Respiratory burst (oroxidative burst) is the rapid release of thereactive oxygen species (ROS),superoxide anion (O−
2) andhydrogen peroxide (H
2O
2), from differentcell types.
This is usually utilised for mammalianimmunological defence, but also plays a role incell signalling. Respiratory burst is also implicated in theovum of animals followingfertilization. It may also occur inplant cells.
Immune cells can be divided intomyeloid cells andlymphoid cells. Myeloid cells, includingmacrophages andneutrophils, are especially implicated in the respiratory burst.[1] They arephagocytic, and the respiratory burst is vital for the subsequent degradation ofinternalisedbacteria or otherpathogens. This is an important aspect of theinnate immunity.
Respiratory burst requires a 10 to 20 fold increase in oxygen consumption throughNADPH oxidase (NOX2 in humans) activity. NADPH is the keysubstrate of NOX2, and bearsreducing power.Glycogen breakdown is vital to produce NADPH. This occurs via thepentose phosphate pathway.
The NOX2 enzyme is bound in thephagolysosome membrane. Post bacterial phagocytosis, it is activated, producing superoxide via itsredox centre, which transfers electrons from cytosolic NADPH to O2 in the phagosome.[2]
2O2 + NADPH —> 2O2•– + NADP+ + H+
The superoxide can thenspontaneously or enzymatically react with other molecules to give rise to other ROS. The phagocytic membrane reseals to limit exposure of the extracellular environment to the generated reactivefree radicals.
There are 3 main pathways for the generation of reactive oxygen species orreactive nitrogen species (RNS) ineffector cells:[3]
Nitric oxide may react with superoxide anions to produceperoxynitrite anion.
The exposure to these reactive species in the respiratory burst results in pathology. This is due tooxidative damage to the engulfed bacteria.
Notably, peroxynitrite is a very strongoxidising agent that can lead tolipid peroxidation, protein oxidation, proteinnitration, which are responsible for its bactericidal effects. It may react directly with proteins that contain transition metal centers, such asFeS, releasing Fe2+ for the Fenton reaction. Peroxynitrite may also react with various amino acids in the peptide chain, thereby altering protein structure and subsequently, protein function. It most commonly oxidisescysteine, and may indirectly inducetyrosine nitration through other generated RNS. Altered protein function includes changes in enzyme catalytic activity, cytoskeletal organisation and cell signal transduction.[4]
Hypochlorous acid reacts with a range of biomolecules, including DNA, lipids and proteins. HClO may oxidise cysteines andmethionines via theirsulfhydryl groups and sulfur groups respectively. The former leads to the formation ofdisulfide bonds, inducing protein crosslinking. Both oxidations result in protein aggregation, and ultimately, cell death.[5] Sulfhydryl groups can be oxidised up to three times by three HClO molecules, forming sulfenic acids, sulfinic acids andR–SO3H, which are increasingly irreversible and bactericidal.[6][7] Meanwhile, methionine oxidation is reversible. HOCl can also react with primary or secondaryamines, producing chloroamines which are toxic to bacteria.[8][9] Protein cross linking and aggregation may also occur, as well as disruption of FeS groups.
Integral to hypochlorous acid formation is myeloperoxidase. Myeloperoxidase is most abundant in neutrophils, wherein phagocytosis is accompanied bydegranulation. This is the fusion of granules with the phagolysosome, releasing their contents, including myeloperoxidase.[10] As many microbicidal products are formed during respiratory burst, the importance of individual molecules in killing invading pathogens is not wholly understood.
Due to the high toxicity of generated antimicrobial products including ROS, neutrophils have a short life span to limit host tissue damage duringinflammation.
