Role Of Bruton’s Tyrosine Kinase In The Pathogenesis Of Acute Lung Injury In Mice With Severe Burn Injury

1. BACKGROUNDAt the early stage following severe burn injury, there are violent changes happen withinternal environment. As results of trauma stress, direct damage by heat power and tissuehypoperfusion, inflammatory cells are activated such as mononuclear macrophage,neutrophils, lymphocyte, platelet, endotheliocyte, pro-inflammatory cytokines are alsoreleased increasingly. Vascular endothelial cells and leucocytes express more adhesionmolecules, which strengthen adhering between them. Activated leucocytes break outbreathing explosion and degranulation, cause release of proteolytic enzyme, active oxygenand metabolic product of arachidonic acid that damage endothelial cell and other cellsextensively. The pathophysiological process described above can be summarized to aninflammatory cascade: severe burn→increased expression of pro-inflammatory cytokines→leucocytes adhere with endothelial cells and activated, releasing a great quantity ofinflammatory mediator→systemic inflammatory response syndrome. To prevent thedevelopment and progress of uncontrolled inflammation, many strategies which act oncytokines such as monoclonal antibody, soluble receptor have been on clinical trial.However, these strategies showed limited success and even increased mortality rate inpatients. There are many kinds of cytokines and their products which constitute a complexnetwork. The strategy which only acts on single or several cytokines does not workeffectively. Although the kinds of cytokines is numerous (over100), the signaltransduction pathways which regulate the production of the cytokines are not too many.The efficacious inhibition and regulation of the pivotal pathways may prevent the progressof inflammatory response and the development of organ injury.Toll like receptors (TLRs) are key molecules for recognizing bacterial components toevoke innate immune response. TLRs (especially TLR4) are pattern recognition receptors(PRRs) that bind not only pathogen-associated molecular patterns (PAMPs) but alsodamage-associated molecular patterns (DAMPs). There is a new molecular pathway inwhich lipopolysaccharide (LPS) induces autophagy in human and murine macrophages bya pathway regulated through Toll interleukin1receptor domain-containingadaptor-inducing interferon-beta (TRIF)-dependent, myeloid differentiation factor88 (MyD88)-independent TLR4signaling. MyD88and TRIF are downstream components ofthis pathway. The former can activate NF-κB and p38mitogen-activated protein-kinase(MAPK) signaling pathway, the latter will activate NF-κB and Interferon regulatory factor3signaling pathway. By means of these signaling pathways, TLR4can produce series ofpro-inflammatory mediators and cytokines which will cause sever inflammatory responseand immunologic reaction. Recent studies show that Toll-like receptor4(TLR4) and itssignaling pathway serves as an important component in the innate immune response andsystemic inflammatory response syndrome which are reduced by infection and non-infectious diseases.Tec is a family of the cytoplasmic non-receptor tyrosine protein kinases. The Tec proteintyrosine kinase is the founding member of a family that includes Btk, Itk, Bmx, and Txk.Bruton’s tyrosine kinase (Btk) is essential for B-cell receptor signaling. The geneexpression profile of Btk is considerably narrow, which is only in myeloid cells but T cellsor plasmocyte. Btk can markedly enhance the TLR signaling transduction and finallyactivate NF-κB and p38mitogen-activated protein-kinase (MAPK) signaling pathways,which result in up-regulation of pro-inflammatory genes. Studies show that Btk deficiencyin macrophages, mastocytes and peripheral blood mononuclear cell evidently inhibit thelipopolysaccharide (LPS)-induced production of tumor necrosis factor alpha, IL-1β andnitric oxide. Further investigations indicate that Btk signaling participates in theregulations of LPS-induced cytokines production mainly through the following p38MAPKand Nuclear Factor-κB signaling pathway. However, the researches about relationshipsbetween the Btk signaling pathway and inflammatory reaction are still limited in vitro, itsregulatory roles in vivo are hardly understood.The