The Mechanism Of Neutrophils Inflitration During Sepsis And The Interventional Effect Of CORM-2

BackgroundSepsis is defined as the systemic inflammatory response to infection,when it became severe that would follow with organ dysfunction,such as tissue hypoperfusion and hypoxia,lactic acidosis,oridiguria,or altered cerebral function.Now with the treatment with antibiotics,provision of adequate fluid resuscitation and technological support of organ function,the mortality rate of sepsis is still high,approximately 35%.The most common pathogens that infect individuals is bacteria and people who undergo sepsis will suffer from lung dysfunction,cardiovascular instability and deterioration renal function.Neutrophils play a pivotal role in the defence against bacterial infections.For example,after chemotherapy,patients would experience neutropenia which increased the susceptibility to infection and even to sepsis.However,overwhelming activation of neutrophils is known to elicit tissue damage.Usually,amount of neutrophils migration to target infection site is needed when the auto-defence system enhanced to anti-infection.In fact,neutrophils are sensitive to inciting stimuli and able to be rapidly and efficiently mobilized in great numbers within minutes to areas of inflammation.In order to finish the effective response,they should detect extracellular chemical gradients and move towards the higher concentration focus.During this process,chemoattractants orchestrate neutrophil migration,cooperating temporally and spatially.In general,neutrophils response t chemoattractants in a hierarchical manner;in other words,they can prioritize among them.Based on this concept,the chemoattractants can be divided into two groups: “end target”,such as f MLP and C5a;“intermediary”,such as lipid mediators and chemokines.And in it,the end target chmoattractants are dominant over the intermediary chemoattractants.Neutrophils migrate to infection areas and display a host-protective role in sepsis via the killing of microorganisms.Besides,there are also some deleterious functions of neutrophils in sepsis.During sepsis,due to the systemic inflammatory response,the circulating neutrophils were activated and sequestered in capillary beds,occluding the lumen and inducing tissue ischemia.Moreover,neutrophils can migrate to distal organs,releasing lytic factors,pro-inflammatory factors,which contribute the organs injury and subsequent multiple organ dysfunction.Examination of autopsy specimens from patients with multiple organ failure reveals that neutrophils are localized from sequestration and aggregation in renal blood vessels to large-scale infiltration of the lung.In the acute respiratory distress syndrome(ARDS),sepsis always accompany with the severe form of acute lung injury.It has been shown that there is a close relationship between the intensity of neutrophil infiltrates and impaired lung function and in the bronchoalveolar lavage,a high concentration of neutrophil-derived proteolytic enzymes is detected.In recent years,several studies have described the mechanisms underlying CXCR2 internalization in circulating neutrophils during sepsis,resulting in failure of migration to infectious focus.Otherwise,in the organ tissue neutrophils follow the CXC chemokine guiding and infiltrate in the lung depended on CXCR2.Based on the data above,the organs damage in sepsis is correlated with neutrophils migration.Indeed,it has been shown that the expression and distribution of chemoattratants receptors play a vital role in neutrophils migration.In the recent study,it has found that the end target chemoattratants signaling fmlp through MAPKs(ERK,p38)pathway regulates neutrophils “go” or “stop”.fMLP interacts with its receptor FPR1 and activates the GPCR,then affecting the neutrophils chemotaxis.In addition,f MLP phosphorylates its receptors through potentiation of GRK2 activity contributing to the receptor desensitization and internalization.Then neutrophils stop migration and the process above also regulated by the MAPKs pathway.Certain CORMs activate the CO release process within a solvent by initiating solvent-induced ligand exchange with the solvent or solutes.CORM-2 is a ruthenium carbonyl compound which is dissolved in dimethyl sulfoxide(DMSO)before being added to aqueous biological solutions and buffers.In septic mice models in which cecal ligation and puncture(CLP)or LPS injection were performed to induce sepsis,the treatment with CORM-2 attenuated the migration and accumulation of polymorphonuclear leukocytes and neutrophils and inhibited the production of nuclear factor kappa B(NF-kB),ICAM-1and reactive oxygen species(ROS).Most importantly,CORM-2 administration increased the survival rate in septic mice.Injection of CORM-2 increased phagocytosis in wild-type mice and improved the survival rate of Hmox1-/-mice.In this study,using LPS injection induced model of septic mice and in vitro neutrophils stimulated with LPS and then administrated with CORM-2.The neutrophils infiltration in distal organ and the chemotaxis change of LPS challenged neutrophils were observed.Moreover,we also investigated the influence of CORM-2 on neutrophil infiltration in sepsis and its potential mechanisms were also explored.MethodsThe present study used LPS-treated septic mice as the in vivo model and the CORM-2 was introduced to treat the septic mice.Survival rate is observed for 5 consecutive days.Pathological HE staining is used to observe the damage of liver and lung,the infiltration of neutrophils in distal organs.MPO assay kit is used to detect the neutrophils accumulation in liver and lung.ELISA assay kit is used to assay the expression of cytokines(TNF-a,IL-1b).MDA detecting kit is used to evaluate the level of MDA in liver and lung.The present study used LPS-treated neutrophils as in vitro model and the CORM-2 was introduced to treat the LPS challenged neutrophils.Neutrophils chemotaxis in single chemoattratant or in two different chemoattractants condition is observed with the using of under agarose chemotaxis experiment.Transcriptome genechip and proteome array are used to detect the change of neutrophils chemoattractants receptors and the activity of MAPKs signaling pathway.RT-PCR,flow cytometry and western blot are used to determine the expression of key signal molecular and the relevant receptors.Laser scanning confocal microscope is used to observe the distribution of chemoattractant receptors and GRK2.Neutrophils apoptosis and phagocytosis are evaluated with flow cytometry.In addition,the optical microscope using the biophase imaging device is used to visualize the neutrophils phase shift.ResultsIn vivo study,it is shown that the pathological injury and inflammatory response(TNF-a,IL-1b,MDA,HE)in liver and lung are sever of septic mice.In addition,neutrophil accumulation(HE,MPO)in liver and lung is also observed in the septic mice.Treatment with CORM-2,the above inflammatory response and the neutrophil infiltration in liver and lung of septic mice are improved.Moreover,the administration of CORM-2 enhances the survival rate of septic mice.In vitro study,it is shown that LPS promotes the hierarchical neutrophil chemotaxis via p38 activation.In a single chemoattract condition,LPS challenged neutrophil chemotaxis increased.The data of genechip indicates the up-regulation of FPR1 in the mRNA level,while the western blot suggests there is no significant change in FPR1 in the protein level.The incompatible expression between mRNA and protein reveals the post-transcriptional or post-translation effects of LPS.Moreover,we evaluated the distribution of FPR1 which is shown that during the chemotaxis FPR1 mainly expresses on the membrance.Treatment with CORM-2 inhibits the chemotaxis of LPS stimulated neutrophils.The results is not related with the neutrophils viability and it is regulated by interfering with FPR1 via p38 MAPK,but not GRK2 pathway.ConclusionsIn LPS induced septic mice,there are excessive neutrophil infiltration in liver and lung and this is correlated with the neutrophils chemotaxis;LPS promotes the hierarchical neutrophil chemotaxis via p38 activation;CORM-2 inhibits sepsis –induced neutrophil infiltration by interfering with FPR1 via p38 MAPK signaling pathway.