between means at a level of P＜0.05 were considered significant.Results1.PGN was added to the apical chambers.TER and permeability to HRP were chosen as indicators of barrier function and determined in T84 monolayers.The results showed that the addition of PGN did not alter TER and HRP in T84 monolayers as compared with naive controls.It is indicate that PGN does not directly alter the barrier function of human intestinal epithelial cell line T84 monolayers.2.Confluent T84 cells were co-cultured with HMC-1 cells in transwell system.Two hours after the addition of PGN to the apical side,histamine levels increased in culture media of the basal chambers in a dose dependent manner.Electron microscopy results showed an extensive degranulation in these mast cells after stimulated by PGN.The TER of T84 monolayers dropped significantly after the addition of PGN that was abolished by the pretreatment with mast cell stabilizer ketotifen.The result show that T84 monolayers transport PGN via HMC-1.3.We hypothesized that TLR2 might mediate PGN absorption in mast cells.HMC-1 cells were cultured in the presence or absence of PGN for 2h.HMC-1 cells were collected and processed for identifying the expression of TLR2.As shown by qPCR,the expression of TLR2 was 0.28±0.06 in HMC-1 cells and 0.29±0.1 in THP-1 cells;HEK293 cells did not show any expression of TLR2 mRNA;the data were also shown by agarose gel electrophoresis bands.The expression of TLR2 in HMC-1 and THP-1 cells(but not in HEK293 cells) was confirmed by western blotting and immunocytochemistry.4.We examined if PGN was absorbed into HMC-1 cells.As observed with confocal microscopy,the absorbed PGN was localized inside HMC-1 and THP-1 cells,but not in HEK293 cells.5.The qPCR results showed that NOD2 mRNA was detected in HMC-1 cells (0.28±0.08 in HMC-1 cells;0.27±0.06 in THP-1 cells;no NOD2 mRNAwas detected in HEK293 cells).As expected,histamine release from HMC-1 cells pretreated with anti-NOD2 antibody or transfected NOD2 siRNA was significantly inhibited as compared with those with controls(naive HMC-1 cells or HMC-1 cells transfected with control siRNA).The results indicate that NOD2 plays a critical role in PGN induced HMC-1 cell activation.6.HMC-1 and T84 cells were co-cultured in transwell system with or without the addition of PGN to the apical chambers in the presence or absence of HMC-1 cells in the basal chambers.HRP flux was carried out and TER was recorded serving as indicators of the epithelial barrier function.The addition of PGN to transwells significantly increased the HRP flux and decreased the TER in T84 monolayers in a dose-dependent manner.The results showed that the increase in HRP recovery at the basal chambers and the decrease in TER were parallel to the number of HMC-1 cells in the basal chambers.Pretreatment with siRNA of TLR2 or NOD2 (but not the control siRNA) abolished the PGN induced T84 monolayer barrier dysfunction.7.Mice were exposed to graded doses of PGN in saline via intragastric garage.The severity of diarrhea appeared in a dose-dependent manner.The results demonstrate that oral PGN is able to induce diarrhea in mice.Mast cell-deficient mice,W/Wv mice,and the +/+ littermate mice were treated with PGN,resulting in diarrhea in +/+ mice but not in W/Wv mice.The result show that mast cell play a important role in PGN-induced diarrhea.8.As determined by electron microscopy,mast cell degranulation was defined as lost granular contents or decreases in density of granules that was clearly observed in intestinal tissue of mice treated with PGN.The ratio of degranulation in mast cells increased significantly after treatment with PGN in a dose-dependent manner. As shown by ELISA,PGN administration resulted in significantly higher levels of serotonin and histamine in culture media as compared with controls.Treatment with the mast cell stabilizer ketotifen or antibodies against TLR2 or NOD1 efficiently inhibited the release of serotonin and histamine as well as mast cell degranulation.9.To probe the pathways involved in PGN-induced mast cell activation,we assessed the effects of exposure to PGN on cytoplasmic IκB levels by immunoblotting.The decrease in IκB levels indicates the release of nuclear factor-κB and its translocation to the nucleus.P815 cells were treated with PGN and tested for the phosphorylation and expression of the unphosphorylated IκB-αand IκB-βusing Western blot assays.10.Mice were pretreated with graded doses of mast cell stabilizer ketotifen and then treated with oral PGN.Diarrhea was inhibited by the pretreatment with ketotifen in a dose-dependent manner.A group of mice was treated with oral PGN first after the diarrhea symptoms occurred;a single dose of ketotifen also resulted in stopping diarrhea.We also found the synergistic effect of antihistamine and antiserotonin attenuates PGN-induced diarrhea.Conclusions1.PGN does not directly alter the barrier function of human intestinal epithelial cell line T84 monolayers and mice intestinal mucosa;2.PGN activates mast cells and dysfunction the T84 monolayers;3.TLR2 and NOD2 mediate PGN absorption in mast cells;4.TLR2 and NOD1 Mediate PGN-Induced Mast Cell Activation;5.Oral PGN induce diarrhea;6.Intestinal epithelial cells absorb PGN via an intracellular pathway,7.Mast cell stabilizers inhibit PGN-induced diarrhea.