lung is the commonest and earliest organ which develops dysfunction after severeburns. Lung not only is the organ of gas exchange, but also is the site of production andinactivation of some cytokines and hormones. Besides, the unique anatomic location of thisfragile organ makes it vulnerable to circulating serum factors created at remote wounds.The lung is one of major target organs injured by postburn inflammation. In considerationthat uncontrolled inflammatory reaction is the pathophysiological foundation of acute lunginjury, as a key upstream signaling transduction pathway, Btk is probably playing animportant role in the development and progression of ALI. Therefore, in this study wefocus on lung to investigate the role of Btk signaling pathway plays in early inflammationregulation and its relation with acute lung injury after severe burn injury. 2. OBJECTIVE(1) To demonstrate the changes of Btk signaling after burn trauma by surveying theactivity of Btk, which are key cytoplasmic mediators of acute lung injury and localinflammatory reactions.(2) By preventive application of LFM-A13, a special inhibitor which can significantlyinhibit the activation of Btk, our further investigations indicate that Bruton’s tyrosinekinase signaling participates in the regulations of acute lung injury postburn mainlythrough the following p38MAPK and Nuclear Factor-κB signaling pathway.3. MATERIALS&METHODSPart one. Activation of Btk in the pathogenesis of acute lung injury in mice withsevere burn injury.252healthy adult male C57BL/6mice were divided into sham burn normal group (Cgroup), burn group (S group), and burn plus LFM-A13group (L group) randomly. Allmice were anesthetized and shaved, the mice of C and S group were subjected an30%totalbody surface area full-thickness scald by being immersed in98℃water for12s on theirdorsal surface. The burn mice were resuscitated intraperitoneally with lactated Ringer’ssolution following Parkland formula. LFM-A13was administered intraperitoneally at4.2mg/kg at1h before scald and11h postburn. At0.5h,1h,3h,6h and12h, six mice fromeach group were sacrificed by exhausting blood through inferior caval vein and their lungstissue were harvested. The mice of C group were underwent the same treatments, but werenot subjected to burn injury and resuscitation. Six mice lungs of each semi-group werekept in10%neutral formalin solution for pathological examination, and the other six lungswere kept in liquid nitrogen for further analysis. Thereafter, Western blot was accessed toexam the protein content and phosphorylation of Btk, Caspase-3and Bcl-2in lung tissue.The lung injury was evaluated by Caraway’s double-blind pathological score. The locationof Btk expression in lung was detected by immunohistochemical staining. The alveolarepithelial apoptosis was tested by the meaning of TUNEL. Pulmonary water contents weredetermined by ratio of dry weight versus wet weight in each group at12h. meanwhile,chromatometry of evans blue was being used to measure microvascular permeability oflung.Part two. Effects of Btk in the expression of pro-inflammatory cytokines and theextent of neutrophil seizure in mice with severe burn injury.The animal model of acute lung injury induced by severely burning was adopted in this study, and the procedure of animal treatment was performed just as the first part describedabove, but they were sacrificed and sera and lungs harvested at12h. Thereafter, sera levelsof TNF-α、IL-1β、IL-6、NO2-/NO3-were determined by enzyme-linked immunosorbantassay. The total RNA was extracted from the lung tissues using Trizol and the expressionof TNF-α, IL-1β, IL-6and iNOS mRNA was tested by the meaning of real time PCR.Activities of myeloperoxidase (MPO) in lung tissues were assayed by using MPO kitaccording to the manufacturer’s instructions.Part three. Molecular mechanism of Btk in mice with severe burn injury.The animal model of acute lung injury induced by severely burning was adopted in thisstudy, and the procedure of animal treatment was performed just as the first part describedabove. At0.5h,1h,3h,6h and12h, six mice from each group were sacrificed byexhausting blood through inferior caval vein and their lungs tissue were harvested.Thereafter, Western blot was accessed to exam the protein