Functional and Mechanistic Contribution of Shp 2 to Oxidative Burst 1 Protein Tyrosine Phosphatase , Shp 2 , Positively Regulates Macrophage Oxidative Burst

Macrophages are vital to innate immunity, and express pattern recognition receptors and http://www.jbc.org/cgi/doi/10.1074/jbc.M114.614057 The latest version is at JBC Papers in Press. Published on December 23, 2014 as Manuscript M114.614057 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Functional and Mechanistic Contribution of Shp2 to Oxidative Burst 2 integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erkand Akt-dependent production of reactive oxygen species (ROS). Shp2, a protein tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt and is crucial for hematopoietic cell function; however, no studies have examined Shp2 function in particulate-stimulated ROS production. Maximal Dectin-1-stimulated ROS production corresponded kinetically to maximal Shp2 and Erk phosphorylation. Bone marrow derived macrophages (BMMs) from mice with a conditionally deleted allele of Ptpn11 (Shp2;Mx1Cre+) produced significantly lower ROS levels compared to control BMMs. Although yellow fluorescent protein (YFP)tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1and CR3stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain-of-function (GOF) Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages (PEMs) from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1and CR3-stimulated ROS which was reduced by pharmacologic inhibition of Erk. Signal regulatory protein  (SIRP) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRP levels and an increased Shp2SIRP interaction compared to YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1and CR3stimulated ROS production in macrophages by de-phosphorylating and thus mitigating the inhibitory function of SIRPand by promoting Erk activation. INRODUCTION Macrophages are phagocytic cells that function as the body’s global first-line defense against invading pathogens. Upon interaction with pathogenproducing stimuli, macrophages internalize large particulate microorganisms and generate microbicidal reactive oxygen species (ROS) produced by activated nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (1). NADPH oxidase is composed of membraneintegrated gp91 and p22 as well as the four cytosolic components p47, p67, p40, and Rac2 (2,3). Individuals with chronic granulomatous disease (CGD) have inherited germline mutations within variable components of the NADPH oxidase complex, and suffer from recurrent, life-threatening bacterial and fungal infections, highlighting the imperative nature of competent ROS production by the innate immunity (4,5). Shp2, a protein tyrosine phosphatase encoded by the PTPN11 gene, promotes activation of Ras-Erk signaling and plays an essential role in hematopoietic cell development (6,7). Genetic disruption of murine Ptpn11 within hematopoietic lineages leads to rapid loss of blood cell production of all lineages (8,9). In humans, gain-of-function PTPN11 mutations are commonly found in children with Noonan syndrome and juvenile myelomonocytic leukemia (JMML) (10,11). Although no PTPN11 mutations have been found to be associated with clinical immune deficiency, Shp2 is a critical signaling component of Leptin Receptor-dependent protection against the parasitic pathogen, Entamoeba histolytica (12), and children bearing germline loss-of-function LEPR mutations are susceptible to respiratory infections (13). Further, previous studies found that Shp2 regulates the phosphorylation of transcription factors HoxA10 and ICSBP, leading to transcriptional repression of the NADPH oxidase components gp91 and p67 and preventing myeloid terminal differentiation (14,15); however, no studies have examined the function of Shp2 phosphatase in ROS production in terminally differentiated macrophages or neutrophils, which may reveal a novel role for Shp2 in innate immunity and ROS production. Macrophages are capable of detecting and responding to pathogen-derived molecules such as fungal glucans and lipopolysaccharides, as they express cell surface pattern recognition receptors (PRRs) such as C-type lectins. Dectin-1 is a C-type lectin expressed on macrophages that responds to by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Functional and Mechanistic Contribution of Shp2 to Oxidative Burst 3 -glucan-containing particles derived from fungal cell walls and stimulates Srcand Syk-dependent signaling (16). Dectin-1 stimulation results in activation of the Ras-Erk pathway, production of microbicidal ROS, and induction of expression of the inflammatory cytokines, TNF and IL6. In humans, loss-of-function mutations in DECTIN-1 confer a state of increased susceptibility to mucocutaneous C. albicans and