Inhibiton And Mechanism Of Bacterial LPS-Indcued Autocrine ATP/P2X1/Ca²⁺ Pathway On Neutrophil Chemotaxis In Sepsis

BackgroundSepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection, which is a common complication of severe trauma, burn and shock. Although markedly progresses have been achieved in the field of anti-infection,fluid resuscitation, wound management and organ support therapy, mortality of sepsis still remains at a high level. Sepsis is the leading cause of death to patients who die in intensive care units (ICU). Patients who suffer sepsis are generally in company with impressed immune function and failed ability to clear infection, which are important causes of uncontrollable infection during sepsis. Usage of antibiotic improves the success rate of emergency treatment for septic patients. However, the long-term application of antibiotics brings problems of bacterial drug resistance and antibiotics are not able to completely solve the infection issue in sepsis. Therefore, how to improve immune function in septic patients so as to control infection is an important topic for the treatment of sepsis. During sepsis, neutrophil function was suppressed in the presence of multiple bacterial toxins and cytokines. Chemotaxis is impaired and the amount of neutrophils in infectious tissues is decreased during sepsis. Failure of neutrophils to clear pathogens effectively is the main cause of death in septic patients.To further clarify the mechanisms of impaired neutrophil chemotaxis and seek specific interventions to improve neutrophil chemotaxis toward infectious tissues may provide theoretical basis for anti-infection treatment of sepsis.Lipopolysaccharide (LPS) is the main component of gram-negative bacterial cell walls.An excess of LPS can lead to the over-activation of innate immune cells and release of excessive inflammatory mediators, which exert its role on whole body tissues and organs through autocrine and paracrine pathway to induce systemic inflammatory response syndrome. ATP is an important energy molecules in cells. Recently, Junger. et al. find that ATP guides neutrophil chemotaxis in a autocrine manner. During chemotactic migration, the autocrine released ATP from front lamellipodium activates local P2Y2 receptor and improves movement function of neutrophil. But when the whole cell receives activation of ATP, neutrophil chemotaxis is impaired which indicates that extracellular ATP may also be a negative regulator of neutrophil chemotaxis. The detailed mechanism remains to be discovered. P2X1 receptor is an ATP-gated cation channels receptor. By combing with extracellular ATP, P2X1 receptor is activated and induces a prompt calcium influx. P2X1 receptor is an important calcium influx channel for neutrophils. Recent reports have proved the expression of P2X1 receptor is peripheral blood neutrophils. ATP increases random movement of neutrophil by activation of P2X1 receptor. But the random movement of neutrophil loses the ability of chemotactic migration, which weakens the specific migration toward infection. Calcium is a critical signaling molecule in cells and involved in the regulation of a variety of biological functions of neutrophils. But excessive influx of calcium inhibits neutrophil chemotaxis. Whether P2X1 receptor is involved in the calcium influx is still unknown.MethodsIn in vivo experiments, lethal dose or sub-lethal dose of escherichia coli (E. coli) is intraperitoneally injected into C57BL/6 mice to induce severe septic infection or mild local infection animal model. Survival rate is observed for 5 consecutive days.Automatic blood cell analyzer is used to detect neutrophil counts in peritoneal lavage fluids and peripheral blood. Plate-counter method is used to detect bacterial load in peritoneal lavage fluids and peripheral blood. Pathological HE staining is used to observe liver and lung injury. ELISA detecting kit is used to determine IL-1? and IL-6 in liver and lung. MDA detecting kit is used to determine MDA content in liver and lung, Transcriptome genechip is used to detect the differences of gene expression in neutrophils.In in vitro experiments, LPS is employed to stimulate primary human blood neutrophil.Under agarose chemotaxis experiment is used to detect neutrophil migration distance.Flow cytometry is used to detect neutrophil chemoattractant receptors expression and apoptosis. High performance liquid chromatograph is used to detect neutrophil ATP release. Western Blot is used to detect phosphorylation and activation of Cx43 protein.Laser scanning confocal microscope is used to observe distribution of P2X1 receptor and calcium influx. Atomic force microscope and field emission scanning electron microscope are used to analysis neutrophil polarization. Laser scanning confocal microscope is used to observe skeleton protein myosin light chain (MLC)phosphorylation and polarization.CRISPR/Cas9 system is used to knockout P2X1 receptor in HL-60 promyelocyte.Incubation with 1.3% dimethyl sulfoxide is performed to differentiate neutrophil-like HL-60 cells. Atomic force microscope and field emission scanning electron microscope are used to analysis neutrophil polarization. Laser scanning confocal microscope is used to observe skeleton protein MLC phosphorylation and polarization.ResultsThe results of in vivo experiments indicate that neutrophil chemotaxis is inhibited and bacterial clearance is impaired. Dramatic pathological injury and inflammatory response are observed in liver and lung of septic mice. Further genechip analysis reveals that neutrophil TLR signaling is activated in septic mice. LPS has a marked regulation on neutrophil chemoattractant receptors via TLR4.The results of in vitro experiments indicate that LPS has a strongest inhibitory effect on neutrophil chemotaxis among the important inflammatory stimuli in sepsis. LPS opens Cx43 hemichannel to release ATP. The released ATP irritates neutrophils' own P2X1 receptor and induces calcium influx to activate MLCK. Activated MLCK induces abnormal phosphorylation and polarity of MLC and inhibits neutrophil chemotaxis.Knockout of P2X1 reverses neutrophil chemotaxis, phosphorylation and polarity of MLC in the presence of LPS.ConclusionsFindings from in vivo and in vitro experiments reveal a previous unknown LPS induced autocrine ATP/P2X1/Ca2+ signaling in inhibiting neutrophil chemotaxis through abnormal phosphorylation and polarity of skeleton protein MLC. It may be the potential mechanism underlying the impaired neutrophil chemotaxis in sepsis.