The Role Of Tyrosine Phosphatase Shp2in Regulation Of Macrophage Activation And Alveolar Homeostasis

Macrophages are highly plastic to external perturbations in the innate immune response. Simultaneously, a range of proteases produced by the alveolar macrophage are capable of degrading elastin and other component of the matrix in alveolar microenvironment.Shp2is a ubiquitously expressed cytoplasmic tyrosine phosphatase, containing two Src homology2(SH2) domains and one catalytic protein tyrosine phosphatase (PTP) domain. It serves important roles on alveolar microenvironment homeostasis.Macrophages acquire functional polarization depending on the microenvironment they received, and this phenotypic plasticity of macrophages is broadly categorized into classically activated (M1) and alternatively activated (M1). In the present work, we utilized transgenic mice carrying LysM-Cre promoter, which selectively disrupts Shp2in mature macrophages. We first demonstrated that Shp2inactivation preferentially activates macrophage M2polarization. Mice lacking Shp2appear to be more sensitive to chitin-induced M2activation, schistosome eggs-induced schistosomiasis and bleomycin (BLM)-induced pulmonary fibrosis. Our further experiments are indicative of negative regulation of IL-4-induced JAK1-STAT6signaling by Shp2via its phosphatase activity. These results suggest that Shp2acts as an important negative regulator of M2polarization and that Shp2function counteracts the development of pulmonary fibrosis.Pulmonary emphysema, the major contributor to morbidity and mortality in patients with chronic obstructive pulmonary disease, is characterized by progressive destruction of the alveolar matrix. Loss of elastic recoil and histologic evidence of damage to elastin fibers implicates elastic degradation as a key feature in the pathogenesis of this disease. An abnormal increase in macrophage metalloelastase (MMP12) breaks extracellular matrix homeostasis and contributes to the pathogenesis pulmonary emphysema. However, the regulation of this protease in vivo is poorly understood. In this study, it reveals that MMP12expression were much higher in alveolar macrophages which were deficient in tyrosine phosphatase Shp2. As expected, these mice develops spontaneous emphysema in an ageing-related manner. As TGFβ1/SMAD signaling is the key mediator on this chronic developed emphysema by properly regulating metalloelastase homeostasis. We found normal amount of TGFβ1in bronchoalveolar lavage fluid (BALF) in Shp2△/△mice, but deficient on inhibition of MMP12expression in Shp2△/△alveolar macrophages. Indeed, TGFβ1-mediated SMAD2/3activation was pharmacologically suppressed by Shp2inhibitor (PHPS1) in vitro, which was also verified in Shp2△/△macrophages in vivo. Furthermore, inhibition or loss of Shp2blocked TGFβ1-mediated SMAD2/3nucleus location. Finally, Shp2positively regulated TGFβ1-mediated transcription. In conclusion, our data demonstrates that the loss of Shp2-meidiated TGFβ1signaling in alveolar macrophages causes age-dependent pulmonary emphysema through alterations of alveolar macrophage Mmpl2expression.Materials and Methods1. Mice and Cell culture;To generate macrophage-specific Shp2-knockout mice, we crossed Shp2flox/flox mice with^mice. LysMCre/+or Shp2flox/flox mice were used as control animals, and LysMCre/+2flox/flox mice designated as shp2△/△) were used as experimental animals Shp2flox/flox mice were generated as described previously.Peritoneal macrophages and alveolar macrophages were harvested directly from treated or untreated mice. Bone marrow-derived macrophages were differentiated with MCSF as described previously. The cells were cultured in DMEM/F12supplemented with10%fetal bovine serum plus50U/mL penicillin and streptomycin. The cells were grown at37℃with 5%CO2in fully humidified air.2. Induction and assessment of M2polarization, MMP12secretion and signaling;IL-4was used for M2induction. Quantitative PCR, Immunofluorescence, Immunoblotting and arginase assay were used to the assessment of M2polarization to IL-4and MMP12expression.3. Animal models.We use BLM and chitin model to assess pulmonary fibrosis and in vivo M2polarization respectively. Briefly, Mice (8to12weeks old) were intratracheally instilled with BLM (2.5mg/kg) then euthanized for analysis of fibrosis21d after BLM administration. Approximately1μg of chitin was intraperitoneally injected into mice, and peritoneal macrophages were collected for analysis2d after the injection. Age-related emphysema: The mice were placed under normal breeding environment and got older.Results1. Shp2down regulates IL-4-induced M2in vitro and in vivo;2. Loss of Shp2aggravates BLM-induced pulmonary fibrosis;3. Shp2down regulates IL-4-mediated JAK1-STAT6activation;4. Shp2inhibites JAK1binding to IL-4Ra;5. An abnormal increase in MMP12in mice of macrophge Shp2deficiency;6. Loss of Shp2blocks TGF01-mediated SMAD2/3signaling;7. Loss of Shp2develops spontaneous emphysema in an ageing-related and MMP12dependent manner.Conclusion1. Shp2acts as an important negative regulator of M2polarization and that Shp2function counteracts the development of pulmonary fibrosis.2. Loss of Shp2-meidiated TGFβ1signaling in alveolar macrophages causes age-dependent pulmonary emphysema through alterations of alveolar macrophage Mmp12expression.