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Loss of NADPH oxidase-derived superoxide skews macrophage phenotypes to delay type 1 diabetes.

ABSTRACT: Macrophages are early islet-infiltrating cells seen in type 1 diabetes (T1D). While proinflammatory M1 macrophages induce T1D, M2 macrophages have been shown to delay this autoimmune disease in nonobese diabetic (NOD) mice, but the environmental cues that govern macrophage polarization and differentiation remain unresolved. We previously demonstrated the importance of reactive oxygen species (ROS) in T1D, as NOD mice deficient in NADPH oxidase (NOX)-derived superoxide (Ncf1(m1J)) were protected against T1D partly because of blunted Toll-like receptor-dependent macrophage responses. We provide evidence that NOX-derived ROS contribute to macrophage differentiation in T1D. During spontaneous diabetes progression, T1D-resistant NOD.Ncf1(m1J) islet-resident macrophages displayed a dampened M1 and increased M2 phenotype. The transfer of diabetogenic T cells into NOX-deficient NOD.Rag.Ncf1(m1J) recipients resulted in decreased TNF-?(+) and IL-1?(+) islet-infiltrating M1 macrophages and a concomitant enhancement in arginase-1(+) M2 macrophages. Mechanistic analysis of superoxide-deficient bone marrow-derived macrophages revealed a marked diminution in a proinflammatory M1 phenotype due to decreased P-STAT1 (Y701) and interferon regulatory factor 5 compared with NOD mice. We have therefore defined a novel mechanistic link between NOX-derived ROS and macrophage phenotypes, and implicated superoxide as an important factor in macrophage differentiation. Thus, targeting macrophage redox status may represent a promising therapy in halting human T1D.

SUBMITTER: Padgett LE

PROVIDER: S-EPMC4338593 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Loss of NADPH oxidase-derived superoxide skews macrophage phenotypes to delay type 1 diabetes.

Padgett Lindsey E LE Burg Ashley R AR Lei Weiqi W Tse Hubert M HM

Diabetes 20141006 3

Macrophages are early islet-infiltrating cells seen in type 1 diabetes (T1D). While proinflammatory M1 macrophages induce T1D, M2 macrophages have been shown to delay this autoimmune disease in nonobese diabetic (NOD) mice, but the environmental cues that govern macrophage polarization and differentiation remain unresolved. We previously demonstrated the importance of reactive oxygen species (ROS) in T1D, as NOD mice deficient in NADPH oxidase (NOX)-derived superoxide (Ncf1(m1J)) were protected ...[more]

PMID: 25288672

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Project description:Superoxide radical plays an important role in redox cell signaling and physiological processes; however, overproduction of superoxide or insufficient activity of antioxidants leads to oxidative stress and contributes to the development of pathological conditions such as endothelial dysfunction and hypertension. Meanwhile, the studies of superoxide in biological systems represent unique challenges associated with short lifetime of superoxide, insufficient reactivity of the superoxide probes, and lack of site-specific detection of superoxide. In this work we have developed 15N-and deuterium-enriched spin probe 15N-CAT1H for high sensitivity and site-specific detection of extracellular superoxide. We have tested simultaneous tracking of extracellular superoxide by 15N-CAT1H and intramitochondrial superoxide by conventional 14N-containing spin probe mitoTEMPO-H in immune cells isolated from spleen, splenocytes, under basal conditions or stimulated with inflammatory cytokines IL-17A and TNFα, NADPH oxidase activator PMA, or treated with inhibitors of mitochondrial complex I rotenone or complex III antimycin A. 15N-CAT1H provides two-fold increase in sensitivity and improves detection since EPR spectrum of 15N-CAT1 nitroxide does not overlap with biological radicals. Furthermore, concurrent use of cell impermeable 15N-CAT1H and mitochondria-targeted 14N-mitoTEMPO-H allows simultaneous detection of extracellular and mitochondrial superoxide. Analysis of IL-17A- and TNFα-induced superoxide showed parallel increase in 15N-CAT1 and 14N-mitoTEMPO signals suggesting coupling between phagocytic NADPH oxidase and mitochondria. The interplay between mitochondrial superoxide production and activity of phagocytic NADPH oxidase was further investigated in splenocytes isolated from Sham and angiotensin II infused C57Bl/6J and Nox2KO mice. Angiotensin II infusion in wild-type mice increased the extracellular basal splenocyte superoxide which was further enhanced by complex III inhibitor antimycin A, mitochondrial uncoupling agent CCCP and NADPH oxidase activator PMA. Nox2 depletion attenuated angiotensin II mediated stimulation and inhibited both extracellular and mitochondrial PMA-induced superoxide production. These data indicate that splenocytes isolated from hypertensive angiotensin II-infused mice are "primed" for enhanced superoxide production from both phagocytic NADPH oxidase and mitochondria. Our data demonstrate that novel 15N-CAT1H provides high sensitivity superoxide measurements and combination with mitoTEMPO-H allows independent and simultaneous detection of extracellular and mitochondrial superoxide. We suggest that this new approach can be used to study the site-specific superoxide production and analysis of important sources of oxidative stress in cardiovascular conditions.

Project description:Hypocretin/orexin-producing neurons, located in the perifornical region of the lateral hypothalamus area (LHA) and projecting to the brain sites of rostral ventrolateral medulla (RVLM), involve in the increase of sympathetic activity, thereby regulating cardiovascular function. The current study was designed to test the hypothesis that the central orexin-A (OXA) could be involved in the cardiovascular dysfunction of acute myocardial infarction (AMI) by releasing NAD(P)H oxidase-derived superoxide anion (O2 (-)) generation in RVLM, AMI rat model established by ligating the left anterior descending (LAD) coronary artery to induce manifestation of cardiac dysfunction, monitored by the indicators as heart rate (HR), heart rate variability (HRV), mean arterial pressure (MAP) and left intraventricular pressure. The results showed that the expressions of OXA in LHA and orexin 1 receptor (OX1R) increased in RVLM of AMI rats. The double immunofluorescent staining indicated that OX1R positive cells and NAD(P)H oxidative subunit gp91phox or p47phox-immunoreactive (IR) cells were co-localized in RVLM. Microinjection of OXA into the cerebral ventricle significantly increased O2 (-) production and mRNA expression of NAD(P)H oxidase subunits when compared with aCSF-treated ones. Exogenous OXA administration in RVLM produced pressor and tachycardiac effects. Furthermore, the antagonist of OX1R and OX2R (SB-408124 and TCS OX2 29, respectively) or apocynin (APO), an inhibitor of NAD(P)H oxidase, partly abolished those cardiovascular responses of OXA. HRV power spectral analysis showed that exogenous OXA led to decreased HF component of HRV and increased LF/HF ratio in comparison with aCSF, which suggested that OXA might be related to sympathovagal imbalance. As indicated by the results, OXA might participate in the central regulation of cardiovascular activities by disturbing the sympathovagal balance in AMI, which could be explained by the possibility that OXR and NAD(P)H-derived O2 (-) in RVLM mediates OXA-induced cardiovascular responses.

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Loss of NADPH oxidase-derived superoxide skews macrophage phenotypes to delay type 1 diabetes.

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Loss of NADPH oxidase-derived superoxide skews macrophage phenotypes to delay type 1 diabetes.

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