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A role for the Sts phosphatases in negatively regulating IFN?-mediated production of nitric oxide in monocytes.

ABSTRACT: INTRODUCTION:The atypical Sts phosphatases negatively regulate signaling pathways in diverse immune cell types, with two of their molecular targets being the related kinases Syk and Zap-70. Mice lacking Sts expression (Sts-/- ) are resistant to infection by the live vaccine strain (LVS) of Francisella tularensis. Although the mechanisms underlying the enhanced resistance of Sts-/- mice have not been definitively established, Sts-/- bone marrow-derived monocytes (BMMs) demonstrate greater clearance of intracellular LVS following ex vivo infection, relative to wild type cells. To determine how the Sts proteins regulate monocyte bactericidal properties, we analyzed responses of infected cells. METHODS:Monocyte bacterial clearance was assayed using ex vivo coculture infections followed by colony-forming unit analysis of intracellular bacteria. Levels of gene expression were quantified by quantitative reverse-transcription polymerase chain reaction, levels of Nos2 protein levels were quantified by Western blot analysis, and levels of nitric oxide (NO) were quantified directly using the Griess reagent. We characterized monocyte cytokine production via enzyme-linked immunosorbent assay. RESULTS:We demonstrate that Sts-/- monocyte cultures produce elevated levels of interferon-? (IFN?) after infection, relative to wild type cultures. Sts-/- monocytes also demonstrate heightened responsiveness to IFN?. Specifically, Sts-/- monocytes produce elevated levels of antimicrobial NO following IFN? stimulation, and this NO plays an important role in LVS restriction. Additional IFN?-stimulated genes, including Ip10 and members of the Gbp gene family, also display heightened upregulation in Sts-/- cells. Both Sts-1 and Sts-2 contribute to the regulation of NO production, as evidenced by the responses of monocytes lacking each phosphatase individually. Finally, we demonstrate that the elevated production of IFN?-induced NO in Sts-/- monocytes is abrogated following chemical inhibition of Syk kinase. CONCLUSION:Our results indicate a novel role for the Sts enzymes in regulating monocyte antibacterial responses downstream of IFN?.

SUBMITTER: Parashar K

PROVIDER: S-EPMC7654413 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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A role for the Sts phosphatases in negatively regulating IFNγ-mediated production of nitric oxide in monocytes.

Parashar Kaustubh K Carpino Nicholas N

Immunity, inflammation and disease 20200825 4

<h4>Introduction</h4>The atypical Sts phosphatases negatively regulate signaling pathways in diverse immune cell types, with two of their molecular targets being the related kinases Syk and Zap-70. Mice lacking Sts expression (Sts<sup>-/-</sup> ) are resistant to infection by the live vaccine strain (LVS) of Francisella tularensis. Although the mechanisms underlying the enhanced resistance of Sts<sup>-/-</sup> mice have not been definitively established, Sts<sup>-/-</sup> bone marrow-derived mon ...[more]

PMID: 32841534

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Project description:Inflammation is regulated by many endogenous factors including estrogen, a steroid hormone that declines with increasing age, leading to excessive inflammation in the elderly. C-reactive protein (CRP) is an acute phase inflammatory protein that exists in two forms, native CRP (nCRP) and monomeric CRP (mCRP), which mediate distinct biological activities. It is unclear how each CRP isoform mediates nitric oxide (NO), a signaling molecule generated by NO synthase (NOS). This study investigated whether CRP isoforms have distinct effects on NO production by unstimulated and lipopolysaccharide (LPS)-activated monocytes/macrophages and whether estrogen mediates CRP-induced NO production in an in vitro model of aging. NO and inducible NOS (iNOS) were measured (n = 12) by the Griess assay and an enzyme-linked immunosorbent assay, respectively following incubation (24 h) of human-derived U937 monocytes/macrophages with CRP isoforms [(nCRP) = 500 and 1,000 µg/ml; (mCRP) = 100 and 250 µg/ml] in the absence or presence of 17 beta-estradiol (1 × 10-7, 1 × 10-8, and 1 × 10-9 M). The response to each CRP isoform and estrogen was dependent on the differentiation and activation status of cells. Monocytes with or without prior LPS-activation significantly increased (P < 0.01) NO/iNOS production when treated with mCRP. The mCRP isoform had no effect (P > 0.05) on NO/iNOS production by unactivated or LPS-activated macrophages, whereas nCRP significantly (P < 0.05) reduced NO/iNOS production by macrophages, with or without prior LPS-activation. The nCRP isoform had opposing actions on monocytes, significantly (P < 0.01) increasing and reducing NO/iNOS by unactivated and LPS-activated monocytes, respectively. Estrogen significantly (P < 0.01) reversed nCRP-mediated NO inhibition by unactivated macrophages but decreased CRP-induced NO by unactivated monocytes treated with nCRP or mCRP and LPS-activated monocytes treated with mCRP. NO was differentially mediated by CRP isoforms in a cell-type/state-specific manner, with production corresponding to concomitant changes in iNOS levels. Collectively, the findings indicate nCRP and estrogen predominantly reduce NO production, whereas mCRP increases NO production. This supports growing evidence that mCRP exacerbates inflammation while nCRP and estrogen dampen the overall inflammatory response. Therapeutic strategies that restore estrogen levels to those found in youth and promote the stability of nCRP or/and prevent the formation of mCRP may reduce NO production in age-related inflammatory conditions.

Dataset Information

A role for the Sts phosphatases in negatively regulating IFN?-mediated production of nitric oxide in monocytes.

Publications

A role for the Sts phosphatases in negatively regulating IFNγ-mediated production of nitric oxide in monocytes.

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