Functions of ROS in Macrophages and Antimicrobial Immunity

Abstract

Reactive oxygen species (ROS) are a chemically defined group of reactive molecules derived from molecular oxygen. ROS are involved in a plethora of processes in cells in all domains of life, ranging from bacteria, plants and animals, including humans. The importance of ROS for macrophage-mediated immunity is unquestioned. Their functions comprise direct antimicrobial activity against bacteria and parasites as well as redox-regulation of immune signaling and induction of inflammasome activation. However, only a few studies have performed in-depth ROS analyses and even fewer have identified the precise redox-regulated target molecules. In this review, we will give a brief introduction to ROS and their sources in macrophages, summarize the versatile roles of ROS in direct and indirect antimicrobial immune defense, and provide an overview of commonly used ROS probes, scavengers and inhibitors.

Keywords: NADPH oxidases; ROS detection; ROS scavenging; antimicrobial defense; immunity; infection; inflammasome; macrophages; mitochondria; reactive oxygen species; redox signaling.

Figure 1

Reduction of O₂ generates O₂^●−, which acts as precursor for all other ROS subspecies produced by cells. O₂^●− quickly dismutates to H₂O₂. H₂O₂ can be converted enzymatically by myeloperoxidase (MPO) to OCl⁻ or by ferric iron (Fe³⁺) to ●OOR or ●OH. The oxidation of H₂O₂ by Fe³⁺ is called Fenton reaction [1] resulting in ●OH as reactive intermediate [2], however, this reaction rarely takes place in cells [3]. Similarly, the excitation of O₂ to ¹O₂ by radiation (ħv) rarely takes place in animals [4,5]. Of note, nitric oxide (NO) and peroxynitrite (ONOO⁻) are reactive nitrogen species (RNS) and not ROS.

Figure 2

The redox status of the cell is influenced by ROS production on the oxidative side and the antioxidant defense system on the reductive side. (1) If ROS producing and eliminating factors are in equilibrium, redox balance is achieved, which represents the normal status in cells. (2) ROS production can be increased and exceed the capacity of the antioxidant defense system. If excess ROS fulfill important cellular functions, this is called oxidative eustress. (3) Continuous ROS production beyond the level required for cellular functions leads to oxidative damage and is called oxidative distress. Thioredoxin reductase (TrxR), glutathione reductase (GR), superoxide dismutase (SOD), catalase (CAT).

Figure 3

The enzyme family of NADPH oxidases consists of Nox1, Nox2, Nox3, Nox4, Nox5, and the dual oxidases Duox1 and Duox2. Different stimuli can trigger ROS production by Nox1, 2 and 3, whereas Nox4 is considered to be constitutively active. Nox5, Duox1 and Duox2 are activated by calcium binding through their cytosolic EF-hand calcium binding domains.

Figure 4

Electrons are shuttled from NADH and FADH₂ through the four complexes I-IV of the electron transport chain (ETC) and the electron carriers coenzyme Q (CoQ) and cytochrome c (Cyt c). Mitochondria generate ROS mainly through complexes I and III. Matrix-derived O₂^●− and H₂O₂ can only reach the cytosol after mitochondrial permeability transition pore (mPTP) opening or damage caused to mitochondrial membranes. From the intermembrane space (IMS), H₂O₂ can reach the cytosol through diffusion or aquaporins (AQP), whereas O₂^●− can cross the outer mitochondrial membrane only through membrane channels such as voltage-dependent anion channels (VDAC).

Figure 5

(1) During infection with L.m., tissue macrophages activate a highly antimicrobial phagocytic pathway called LC3-associated phagocytosis (LAP). LAP induction strictly depends on production of extracellular ROS by Nox2, which induces the eponymous recruitment of LC3 to phagosomes. These so-called LAPosomes show enhanced fusion with lysosomes, leading to improved killing of L.m. by macrophages. (2) S.t. can evade degradation in the phagosome by using oxidative stress for their benefit. ROS production by Nox2 contributes to S.t. killing but, on its own, is not sufficient for complete eradication of the pathogen. Mitochondria are recruited as additional ROS source via Toll-like receptor (TLR) signaling to overcome the antioxidative capacity of S.t. resulting in its degradation. (3) Mtb inhibits Nox2-mediated ROS production and subsequent LAP induction through its virulence factor CpsA and quickly escapes into the cytosol. Macrophages activate excessive production and release of mtROS through the mitochondrial permeability transition pore (mPTP) via TNF receptor 1 (TNFR1) signaling to combat cytosolic Mtb.

Figure 6

(1) L.m. infection induces cytosolic mtROS production by complex III of the electron transport chain (ETC) in a Toll-like receptor (TLR)-dependent manner. These cytosolic mtROS covalently link the Iκb kinase (IKK) complex subunit NF-kappa-B essential modulator (NEMO) via disulfide bonds. This covalent linkage of NEMO is crucial for proinflammatory signaling and cytokine secretion. (2) After phagocytosis, Nox2-mediated ROS production is induced and the phagosomal ROS inactivate the cathepsins L and S in a redox-dependent manner. This inhibits excessive proteolysis of engulfed peptides and promotes proper presentation of antigens by major histocompatibility complex (MHC)-class II molecules. (3) mtROS disrupt the interaction of thioredoxin-interacting protein (TXNIP) with thioredoxin, enabling its interaction with inflammasome subunit NLRP3. This step is crucial for assembly of the NRLP3 inflammasome and subsequent caspase-1 activation and cleavage of IL-1β and IL-18.

Figure 7

(1) Diffusible ROS probes are not retained or targeted to a specific cellular compartment. Luminol is a luminophore that reacts with all types of ROS. Luminol-based chemiluminescence represents a quantitative value for the amount of ROS generated but since this probe is diffusible, both extra- and intracellular ROS, i.e., total cellular ROS, are detected. H₂DCF-DA is a cell-permeable derivative of fluorescein and one of the most commonly used ROS probes. H₂DCF-DA is often referred to as intracellular ROS probe. However, it is diffusible and therefore does not only remain in the cytosol but also reaches cell organelles and diffuses back into the extracellular space. Therefore, like with luminol, only total cellular ROS can be detected with this probe. DHE is a red fluorescent probe that rather specifically reacts with cellular O₂^●− resulting in red fluorescence. However, it easily crosses cellular membranes and therefore can be oxidized by O₂^●− anywhere in and outside of the cell. Therefore, with this probe only total cellular O₂^●− production can be detected. Oxidation of DHE generates two products, 2-hydroxyethidium and ethidium. Both products bind to DNA, which highly increase their fluorescence. This may lead to false interpretations regarding the localization of ROS production, namely DNA containing organelles such as the nucleus and mitochondria, when fluorescence microscopy is used to analyze ROS production. (2) Some ROS probes are either cell-impermeable, targeted to or retained in a specific cellular compartment. Isoluminol is a cell-impermeable derivative of luminol. This makes it ideal for exclusive measurement of extracellular ROS, which also includes ROS produced into the lumen of endosomes and phagosomes. 5/6-Carboxy-DCF is a derivative of H₂DCF-DA that contains two additional carboxyl groups that enhance its hydrophilicity and therefore strongly increase its retention in the cytosol. Therefore, 5/6-Carboxy-DCF can be used to specifically detect cytosolic ROS levels. MitoSOX is accumulated in the mitochondrial matrix and specifically detects O₂^●−.