Abstract
Macrophages are vital to innate immunity and express pattern recognition receptors and integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erk- and 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(flox/flox);Mx1Cre+) produced significantly lower ROS levels compared with control BMMs. Although YFP-tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1- and CR3-stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain of function Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1- and CR3-stimulated ROS, which was reduced by pharmacologic inhibition of Erk. SIRPα (signal regulatory protein α) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRPα levels and an increased Shp2-SIRPα interaction compared with YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated ROS production in macrophages by dephosphorylating and thus mitigating the inhibitory function of SIRPα and by promoting Erk activation.
Highlights
Innate immune cell oxidative burst is needed to combat pathogens
Given that the protein-tyrosine phosphatase Shp2 positively regulates Ras-Erk pathway signaling and is known to be crucial for normal hematopoietic cell development and function [6, 7], we hypothesized that Shp2 functions in the Dectin-1 signaling pathway to promote particulate-stimulated reactive oxygen species (ROS) production in macrophages
We used peritoneal exudate macrophages (PEMs) to examine the kinetics of Shp2 phosphorylation at tyrosine 580 [34] in response to zymosan and correlated Shp2 phosphorylation with peak ROS production and activation of the known Dectin-1-responsive signaling molecules, Syk, Erk, and Akt
Summary
Innate immune cell oxidative burst is needed to combat pathogens. Results: Loss of Shp phosphatase reduces, whereas increased Shp phosphatase function enhances, ROS production. Based on the known high expression of Shp in macrophages and its well defined role as a positive regulator of the Ras-Erk pathway, we hypothesized that Shp promotes normal innate immunity by positively up-regulating particulate-stimulated NADPH oxidase activation and abrupt production of ROS, known as oxidative burst. To address this hypothesis, we examined the correlation of Shp activation to peak ROS production in zymosan-stimulated peritoneal exudate macrophages (PEMs) and examined the putative placement of Shp in the Dectin-1-stimulated pathway employing genetic studies and pharmacologic studies using the Syk inhibitor R406 and the Erk inhibitor SCH772984. Mechanistic studies demonstrated that Shp exerts its positive effect on ROS generation by dephosphorylating the myeloid inhibitory immunoreceptor, SIRP␣ (signal regulatory protein ␣), and by promoting Erk activation
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