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Review
. 2014 Mar 1;20(7):1126-67.
doi: 10.1089/ars.2012.5149. Epub 2013 Oct 22.

Reactive oxygen species in inflammation and tissue injury

Manish Mittal  1 , Mohammad Rizwan Siddiqui, Khiem Tran, Sekhar P Reddy, Asrar B Malik
Affiliations
Review

Reactive oxygen species in inflammation and tissue injury

Manish Mittal et al. Antioxid Redox Signal. .

Abstract

Abstract Reactive oxygen species (ROS) are key signaling molecules that play an important role in the progression of inflammatory disorders. An enhanced ROS generation by polymorphonuclear neutrophils (PMNs) at the site of inflammation causes endothelial dysfunction and tissue injury. The vascular endothelium plays an important role in passage of macromolecules and inflammatory cells from the blood to tissue. Under the inflammatory conditions, oxidative stress produced by PMNs leads to the opening of inter-endothelial junctions and promotes the migration of inflammatory cells across the endothelial barrier. The migrated inflammatory cells not only help in the clearance of pathogens and foreign particles but also lead to tissue injury. The current review compiles the past and current research in the area of inflammation with particular emphasis on oxidative stress-mediated signaling mechanisms that are involved in inflammation and tissue injury.

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Figures

<b>FIG. 1.</b>
FIG. 1.
Sources of reactive oxygen species (ROS) and antioxidant defense system. The major ROS include superoxide (O2•−), hydrogen peroxide (H2O2), hydroxyl anions (OH−), hydroxyl radicals (OH•), and hypochlorous acid (HOCl). Superoxide is produced by NADPH oxidase/xanthine oxidase-derived reduction of molecular oxygen, uncoupled endothelial nitric oxide synthase (eNOS), or mitochondrial electron transport chain (ETC). Superoxide is rapidly dismutated to H2O2 by superoxide dismutase (SOD). However, in the presence of nitric oxide (NO), O2•− rapidly reacts with NO, resulting in the formation of highly reactive peroxynitrite (ONOO−), which is three to four times faster than dismutation of O2•− to H2O2. H2O2 can change to highly reactive HOCl at the inflammatory sites by an enzyme known as myeloperoxidase (MPO), which is abundantly expressed in neutrophils. H2O2 can also change to the highly toxic OH• in presence of Fe2+ by Fenton's reaction. H2O2 is scavenged to H2O and O2 by catalase, glutathione peroxidase (GPX), or peroxiredoxins (Prx) antioxidant enzymes. Prx uses thioredoxin (Trx) to detoxify H2O2. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 2.</b>
FIG. 2.
Activation mechanism and assembly of different NADPH oxidase homologs. (a) Two independent events are required for the activation of gp91phox, resulting in the assembly of the cytosolic regulatory proteins (p40phox, p47phox, and p67phox) with the flavocytochrome b558 (made up of the membrane-associated catalytic subunit gp91phox and p22phox). One of the two events is the activation of protein kinases such as protein kinase C (PKC) and AKT, which phosphorylate the autoinhibitory region (AIR) of p47phox, thus relieving its inhibition from the autoinhibitory loop and enabling p47phox to bind with p22phox. The second event results in the replacement of GDP residue with GTP by a guanine nucleotide exchange factor (GEF), resulting in a conformational change of Rac protein by relieving inhibition from Rho GDP-dissociation inhibitor (RhoGDI), promoting its binding with p67phox, and finally, resulting in the formation of active complex. The pink hexagon represents heme. (b–e) differential assembly of NADPH oxides homologs. NOX1, NOX3 (not shown), and NOX4 share a similar topological structure of the catalytic core of gp91phox. NOX5 carries an additional intracellular N-terminal calcium-binding domain, whereas Duox1 and 2 are built on NOX5 structure that carries another additional N-terminus transmembrane α-helix which possesses a peroxidase homology domain. ROS generation by NOX1 is induced by assembly with cytosolic subunits NOXO1, NOXA1, and Rac. NOX4 does not require cytosolic subunits for ROS generation but requires p22phox. ROS generation by NOX5 and DUOX can be induced by calcium and is independent of p22phox subunit. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 3.</b>