Chronic Granulomatous Disease is an inherited disease of humanneutrophils, wherein NOX2 is defective. Phagocytosis may still occur, but without proper functioning NOX2, there is no superoxide production, and therefore no respiratory burst. The bacterialinfection is not cleared.[11]
In non-phagocytic cells, oxidative burst products are used in intracellular signalling pathways. The generated ROS achieve this via shifting the cellredox state. This may be monitored by the ratio of the antioxidant enzymeglutathione to its oxidised product,glutathione disulphide (GSH:GSSG).[12] Antioxidant enzymes counterbalance redox signalling by eliminating the involved molecules, importantly superoxide anion and nitric oxide. Redox signalling is critical for normal processes such as proliferation, differentiation, as well as vascular function and neurotransmission. It is also involved in disease states such ascancer.
The NADPH oxidase isoformNOX1 transiently produces a burst of superoxide in response togrowth factor (e.g.EGF) stimulation of respective receptors.[13] Superoxide is dismutated to hydrogen peroxide at a rate close to the diffusion-limited rate. This spatial restriction for superoxide‘s dismutation allows for specificity of redox signalling. Specificity is also ensured by NOX1 localisation in specific microdomains in the cell’s plasma membrane. Through channels such asaquaporin or diffusion, hydrogen peroxide enters the cytosol. There, it oxidises the cysteine groups of redox-sensitive proteins, which can then transduce signals.[14]
Oxidative burst in phagocytes is most commonly associated with bacterial killing. However, macrophages, especiallyalveolar macrophages, usually produce far lower levels of ROS than neutrophils, and may require activation for their bactericidal properties. Instead, their transient oxidative burst regulates the inflammatory response by inducingcytokine synthesis for redox signalling, resulting in an influx of neutrophils and activated macrophages.[15]
Cancer cells can manipulate cell signalling by producing excess levels of ROS, thereby constitutively activating pathways to promote their cellular growth and proliferation.[16] Implicated pathways includeNF-κB,PI3K,HIFs andMAPKs. In humans, mitochondrial ROS is required alongside those released in the oxidative burst formitogenic pathway stimulation inoncogenicKRAS cells. However, in oncogenic Kras micefibroblasts, NADPH oxidase inhibitors have been shown to be sufficient to block these growth factor pathways.[17] Tumorigenic cells also simultaneously maintain high levels of antioxidants to protect against cancer cell death.[18]
Most notably, oxidative burst post fertilisation can be seen in thesea urchin egg. This is believed to be evolutionally divergent from that in neutrophils.
Hydrogen peroxide is produced byegg oxidase activity following an increase in oxygen consumption.[19] This is essential for thecross-linking of the ovum proteins to prevent lethalpolyspermy. Hydrogen peroxide itself is also spermicidal. However, the generated reactive species are maintained at lower levels than in immunity to protect the fertilised egg itself from oxidative damage. This is achieved by the elimination of hydrogen peroxide primarily through the dual function of the same egg oxidase, and secondarily through cytoplasmic ROS scavengers, such ascatalase andglutathione.[20]
Oxidative burst acts as a defence mechanism to pathogen infection in plants. This is seen postPAMPs detection by cell-surface located receptors (e.g.FLS2 orEFR).[21][22][23] As in animals, the production of reactive oxygen species in plants is mediated byNADPH oxidase. Inplant immunity, the NADPH oxidase subunits RbohD and RbohF have overlapping functions are expressed in different tissues and at different levels.[24][25] However, in contrast to animal phagocytes, wherein generated ROS are contained in the sealed phagolysosome, oxidative burst in plants is not contained. Consequently, generated ROS bear additional effects alongside pathogen toxicity. Hydrogen peroxide induces oxidative cross-linking of the plant’s cell wall glycoproteins.[26][27] This reduces susceptibility to enzymatic degradation by pathogens.[28]Systemic acquired resistance, which is analogous to innate immunity in animals, is also induced in the exposed plant cells.[29] Hydrogen peroxide exposure may also result inhypersensitive response, which is the death of a small number of host cells at the site of infection, for the purpose of limiting pathogenic infection.[30][31] ROS production in plants can be used as a readout for successful pathogen recognition via aluminol-peroxidase based assay.[32]