Research On The Role Of Transient Receptor Potential Melastatin2in Sepsis And Underlying Mechanism

Sepsis is characterized as a harmful and dysregulated inflammatory response to infection. Sepsis remains the chief cause of death in intensive care units, with overall mortality rate approximately30%～50%in severe or septic shock patients. The failure of anti-lipopolysaccharide, anti-proinflammatory cytokine, interfere with dysregulated cogulation, and anti-oxidantive stress strategies for treatment of sepsis in clinical trials led researchers to state that other key pathophysiological mechanisms of sepsis may exist. Increasing evidence supports that immunosuppression exits in septic patients and plays a central role in sepsis. The immune cells of both innate and adaptive system are severely suppressed in septic patients, resulting in reduced capability of immune cells to eradicate invading pathogens. Despite antimicrobial therapy used, the septic patients frequently have ongoing infectious foci. Macrophages serve as the first line of host defense for killing invading microorganisms. However, in septic patients, macrophage function is severely impaired, which may promote uncontrolled microbial growth.Transient Receptor Potential Melastatin2(TRPM2), a nonselective Ca2+-permeable channel, is expressed abundantly in macrophages and its expression could be up-regulated under lipopolysaccharide stimulation. Accumulated studies suggest an important role of TRPM2in macrophage function, including imflammatory cytokines production and phagocytic capacity. Disruption of TRPM2attenuates inflammatory mediator production and phagocytic activity. These findings suggest that TRPM2may involve in the pathogenesis of sepsis by immnuoregulating the macrophage function. However, the role and the underlying mechanism of TRPM2in polymicrobial sepsis remain insufficiently understood thus far.Heme oxygenase-1(HO-1), a stress-responsive enzyme, plays a key role in protecting the host against inflammation, oxidantive stress, and apoptosis. Furthermore, HO-1also plays a protective role in host defense against microbial sepsis by enhancing bacterial clearance. Recent studies indicate that calcium influx is critical for HO-1induction. Whether TRPM2-mediated calcium influx plays a role in bacterial clearance within macrophage by regulating HO-1expression is unknown. Whether TRPM2-mediated HO-1expression protects mice against sepsis by enhancing bacterial clearance is also needed to be explored.Here we sought to investigate the effect and underlying mechanism of TRPM2on bacterial clearance within murine bone marrow-derived macrophages (BMDM) and in cecal ligation and puncture (CLP)-induced polymicrobial sepsis model. We aimed to explore the role of TRPM2in the sepsis and underlying mechanism. Furthermore, we also investigated whether TRPM2expression correlates with HO-1expression in monocytes from septic patients and contribute to the outcome. Our data may offer additional insight into the role and mechanism of TRPM2in the pathogenesis of sepsis, which provides a new target for the prevention and treatment of sepsis. Part OneEffects of TRPM2on Bacterial Clearance within Murine Macrophages and Underlying MechanismObjective:To investigate the effect of transient receptor potential melastatin2(TRPM2) on bacterial clearance within macrophages and the underlying mechanism.Methods:Using Bone marrow-derived macrophages (BMDMs) from wildtype (WT) and Trpm2-knockout (KO) mice, we investigated the role of TRPM2in regulating heme oxygenase-1(HO-1) expression in BMDMs stimulated with100ng/ml lipopolysaccharide (LPS) for12h. Electrophysiology, intracellular calcium concentration detection, and immunoblot were used to explore the mechanism of TRPM2in regulating HO-1expression. Intracellular bacterial killing assay was performed to investigate the effect of TRPM2on bacterial clearance within macrophages and the possible mechanism.Results:Among BMDMs tested from WT mice, cells displayed LPS-induced whole cell currents with characteristic of TRPM2channels. In BMDMs from Trpm2-KO mice, this LPS-induced current was abolished. At12hours after LPS, HO-1expression in Trpm2-KO BMDMs was significantly