invasive aspergillosis (17,18). Based on the known high expression of Shp2 in macrophages and its well-defined role as a positive regulator of the Ras-Erk pathway, we hypothesized that Shp2 promotes normal innate immunity by positively upregulating particulate-stimulated NADPH oxidase activation and abrupt production of ROS, known as oxidative burst. To address this hypothesis, we examined the correlation of Shp2 activation to peak ROS production in zymosanstimulated peritoneal exudate macrophages (PEMs) and examined the putative placement of Shp2 in the Dectin-1-stimulated pathway employing genetic studies and pharmacologic studies using the Syk inhibitor, R406, and the Erk inhibitor, SCH772984. Genetic disruption of Ptpn11 resulted in reduced macrophage ROS production in response to both zymosan (Dectin-1 stimulation) and serum opsonized zymosan (SOZ, complement receptor 3 stimulation), indicating a positive function of Shp2 in oxidative burst. Structure-function studies using various Shp2 loss-of-function and gain-of-function constructs indicated that the phosphatase function of Shp2 is specifically required for positive regulation of particulate-stimulated oxidative burst. Mechanistic studies demonstrated that Shp2 exerts its positive effect on ROS generation by dephosphorylating the myeloid inhibitory immunoreceptor, SIRP (signal regulatory protein ), and by promoting Erk activation. EXPERIMENTAL PROCEDURES Reagents. Chemicals were purchased from SigmaAldrich (St. Louis, MO) unless otherwise stated. Phosphate-buffered saline (PBS) pH 7.2, penicillin/streptomycin, neomycin, IMDM and DMEM were from Invitrogen Life Technologies (Carlsbad, CA); fetal calf serum (FCS) was from HyClone Laboratory (Logan, UT). The ECL detection kit came from Pierce (Rockford, IL). Rabbit polyclonal antibody against p40 and mouse monoclonal antibody T-Syk were from Upstate Biotechnology (Lake Placid, NY), rabbit polyclonal antibody against p47 was from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal antibody against p67phox was from BD Biosciences (Franklin Lakes, NJ). Rabbit polyclonal and mouse monoclonal antibodies against Shp2 was from Santa Cruz Biotechnology (SC-280) and BD Bioscience (#610621), respectively. Anti-SIRP polyclonal antibody was from Abcam (#53721) and mouse dectin1/CLEC7A antibody (Clone 218838) was from R&D Systems, Inc. (Minneapolis, MN). Antiphospho-SIRP was generated as previously described (19). Anti-gp91 mAb 54.1 and antip22 mAb NS2 were kindly provided by A. J. Jesaitis (Montana State University) (20). All the other antibodies, including anti-phosphoantibodies, were obtained from Cell Signaling Technology (Beverly, MA) unless otherwise stated. Zymosan A from Saccharomyces cerevisiae was from Sigma (Z-4250). The fluorescein isothiocyanate (FITC)–labeled zymosan particles were from Life Technologies. Phycoerythrin (PE)Mac-1 and allophycocyanin (APC)-F4/80 were from BD Pharmingen. Animal Husbandry. Mice bearing a conditional gain-of-function (GOF) Ptpn11 allele (LSLShp2) or a conditional floxed Ptpn11 allele (Shp2) have been described (9,21,22) and were crossed with mice bearing the Mx1Cre transgene to generate experimental (Shp2D61Y;Mx1Cre+ or Shp2;MxCre+) and negative control (Shp2D61Y;Mx1Creor Shp2;MxCre-) animals. All animals received three intraperitoneal injections with 300 μg polyI:polyC (GE Healthcare) to induce Ptpn11 recombination. All mice were maintained under specific pathogen-free conditions at the Indiana University Laboratory Animal Research Center (Indianapolis, IN) and this study was approved by the Institutional Animal Care and Use Committee of the Indiana University School of Medicine. Plasmid construction. The cDNAs encoding wildtype (WT) Shp2 or Shp2-R32K and Shp2C463A mutants were cloned into EcoRI and KpnI sites of pEYFP-N1 (Clontech; San Diego, CA) to generate yellow fluorescent protein (YFP)-tagged by gest on O cber 5, 2017 hp://w w w .jb.org/ D ow nladed from Functional and Mechanistic Contribution of Shp2 to Oxidative Burst 4 Shp2 constructs, and the constructs were confirmed by sequencing. The YFP-tagged Shp2 cDNAs were then ligated into pMSCV (Clontech) for use in generation of retroviral supernatants. Preparation and characterization of the pMIEG3WT Shp2, Shp2-D61Y, and Shp2-E76K was described previously (23). Retroviral transduction of bone marrow (BM) and macrophage differentiation. Retroviral transduction of murine bone marrow low density mononuclear cells (LDMNCs, C57Bl/6 background) with MSCV-WT Shp2-YFP, Shp2R32K-YFP, or Shp2-C463A-YFP or with MIEG3WT Shp2, Shp2-D61Y, or Shp2-E76K was performed as described (23,24). Transduced cells were sorted by fluorescence activated cell sorting (FACSCalibur, BD; Franklin Lakes, NJ) prior to macrophage differentiati