FIG. 3.
Schematic presentation of mitochondrial ETC and site of ROS generation. The Mitochondrial ETC consists of a series of electron carriers that are arranged spatially in the order of their increasing redox potential and organized into four complexes. Arrows in the region of complexes I-IV show pathways of electron transfer between flavins (FMN-H2, FADH2), iron-sulfur centers (Fe-S), coenzyme Q (Q-QH2), cytochromes (c1, c a, and a3), and molecular oxygen (O2), resulting in the formation of H2O. The main sites of ROS generation are complex I and III. Pharmacological inhibitors of complex I (such as rotenone) and complex III (myxothiazol and antimycin A) are known to enhance mitochondrial ROS generation because of uncoupling of ETC. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 4.</b>
FIG. 4.
Inflammasome activation by ROS. Increased ROS production inside the cell either through NADPH oxidase or mitochondrial ETC is sensed by a complex of Trx and thioredoxin interacting protein (TXNIP), which dissociates and enables the binding of TXINP with NLRP3. This is followed by activation of NLRP3 and recruitment of Asc and Pro-caspase1/12 proteins, leading to formation of active inflammasome. Active NLRP3 inflammasome cleaves pro-interleukin-1 (IL-1) beta and pro-IL-18 to active IL1 beta and IL-18, which are subsequently secreted by the inflammatory cells. PAMP, pathogen-associated molecular patterns; DAMP, danger-associated molecular patterns; ASC, apoptosis-associated speck-like protein. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 5.</b>
FIG. 5.
Schematic presentation of ROS generation by uncoupling of eNOS. (a) eNOS is a homodimeric enzyme consisting of a reductase and an oxygenase domain. The dimer interface of eNOS carries the binding sites for the cofactor tetrahydrobiopterin (BH4) and the substrate L-arginine. eNOS carries out two successive monoxygenation of L-arginine, resulting in formation of L-citruline and NO as a by product. In this reaction, BH4 is oxidized to form trihydropterin radicals protonated at N5 (BH3H+), which are recycled to BH4 by endothelial NO synthase itself (using an electron supplied by the flavins). (b) Increased levels of peroxynitrite as a result of oxidative stress leads to oxidation of BH4 to biologically inactive products BH2, which cannot be recycled back by cellular machinery. This creates an “uncoupled” enzymatic state that reduces oxygen to superoxide and no longer synthesizes NO. ONOO−, Peroxynitrite; O2, molecular oxygen; O2•−, superoxide anion. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 6.</b>
FIG. 6.
NF-E2-related factor 2 (Nrf2)-dependent antioxidant mechanism. (a) Nrf2 dissociates from Kelch-like ECH-associated protein 1 (Keap1) on exposure to oxidants and translocates to the nucleus where it binds to the promoter region of antioxidant enzymes containing antioxidant response element (ARE) such as Gpx2, NQO1, and GCLC. M/J/A: Maf, Jun, or ATF family of proteins. (b) Nrf2-dependent effector mechanism involves transcription of antioxidant enzymes and attenuation of injury-related genes that provide protection against oxidant-induced acute lung injury. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 7.</b>
FIG. 7.
Schematic representation of transendothelial migration of leukocytes. (a) Different classes of cell adhesion molecules (CAMs) expressed in endothelial and inflammatory immune cells. There are three main classes of CAMs: selectin family, integrin family, and immunoglobulin superfamily. (b) The initial binding of leukocytes on endothelial surface is mediated by low-affinity binding of selectins, which enables rolling of neutrophils on the endothelium. The selectin-mediated bonds are formed between P- and E-selectin on endothelial cells and L-selectin, E-selectin ligand-1 (ESL-1) and P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes. The affinity of PSGL-1 to P-selectin is much higher compared with E-selectin; whereas