Mechanism Of Neutrophil Migration And Inflammation

Cell migration is one of the fundamental function of the cell and plays a critical role in many physiological and pathological processes,such as embryonic development,angiogenesis,wound healing,host defense,inflammation and cancer.As the front line of host defense,neutrophils rapidly recruit into the infected tissue induced by the chemoattractant and then digest the invaders.Neutrophil is the fastest mammalian cell and regarded as one of the best models for the mechanism study of cell migration.Inappropriate tissue sequestration of neutrophils during inflammation can cause severe tissue injury and lead to chronic inflammation.The tissue infiltration of neutrophils is tightly regulated;however,the signaling mechanisms that prevent excessive neutrophil infiltration remain mostly unclear.Here,we demonstrate the mechanism of neutrophil migration and inflammation on the level of molecular,cellular and animal studies.Following please find the result we got.（1）Differential roles of integrin β₁ and integrin β₂ was observed during neutrophil migration.First,we obtained myeloid cell lineage-specific knockout of integrin β₁ via crossing of LyzCRE and LoxP-β₁ mice after several backcrossing and screening.There’re no bands in neutrophils isolated from integrin β₁ and integrin β₂ knockout mice by Western blot analysis.Function blocking antibodies-treated HL-60 cells showed decreased adhesion on fibronectin and fibrinogen,furthermore neutrophils isolated from integrin β₁ and integrin β₂ knockout mice also decreased adhesion to fibrinogen,which means both integrin β₁ and integrin β₂ were involved in neutrophil adhesion.In transwell migration analysis,inhibition or ablation of integrin β₁ enhanced neutrophil migration,while the number of transmigrating cells was decreased after blocking or deletion of integrin β₂.Also,in EZ-TAXIScan directional migration assay,the speed of cell migration was increased after blocking or deletion of integrin β₁,in contrast,loss of integrin β₂ leaded to long tails and decreased migration.To further comfirm the phenomenon we observed in in vitro cell migration assay,we used two in vivo models to study the function of integrins in neutrophil migration.10 nmol/L of fMLP was intraperitoneal injected into mice,knockout of integrin β₂ decreased the infiltration of neutrophils in peritoneal cavity,whereas depletion of integrin β₁ augmented the number of infiltrated neutrophils.In another model,to induce local Shwartzman reaction we injected LPS and then TNF-α into mice skin of back.MPO activity in wildtype was increased,however it was significantly greater in ablation of integrin β₁ mice and less in integrin β₂ knockout mice.Since the injury of microvascular vessel is neutrophil-dependent,deletion of integrin β₁ exacerbated vascular injury and hemorrhage while loss of integrin β₂ attenuated these phenomenons compare to wildtype.fMLP-induced ROS production was decreased in integrin β₂ knockout neutrophils than wildtype,whereas neutrophils deficient in integrin β₁ produced more ROS.In summary,integrin β₁ and β₂ played differential roles in regulating neutrophil migration.（2）In our study,we found that TRPM2 played a negative role in modulating neutrophil migration.Oxidant sensing by TRPM2 downregulated neutrophil migration and prevented subsequent neutrophil-mediated inflammation.While basal MPO activity in TRPM2-/-lungs remained the same as in wildtype,fMLP induced significantly greater MPO activity in TRPM2-/-than wildtype lungs.In histological sections,fMLP treatment increased markedly higher sequestered number of neutrophils within pulmonary vasculature and interstitium as compare to wildtype mice.Vascular inflammation induced in modified LSR（local Schwartzman reaction）is mediated by neutrophils,TRPM2-/-mice displayed increased neutrophil infilatration and enhanced vascular response as compare to wildtype.How did TRPM2 regulate neutrophil migration?Deletion of TRPM2 didn’t affect neutrophil polarization and adhesion,actin could still recruit to the cell membrane and translocate to the leading edge of migrating neutrophil of TRPM2-/-mice.Comparing to wildtype neutrophils,TRPM2-/-neutrophils showed increased chemotaxis index and migration speed in EZTAXIScan assay.FPR1 belongs to the family of G protein receptors,it can recognize the formyl peptide produced by bacteria and sense the migration direction.The internalization of FPR1 was decreased in TRPM2-/-neutrophils as compare to wildtype after fMLP stimulation.The direct interaction between N-ternimus of TRPM2 and FPR1 was approved via immunoprecipitation and FRET analysis.Binding of TRPM2 to FPR1 protomed membrane recruitment of GRK2 and inhibited the phosphorylation of p38 MAPK.Loss of TRPM2 in neutrophils resulted in higher activation of p38 and less recruitment of GRK2 onto membrane,which stabilized the FPR1 receptors on cell membrane and increased direction sensing and then enhanced migration.Function blocking of TRPM2 channel by NMDG didn’t affect neutrophil migration and depletion of TRPM2 didn’t influence the calcium release and influx,therefore the regulation of neutrophil migration is TRPM2 channel function independent.Inhibition of ROS release reduced TRPM2-medicated FPR1 internalization,meanwhile,both DPI-treated neutrophils and GP91-/-neutrophils showed decreased internalization of FPR1 and enhanced neutrophil migration,so we think TRPM2 regulates neutrophil migration by sensing of ROS.Later,we found stable overexpression of TRPM2 N-terminus promoted GRK2 membrane recruitment and FPR1 internalization and then attenuated neutrophil migration while TRPM2 C-terminus didn’t play a role.Taken together,oxidation of TRPM2 in N-terminus by ROS induced the conformational changes of TRPM2 and promoted the binding to FPR1 and internalization to inhibit receptor signaling and thereby cell migration.（3）PARP-1 is one of DNA repair enzymes,which is involved in inflammation responses.In this study,we use a selective PARP-1 inhibitor DPQ to attenuate LPS-induced acute lung injury and inflammation.After DPQ treatment,the number of infiltrated neutrophils in lung tissue induced by LPS was significantly decreased compare to control.Histological section staining displayed better integrity of pulmonary structures in DPQ-treated group than control.To our surprise,enhanced migration was observed after DPQ treatment.Therefore the decreased infiltration of neutrophils should be ascribed to less chemokines.Quantitive realtime PCR showed less expression of pro-inflammatory cytokines of TNF-α,IL-1β,IL-6,iNOS and chemokines of CXCL-1 and MIP-2,which were produced by macrophages.LPS-elevated vascular permeability was decreased by DPQ treatment,accompanied by the inhibition of apoptotic cell death in mice lungs.In addition,we isolated mice peritoneal macrophages and showed pretreatment with DPQ at 10 μmol/L inhibited the production of cytokines in the macrophages following LPS stimulation.DPQ treatment also inhibited the phosphorylation and degradation of IκB-α,subsequently blocked the activation of nuclear factor NF-κB induced by LPS in vivo and in vitro.Taken together,our results show that DPQ treatment inhibits NF-κB signaling in macrophages and protects mice against ALI induced by LPS,suggesting inhibition of PARP-1 may be a potential and effective approach to resolve inflammation for the treatment of ALI.