Alkaline pH Promotes NADPH Oxidase-Independent Neutrophil Extracellular Trap Formation: A Matter of Mitochondrial Reactive Oxygen Species Generation and Citrullination and Cleavage of Histone.

pH is highly variable in different tissues and affects many enzymatic reactions in neutrophils. In response to calcium ionophores such as A23187 and ionomycin, neutrophils undergo nicotinamide adenine dinucleotide phosphate oxidase (NOX)-independent neutrophil extracellular trap (NET) formation (NETosis). However, how pH influences calcium-dependent Nox-independent NET formation is not well understood. We hypothesized that increasing pH promotes Nox-independent NET formation by promoting calcium influx, mitochondrial reactive oxygen species (mROS) generation, histone citrullination, and histone cleavage. Here, we show that stimulating human neutrophils isolated from peripheral blood with calcium ionophore A23187 or ionomycin in the media with increasing extracellular pH (6.6, 6.8, 7.0, 7.2, 7.4, 7.8) drastically increases intracellular pH within in 10-20 min. These intracellular pH values are much higher compared to unstimulated cells placed in the media with corresponding pH values. Raising pH slightly drastically increases intracellular calcium concentration in resting and stimulated neutrophils, respectively. Like calcium, mROS generation also increases with increasing pH. An mROS scavenger, MitoTempo, significantly suppresses calcium ionophore-mediated NET formation with a greater effect at higher pH, indicating that mROS production is at least partly responsible for pH-dependent suppression of Nox-independent NETosis. In addition, raising pH increases PAD4 activity as determined by the citrullination of histone (CitH3) and histone cleavage determined by Western blots. The pH-dependent histone cleavage is reproducibly very high during ionomycin-induced NETosis compared to A23187-induced NETosis. Little or no histone cleavage was noted in unstimulated cells, at any pH. Both CitH3 and cleavage of histones facilitate DNA decondensation. Therefore, alkaline pH promotes intracellular calcium influx, mROS generation, PAD4-mediated CitH3 formation, histone 4 cleavage and eventually NET formation. Calcium-mediated NET formation and CitH3 formation are often related to sterile inflammation. Hence, understanding these important mechanistic steps helps to explain how pH regulates NOX-independent NET formation, and modifying pH may help to regulate NET formation during sterile inflammation or potential damage caused by compounds such as ionomycin, secreted by Streptomyces, a group of Gram-positive bacteria well known for producing antibiotics.

Administration of α-Galactosylceramide Improves Adenine-Induced Renal Injury.

Natural killer T (NKT) cells are a subset of lymphocytes that reacts to glycolipids presented by CD1d. Invariant NKT cells (iNKT) correspond to >90% of the total population of NKTs and reacts to α-galactosylceramide (αGalCer). αGalCer promotes a complex mixture of Th1 and Th2 cytokines, as interferon (IFN)-γ and interleukin (IL)-4. NKT cells and IFN-γ are known to participate in some models of renal diseases, but further studies are still necessary to elucidate their mechanisms. The aim of our study was to analyze the participation of iNKT cells in an experimental model of tubule-interstitial nephritis. We used 8-wk-old C57BL/6j, Jα18KO and IFN-γKO mice. They were fed a 0.25% adenine diet for 10 d. Both adenine-fed wild-type (WT) and Jα18KO mice exhibited renal dysfunction, but adenine-fed Jα18KO mice presented higher expression of kidney injury molecule-1 (KIM-1), tumor necrosis factor (TNF)-α and type I collagen. To analyze the role of activated iNKT cells in our model, we administered αGalCer in WT mice during adenine ingestion. After αGalCer injection, we observed a significant reduction in serum creatinine, proinflammatory cytokines and renal fibrosis. However, this improvement in renal function was not observed in IFN-γKO mice after αGalCer treatment and adenine feeding, illustrating that this cytokine plays a role in our model. Our findings may suggest that IFN-γ production is one of the factors contributing to improved renal function after αGalCer administration.

The Macrophage Switch in Obesity Development.

Immune cell infiltration in (white) adipose tissue (AT) during obesity is associated with the development of insulin resistance. In AT, the main population of leukocytes are macrophages. Macrophages can be classified into two major populations: M1, classically activated macrophages, and M2, alternatively activated macrophages, although recent studies have identified a broad range of macrophage subsets. During obesity, AT M1 macrophage numbers increase and correlate with AT inflammation and insulin resistance. Upon activation, pro-inflammatory M1 macrophages induce aerobic glycolysis. By contrast, in lean humans and mice, the number of M2 macrophages predominates. M2 macrophages secrete anti-inflammatory cytokines and utilize oxidative metabolism to maintain AT homeostasis. Here, we review the immunologic and metabolic functions of AT macrophages and their different facets in obesity and the metabolic syndrome.