E-selectin binds predominantly to ESL-1. The secretion of chemokine CXCL8 from WPB binds to chemokine receptor on neutrophils and activates integrin by changing its conformation from bend to a fully extended form. The activated integrins then subsequently bind to intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expressed in endothelial cells and cause neutrophil arrest on the endothelial surface. leukocyte function–associated antigen 1 (LFA-1 or αLβ2) and αMβ2 (Mac1) are the principal integrins that are expressed in neutrophils and bind to ICAM-1 in endothelial cells. The Mac-1 integrins enable slow crawling of neutrophils toward the endothelial junctions. At the site of endothelial junctions, ICAM-1 and VCAM-1 are gathered in transmigratory cups that hold neutrophils and facilitate their transmigration. The binding of JAM molecules further enables the deep penetration of neutrophils between endothelial cells. Platelet endothelial cell adhesion molecule (PECAM1) is recruited to sites of transmigration from the lateral border recycling compartment (LBRC), which stores 30% of the total PECAM-1. CD99L plays a similar role to PECAM-1 in enabling neutrophil exit across endothelial junctional compartments. The transcellular route of migration is adopted by 10%–15% of neutrophils that can greatly increase in the absence of Mac-1 integrin. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 8.</b>
FIG. 8.
Structure of endothelial junctional proteins. Interaction between two adjacent endothelial cells is regulated by Adherens junction (AJ) and tight junction (TJ) proteins. AJs are formed by a homophilic interaction of transmembrane cadherin in a calcium-dependent manner that is linked to the actin cytoskeleton by supporting p120-, α-, β-, and γ-catenins. TJs consist of integral transmembrane proteins, claudin, occludin, and (JAM), form integral TJs between adjacent endothelial cells. Other accessory proteins, such as zonula occludens (ZO-1/2/3, etc.), are involved in structure support. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 9.</b>
FIG. 9.
Dynamic behavior of actin filament. Actin tread-milling is driven by ATP-hydrolysis. ATP-actin polymerizes at the plus end of filaments, while ADP-actin dissociates from the minus end. ADF and cofilin sever actin filaments at point end and thus promote the release of ADP-actin monomers. In the presence of Ca2+ gelsolin caps (+) end to block the filament growth. By inducing ATP/ADP exchange, profilin increases actin-ATP and promotes polymerization. Actin depolymerizing factor (ADF). To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 10.</b>
FIG. 10.
Signaling mechanisms of ROS-mediated increase in leukocyte migration and junctional permeability. During neutrophil migration, clustering of ICAM-1 by integrins leads to the activation of Rac, which induces intracellular ROS generation by NADPH oxidases. Increased ROS generation activates tyrosine kinase activity of c-Src and PYK2, which phosphorylates VE-cadherin at Y645, Y658, Y731, and Y733. The phosphorylation of these residues destabilizes the AJs by preventing interactions with the cytoplasmic proteins such as beta catenin and p120 catenin. Increased ROS enhances the expression of P-selectin on the endothelium by enabling secretion from Weibel–Palade bodies (WPB). In addition, ROS is known to activate NF-κB, which induces the expression of cell adhesion molecules such as ICAM-1, VCAM-1, and E-selectin; enhances neutrophil binding on the endothelium; and increases paracellular migration. Acute exposure of pro-inflammatory factors such as thrombin, lipopolysaccharide (LPS), and VEGF trigger NOX-mediated ROS formation and PLC-γ/cADPR activation, which resulted in an increase of [Ca2+]i by TRPC/TRPM. Increase in [Ca2+]i activates calmodulin and kinases, which, in turn, modified constituents of AJs and reorganized actin cytoskeleton and resulted in junction disassembly. Agonist-mediated ROS formation also induces transcription of pro-inflammatory genes by canonical NFκB pathway. ROS are also involved in activation of different kinases, including c-Src, PYK2, FAK, and PKC, and thus induce phosphorylation of AJ proteins and destabilize AJs. E-Sln, E-Selectin; P-Sln, P-Selectin; WPB, Weibel-Palade bodies; CaM, calmodulin; cADPR, cyclic ADP Ribose; DAG, diacylglycerol; FAK, focal adhesion kinase; GPCR, G protein coupled receptor; MLCK, myosin light-chain kinase; PLC, phospho lipase C; PKC, protein Kinase C; PYK2, protein tyrosine kinase; RTK, receptor tyrosine kinase; TRPC/M, transient receptor potential canonical/melastatin channel; TLR, toll-like receptor. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 11.</b>
