Mechanism Of Protein Tyrosine Phosphatase SHP2in The Regulation Of Pulmonary Inflammation And Bacterial Clearance

Background and objectiveChronic Obstructive Pulmonary Disease (COPD) featured by persistent respiratory airflow limition, is a common systemic disease which is treatable and preventable. The airflow limitation often shows progressively severe. Repeated infection in thelower respiratory tract(LRT) is the main cause of acute exacerbation of COPD, and Haemophilus influenzae is themost important pathogen to lead to the LRT recurrence. Non-typeable Haemophilus Influenzae (NTHi) a kind of polymorphic gram-negative bacillus, is the most common colonized bacteria in the upper respiratory tract. The invasion of NTHi into lower respiratory tract will impair the ability of airway mucosa cilia clearance, leading to the increased airway mucus secretion and epithelial damage. These pathological changes then accelerate bacteria colonization and infection in the respiratory tract, promoting lung parenchyma damage and progressive small airway obstruction, and subsequently the development of COPD.Macrophages play key roles in clearance of pathogenic microorganisms and senescent cells, promotion of inflammation, induction of adaptive immune response and the reconstruction and repairment of the injured tissue.Under the influence of different micro-environment, macrophages can be divided into two types:M1and M2type. M1macrophages secrete a large number of inflammatory factors, effectively present antigens, activate the Thl cell response, thus playing main roles in resistanting microorganisms and killing tumor cells. M2macrophages, which have weaker sterilization ability, can inhibit Thl cellular immune response and promotetissue repair, reconstruction, and the growth of blood vessel.However, in COPD patients, the ability of alveolar macrophages in engulfing bacteria, secreting the cytokines and removing bacteria has been severely damaged,.Therefore, clarifying the cellular and molecular mechanisms of lung inflammation and bacterial clearance upon NTHi infection will provide new strategies of preventing, controlling and treating NTHi infection-related diseases. Moreover, it may also provide new molecular targets for drug development.SHP2(Src homology phosphotyrosyl phosphatase2), belonging to protein tyrosine phosphatase (protein tyrosine phosphatase, inhibits PTP) family, is encoded by P T P N11gene and widely expresses in human cells and tissues. Shp2participates in the regulation of a variety of intracellular signal transduction pathways. In addition, SHP2is closely associated with multiple respiratory system diseases..However, the effect of SHP2gene on acute inflammation upon bacterial infection in lung and its related molecular mechanism have not been elucidated. Therefore, we try to establish NTHi infection models both in vitro and in vivo that we use NTHi to infect bone marrow-derived macrophages (BMDMs) with Shp2knocked-down and mice with Shp2specifically knocked-out in myeloid-cell lines, to explore the functions of Shp2gene and its related signaling pathways in regulating macrophage inflammatory response and eliminating the bacterials. Our research will contribute to state the function of SHP2in regulating macrophage differentiation and the molecular mechanism of strengthening anti-bacterial immunityoffering a new strategy for immunological treatment of bacterial infection diseases in lung.MethodIn vivo1. Established the acute lung infection model of NTHi:C57/BL6mice were intratracheally challenged with107colony-forming units (CFU) of NTHi with total vovume of40μL then collect the lung tissue after6hours,12hours,24hours and measure the level of inflammation cytokines such as IL-6,TNF-α, we also measure the expression level of SHP2at the same time.To investigate the effects of PHPS1(SHP2specific inhibitors) on airway inflammation, mice were pretreated with PHPS1by an i.p. injection at concentrations of3mg/kg0.5h before NTHi infection. Hematoxylin-eosin staining of lung tissues, RNA and bronchoalveolar lavage fluid collected from WT and PHPS1pretreated mice were used to evaluate the different inflammatory reaction between two group.2. Mononuclear macrophages SHP2knockout mouse animal infection model: Endogenous disruption of the Shp2enzyme in macrophages was generated using the Cre-loxP technology:Shp2flox/flox mice were crossed with LysMCre/+mice to generate conditional Shp2knockout animals. LysMCre/+or Shp2flox/flox and LysMCre/+:Shp2flox/flox (Shp2△/△) mutant mice were used in the experiments. We measure the bacterial burden in bronchoalveolar lavage fluid (BALF) and lung tissue, the level of imflamatory madiator and Hematoxylin-eosin staining of lung tissue after NTHi infection.In vitro1.Extraction of C57/BL6mice bone marrow derived macrophages (BMDM):Bone marrow-derived macrophages (BMDMs) were differentiated with M-CSF. The expression level of SHP2was measured after NTHi infection. Moreover, we detected the macrophage phenotype change, bactericidal capability and phagocytosis when BMDM treated with NTHi with or without PHPS1pretreated. To exam the role of SHP2in skewing macrophage phenotype,we also measure the macrophage surface molecules marker from SHP2A/mouse.2. To elucidate the molecular mechanism responsible for SHP2-mediated regulation of Ml skewing, we initially analyzed the activities of STAT1, STAT6, AKT, p65when the macrophages treated with NTHi.Result1. We found the expression of SHP2was elevated and the inflammatory factor such as TNF-a,IL-6,MIP-2were remarkable increased in C57/BL6after NTHi infection. Further, Histopathological examination revealed that NTHi infected lungs were apparently damaged. H&E staining of sliced lung tissues presented with prominent thickening of alveolar walls and accumulation of inflammatory cells.2. Endogenous inactivation of Shp2exhibits decreased inflammation in NTHi infection model:the inflammatory factor and cells were decreased when mouse was treated PHPSl.3. The gene and protein level of SHP2were remarkable increased in BMDM from C57/BL6after NTHi infection, PHPSl can inhibit NTHi-induced Ml polarization and bactericidal capability, meanwhile, the M2genes:YM-1,ARG were highly increased.4. Inflammatory cells were decreased in Shp2A/A mouse, however, bacterial burden in bronchoalveolar lavage fluid (BALF) or lung tissue were elevated (no statistical significance), The data indicated that the prototypical Ml genes was decreased in macrophage from SHP2A/A mouse following NTHi exposure.5. Our results showed that p65phosphorylation induced by NTHi P65was decreased by SHP2deficiency, and Shp2seems to mediate inflammation response through NF-kB signal pathway.ConclusionOur research indicated that SHP2can mediate lung inflammation induced by NTHi and SHP2also can mediate inflammation response of macrophage through p65-NF-icB signal pathway. SHP2deficiency can decrease the M1polarization and bactericidal capability of macrophages.