NOX2 deficiency alters macrophage phenotype through an IL-10/STAT3 dependent mechanism: implications for traumatic brain injury

Abstract

BackgroundNADPH oxidase (NOX2) is an enzyme system that generates reactive oxygen species (ROS) in microglia and macrophages. Excessive ROS production is linked with neuroinflammation and chronic neurodegeneration following traumatic brain injury (TBI). Redox signaling regulates macrophage/microglial phenotypic responses (pro-inflammatory versus anti-inflammatory), and NOX2 inhibition following moderate-to-severe TBI markedly reduces pro-inflammatory activation of macrophages/microglia resulting in concomitant increases in anti-inflammatory responses. Here, we report the signaling pathways that regulate NOX2-dependent macrophage/microglial phenotype switching in the TBI brain.MethodsBone marrow-derived macrophages (BMDMs) prepared from wildtype (C57Bl/6) and NOX2 deficient (NOX2−/−) mice were treated with lipopolysaccharide (LPS; 10 ng/ml), interleukin-4 (IL-4; 10 ng/ml), or combined LPS/IL-4 to investigate signal transduction pathways associated with macrophage activation using western immunoblotting and qPCR analyses. Signaling pathways and activation markers were evaluated in ipsilateral cortical tissue obtained from adult male wildtype and NOX2−/− mice that received moderate-level controlled cortical impact (CCI). A neutralizing anti-IL-10 approach was used to determine the effects of IL-10 on NOX2-dependent transitions from pro- to anti-inflammatory activation states.ResultsUsing an LPS/IL-4-stimulated BMDM model that mimics the mixed pro- and anti-inflammatory responses observed in the injured cortex, we show that NOX2−/− significantly reduces STAT1 signaling and markers of pro-inflammatory activation. In addition, NOX2−/− BMDMs significantly increase anti-inflammatory marker expression; IL-10-mediated STAT3 signaling, but not STAT6 signaling, appears to be critical in regulating this anti-inflammatory response. Following moderate-level CCI, IL-10 is significantly increased in microglia/macrophages in the injured cortex of NOX2−/− mice. These changes are associated with increased STAT3 activation, but not STAT6 activation, and a robust anti-inflammatory response. Neutralization of IL-10 in NOX2−/− BMDMs or CCI mice blocks STAT3 activation and the anti-inflammatory response, thereby demonstrating a critical role for IL-10 in regulating NOX2-dependent transitions between pro- and anti-inflammatory activation states.ConclusionsThese studies indicate that following TBI NOX2 inhibition promotes a robust anti-inflammatory response in macrophages/microglia that is mediated by the IL-10/STAT3 signaling pathway. Thus, therapeutic interventions that inhibit macrophage/microglial NOX2 activity may improve TBI outcomes by not only limiting pro-inflammatory neurotoxic responses, but also enhancing IL-10-mediated anti-inflammatory responses that are neuroprotective.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-017-0843-4) contains supplementary material, which is available to authorized users.

Melatonin protects against ischemic stroke by modulating microglia/macrophage polarization toward anti‐inflammatory phenotype through STAT3 pathway

“…STAT3 plays a critical role in macrophage polarization . In order to elucidate the mechanism underlying melatonin's regulation on microglia polarization, we examined the protein levels of STAT3 and p‐STAT3 in BV2 cells.…”

“…Microglia/macrophage polarization is modulated by many transcription factors, such as STATs and PPAR‐γ STAT3 was demonstrated to play a dominant role in IL‐10‐mediated anti‐inflammatory effects in human macrophages . In addition, STAT3 appeared to be critical in regulating macrophage/microglia polarization to anti‐inflammatory phenotype in the TBI brain . Besides, resveratrol was reported to suppress the LPS‐induced pro‐inflammatory response in microglia cells through the activation of STAT3 pathway .…”

Combination of Fluorescent in situ Hybridization (FISH) and Immunofluorescence Imaging for Detection of Cytokine Expression in Microglia/Macrophage Cells

“…Details regarding quantification of immunofluorescence and in situ mRNA hybridization has been provided by (Allen et al, 2016; Barrett et al, 2017; Villapol et al, 2017). Briefly:

Positive in situ mRNA hybridization signal consists of a punctate signal representing a single mRNA transcript (Wang et al, 2012).

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“…Our data demonstrate that a mixed pattern of both pro-inflammatory and anti-inflammatory cytokine expression occurred in microglia/macrophages in the first week after TBI. Also, we have previously shown that IL-10 levels are significantly increased in microglia/macrophages in the injured cortex of NOX2 −/− mice (Barrett et al, 2017). …”

“…We would like to say thanks to Dr. Kathy Maguire-Zeiss for letting us use her fluorescence microscope, and to Lucas Djavaherian for his help recording the video. This protocol has been adapted from (Barrett et al, 2017, Villapol et al, 2017). …”