FIG. 11.
H2O2-mediated ionic current and increase in endothelial barrier permeability depends on TRPM2-L expression. (a) Current–voltage relationship for membrane current induced by H2O2 in untransfected, TRPM2-L–overexpressing and TRPM2 siRNA–transfected cells. (b) Bar graph quantifying peak currents (in pA) of (a). (c, d) human arterial endothelial cells were plated to confluence on gold electrodes without or with control/TRPM2 siRNA and then treated with 300 μM H2O2. H2O2-mediated decrease in trans-endothelial electrical resistance (TER) was significantly attenuated in cells transfected by TRPM2-specific siRNAs compared with untreated control or control group transfected with scrambled siRNA. The abscissa indicates time in hours; the ordinate, normalized resistance (relative to basal value). Modified from Hecquet et al. (171). *p<0.05. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
<b>FIG. 12.</b>
FIG. 12.
Schematic representation of ROS-mediated cell death and cell survival signaling pathways. The intricate balance between cell death and cell survival is largely modulated by intracellular ROS generation. In general, high intracellular ROS generation causes cell death by activation of cell death pathways (mitochondrial dependent and independent), whereas low levels of ROS acts as a signaling molecules that help in cell survival. The major cell death receptors such as TNF receptor–1 (TNFR1) and FasR cause cell death by enhancing high intracellular ROS production. The binding of TNF alpha to TNFR1 recruits proteins such as TNFR1-associated death domain (TRADD) and TNFα-receptor-associated factor 2 (TRAF2) to activate the TNFR1 signaling. The associated TRADD further recruits Fas-associated death domain (FADD) and pro-caspase-8, and this whole signaling complex known as death-inducing signaling complex (DISC) is endocytosed, resulting in caspase-8 activation. Similar to TNFR1, FasR recruits FADD and activates caspase-8. The extent of caspase-8 activation determines whether a cell will follow a mitochondrial-dependent pathway or an independent pathway. Low caspase-8 activation follows a mitochondrial-dependent amplification loop that is accompanied by caspase-8-mediated cleavage of pro-apoptotic bid (truncated bid [t-bid]), which causes Cyt-c release via oligomerization of Bax/Bak proteins in mitochondria. The released cytochrome c then subsequently binds apoptosis activation factor-1 (Apaf-1) and caspase-9 and activates effector caspases such as caspase-3, which causes cell death. In the event of significant caspase-8 activation, it directly activates casapse-3 independent of mitochondria. High intracellular ROS induces sustained JNK activation and causes mitochondrial cytochrome c release-dependent cell death. High intracellular ROS also causes cardiolipin (CL) oxidation, which is translocated to the outer membrane and provides the docking site for t-bid, facilitating the cytochrome c-release. In contrast, low levels of intracellular ROS mediate a transient JNK activation that helps in cell survival by activating AP-1 transcription factor and anti-apoptotic genes. TNFR1 signaling also activates NF-κB transcription factor by recruiting TRAF-2 and receptor-interacting kinase 1 (RIP1) proteins. NF-κB activation is involved in the transcription of manganese SOD (MnSOD) and anti-apoptotic signaling proteins such as cIAP-2 and Bcl-xL. MnSOD puts a negative feedback loop on enhanced ROS generation and switches off the apoptotic pathway. To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars
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