content and phosphorylation ofp38, JNK, ERK, IκBα and p-IκBα in lung tissue to evaluate the molecular mechanism ofBtk in acute lung injury of mice with severe burn injury.4. RESULTSPart one.The lung tissue showed significant inflammatory injury from six hours after burning,and administration of LFM-A13made the situation much more attenuated. Results ofCaraway’s double-blind pathological scores evaluation showed that, the sores of S and Lgroup were significant higher than the C group. After administration of LFM-A13, thescores of the L group markedly decreased and significant lower than S groups. Theexpression of Btk and phospho-Btk at0.5h and the following time-nodes in S group wereall significantly higher than the C group by western blot analysis. Btk content in lungs wasremarkably decreased than S group at every time-node in L group by administration withLFM-A13, meanwhile Btk activity was also abolished at every time-node in L group.According to the IHC examination results, Btk had been found located in mononuclearmacrophages and infiltrated neutrophil cells in S and L group.Results of TUNEL technology showed that, the Apoptosis Index of S and L group weresignificant higher than the C group. After administration of LFM-A13, the AI of the Lgroup markedly decreased and significant lower than S groups. According to western blotanalysis, Caspase-3and Bcl-2content in S group increased remarkably and were bothsignificantly higher than the C group’ s level. After administration of LFM-A13, the content of Caspase-3markedly decreased and significant lower than S group, but Bcl-2content were much higher than S group.In addition, chromatometry of evans blue was being used to measure microvascularpermeability and pulmonary edema. According to these examinations, burns resulted inworsen pulmonary edema by promoting microvascular permeability at twelve hours afterburning, administration of LFM-A13made the situation much more attenuated.Part two.Twelve hours after injury, ELISA showed that serum TNF-α, IL-1β, IL-6and NO2-/NO3-in both of S group and L group increased remarkably compared to C group, administrationof LFM-A13significantly decreased the serum of TNF-α, IL-1β, IL-6and NO2-/NO3-.At twelve hours after burning, TNF-α, IL-1β, IL-6and iNOS mRNA from the S groupexpressed significantly higher than the C group did. In vivo administration of LFM-A13markedly decreased the expression of TNF-α, IL-1β, IL-6and iNOS mRNA in L groupthan S group, but still showed significant higher than the C group did.In addition, activities of Myeloperoxidase (MPO) increased significantly in S groupcompare to C group at twelve hours postburn, while pretreatment of LFM-A13couldmodestly diminish it, but still higher than C group.Part three.We compared the expression of phosphorylated p38MAPK, phosphorylated JNK,phosphorylated ERK, IκBα and phosphorylated IκBα by western blot analysis. Accordingto the results, we found that the p38MAPK content, NF-κB content and the expression ofphosphorylated ERK were increased at0.5h and the following time-nodes after injury,whereas the expression of phosphorylated JNK showed significant difference at3h. In vivoadministration of LFM-A13markedly decreased the expression of phosphorylated p38MAPK and phosphorylated IκBα in L group than S group at the early stage of postburn,meanwhile, both JNK and ERK were not markedly changes in activities by administrationwith LFM-A13.5. CONCLUSIONS(1) Preventive application of LFM-A13, a special inhibitor of Bruton’s tyrosine kinase(Btk), could significantly inhibit the activation of Btk and diminish its expression inmononuclear macrophages and infiltrated neutrophil cells, which would reduce theextent of alveolar epithelial apoptosis, pulmonary edema and neutrophil seizure. At thesame time, this inhibitory effect could distinctly reduce the serum levels and mRNA expressions of pro-inflammatory cytokines in postburn mice, thereby effectivelyattenuating the acute lung injury.(2) Further investigations indicate that Bruton’s tyrosine kinase signaling participates in theregulations of acute lung injury postburn mainly through the following p38MAPK andNuclear Factor-κB signaling pathway, while JNK and ERK were not markedly changesin activities by administration with LFM-A13.