less than in WT BMDMs (P<0.01). Removal of the extracellular Ca2+dose-dependently reduced the increased expression of HO-1. The intracellular Ca2+concentration decreased in Trpm2-KO BMDMs compared with WT BMDMs at1,2,12, and24hours after LPS stimulation. The bacterial burden was greater in Trpm2-KO BMDMs than in WT BMDMs (P<0.05). HO-1inducer (hemin) could markedly increase LPS-induced HO-1expression and decrease bacterial burden in the WT BMDMs compared with WT BMDMs treated with vehicle control (P<0.001). Treatment of Trpm2-KO BMDMs with hemin markedly increased LPS-induced HO-1expression and decreased bacterial burden compared with Trpm2-KO BMDMs treated with vehicle control (P<0.01).Conclusion:TRPM2-mediated Ca2+influx may play an important role in regulation of LPS-induced HO-1expression in macrophages. TRPM2may critical for bacterial killing by regulating HO-1expression in macrophages. Part TwoThe Role of TRPM2in Sepsis and Underlying MechanismObjective:To investigate the role of transient receptor potential melastatin2(TRPM2) in sepsis and the underlying mechanism.Methods:Using cecal ligation and puncture (CLP)-induced polymicrobial sepsis model, we investigated the role of TRPM2in sepsis through comparing the mortality, bacterial clearance, organ injury, systemic inflammation, and heme oxygenase-1(HO-1) expression in macrophages from peritoneal cavity between septic Trpm2-KO and WT mice. To explore the possible mechanism of TRPM2in controlling bacterial reproduction during polymicrobial sepsis via HO-1expression, we examined whether the HO-1inducer improved bacterial clearance and decreased the mortality, organ injury, and systemic inflammation in the septic WT and Trpm2-KO mice.Results:After CLP, Trpm2-KO mice had increased mortality compared with wild-type (WT) mice (P<0.05). The increased mortality was associated with increased bacterial burden, organ injury, and systemic inflammation. Up-regulation of HO-1expression in peritoneal macrophages could decrease bacterial burden in CLP-induced septic WT mice. The HO-1expression in peritoneal macrophages after CLP decreased significantly in Trpm2-KO mice compared with WT mice (P<0.05). Pretreatment of Trpm2-KO mice with HO-1inducer markedly increased HO-1expression in peritoneal macrophages (P<0.05) and decreased bacterial burden after CLP, resulting in decreased mortality, organ injury, and systemic inflammation. Conclusion:Our data demonstrate a protective role of TRPM2in controlling bacterial reproduction during polymicrobial sepsis, possibly by regulating HO-1expression. Part ThreeThe Expression of TRPM2and HO-1in Peripheral Blood Monocytes from Septic PatientsObjective:To further confirm the relationship between the transient receptor potential melastatin2(TRPM2) and heme oxygenase-1(HO-1), and evaluate whether TRPM2or HO-1expression contributes to the outcome of septic patients.Methods:Using peripheral blood monocytes separated from septic patients, we examined the TRPM2and HO-1mRNA expression by real-time PCR. The demographics, APACHE II and SOFA score, and30-days mortality of septic patients were also recorded.Results:No significant differences were found between severe septic or septic shock patients and non-septic patients with respect to age, gender, ICU stay. Severe septic or septic shock patients had higher APACHE II socre (P=0.001), SOFA score (P<0.05), and28-day mortality (P<0.05) compared with non-septic patients. Real-time PCR analysis showed that monocytic TRPM2mRNA levels in unsurvival septic patients were lower than non-septic patients (P<0.05) or survival septic patients (P=0.001). Monocytic HO-1mRNA levels in unsurvival septic patients were lower than non-septic patients (P<0.05) or survival septic patients (P<0.05). No differences were found in TRPM2and HO-1mRNA levels between non-septic patients and recovered septic patients. The TRPM2mRNA levels were significantly correlated with HO-1mRNA levels (r=0.675, P=0.001). Conclusion:Monocytic TRPM2and HO-1expression were significantly lower in patients with severe sepsis or septic shock who died than in those who survived. TRPM2expression correlates with HO-1expression in peripheral blood monocytes from septic patients.