Review ArticleNADPH oxidase- and mitochondria-derived reactive oxygen species in proinflammatory microglial activation: a bipartisan affair?
Graphical abstract
Introduction
Microglia are macrophage-like immune cells of the central nervous system that play major roles in both health and disease [1], [2]. In health, microglia are highly motile cells that survey their surrounding environment to maintain homeostasis [3], promote synaptogenesis, and regulate the integrity of the blood–brain barrier [4], [5]. However, microglia become activated and proliferate after acute brain injury or in chronic neurodegenerative diseases such as Parkinson disease and Alzheimer disease [1], [2]. Activated microglia undergo drastic morphological changes, transforming from a ramified to an amoeboid morphology [1], [2]. This change in morphology is thought to favor mobility and increase phagocytic activity [6]. Microglia cordon off damaged neurons and assist in the repair and regrowth of compromised cells by releasing various metabolites, growth factors, and cytokines. However, prolonged or excessive microglial activation becomes maladaptive [7]. This is partly because activation of microglia takes multiple shapes. Classical M1 microglial activation results in the proinflammatory state implicated in neurotoxicity, for instance, the degeneration of dopaminergic neurons in Parkinson disease [3], [8], [9], [10], [11] or of motor neurons in amyotrophic lateral sclerosis [12], [13]. In addition to the creation of a toxic proinflammatory milieu, activated microglia may also contribute to cell death after brain injuries via phagocytosis of live neurons [14], [15], [16]. In contrast to M1 activation, alternative microglial activation states, generally classified as M2 with further subclasses, counteract proinflammatory mediators and promote repair processes such as remodeling of the extracellular matrix and angiogenesis [17]. Impairing excessive M1 microglial activation and/or promoting a transition toward alternative M2 activation are promising neuroprotective treatment strategies receiving considerable attention [18], [19]. It is important to understand the M1 activation process in greater detail to successfully achieve this goal.
To this end, extensive progress has been made in elucidating the initial molecular signaling pathways underlying microglial activation, especially through activation of Toll-like and cytokine receptors (see [9], [20] for detailed reviews). Treatment with the bacterial endotoxin lipopolysaccharide (LPS)1 alone or in combination with the proinflammatory cytokine interferon-γ (IFN-γ) is frequently used to induce and study M1 microglial activation in vitro. These factors activate Toll-like receptor 4 (TLR4, also called CD14) and IFN-γ receptor, respectively. LPS also induces TLR4-independent signaling by binding phagocytic scavenger receptors [21] and by binding macrophage antigen complex I (MAC1, also called CD11b/CD18), a pattern recognition receptor linked to the superoxide-generating enzyme NADPH oxidase (alternatively called Phox, for phagocyte oxidase) [22]. Reactive oxygen species (ROS) production, activation of kinase cascades, and changes in gene transcription occur subsequent to receptor ligation. ROS such as hydrogen peroxide (H2O2) are suggested to act as second messengers in cytokine responses [23], [24]. H2O2 activates mitogen-activated protein kinase (MAPK) cascades, partly through oxidation of catalytic cysteines on MAPK-inactivating phosphatases [25], [26], [27], and induces nuclear factor κB (NF-κB) translocation from the cytosol to the nucleus [28], [29]. Initiation of NF-κB-dependent gene transcription is a key step in the production of proinflammatory mediators [30], [31].
Here, we review evidence linking ROS to the proinflammatory M1 microglial activation state and the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome complex, a mediator of caspase-1-dependent interleukin-1β (IL-1β) secretion. We also discuss the extent to which mitochondrial dysfunction can be incriminated. Importantly, although much can be extrapolated from macrophages and other immune cells that share many features of microglial activation, precise roles for ROS and the NLRP3 inflammasome in the activation of primary microglia require further elucidation.
Section snippets
A role for NADPH oxidase in the proinflammatory state
Early work linking ROS to the M1 microglial activation state used a pharmacological approach (Fig. 1, i). Kang et al. demonstrated that the superoxide dismutase (SOD)/catalase mimetic Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP) or the NADPH oxidase/flavoprotein inhibitor diphenylene iodonium (DPI) reduced NF-κB activation and IL-1β transcription in the mouse microglial cell line BV2 in response to treatment with an Alzheimer disease-relevant amyloid-β (Aβ) peptide [32]. Additional
Reactive oxygen species, nitric oxide, and reactive microgliosis
Microglia in vitro continue to produce proinflammatory factors after removal of activating agent [47], and microglia in vivo frequently remain activated over long time periods after injury or onset of disease [50], [51]. This continued activation is probably due to a phenomenon termed reactive microgliosis, a self-propelling cycle mediated by intracellular ROS and NO that serves to maintain the M1 phenotype [47]. Hence, ROS play a vital role in both the initial activation of microglia and their
MAPK and NF-κB as hydrogen peroxide-sensitive regulators of microglial activation
The precise mechanisms by which ROS, and H2O2 in particular, promote microglial proliferation and the M1 transition remain incompletely characterized. Much of the work on the proinflammatory role of ROS, in both nonmicroglial and microglial cell types, has focused on the MAPK signaling pathway and the downstream transcription factor NF-κB (Fig. 1, iii). Although most reports do not examine exactly how ROS increase MAPK phosphorylation during the activation process, oxidation of catalytic
Rotenone and microglial activation—is mitochondrial superoxide involved?
To this point we have focused on studies pointing to a role for NADPH oxidase in microglial activation. However, mitochondria are a possible source of the component of LPS-induced intracellular ROS that was not eliminated by gp91phox knockout [8]. Superoxide anion is the primary ROS produced by mitochondria, mostly in the matrix, but it does not readily cross biological membranes [79]. MnSOD (SOD2) efficiently converts mitochondrial matrix superoxide into H2O2, which diffuses into the
A proinflammatory amplification loop initiated by mitochondrial superoxide? Lessons from macrophages
Not long ago, Kasahara et al. [101] took an interesting alternative approach to address whether there is a mitochondrial contribution to the activation phenotype in macrophages, the peripheral “cousins” of microglia. Rho zero (ρ0) cells lacking mitochondrial DNA were created from the RAW 264.7 macrophage cell line. The cells exhibited defective oxygen consumption, diminished ROS production in response to LPS, and attenuated TNF-α and IL-6 secretion [101]. An H2O2-generating system consisting of
NADPH oxidase-independent reactive oxygen species and NLRP3
Our discussion to this point has centered mostly on TNF-α; however, IL-1β is also one of the major proinflammatory cytokines in the brain. Release of IL-1β, in contrast to TNF-α, is regulated enzymatically through caspase-1 activity. The NLRP3 (also called NALP3) inflammasome is a protein scaffolding complex that induces the secretion of the cytokines IL-1β and IL-18 from their proforms by activating caspase-1 (Fig. 1, iv) [111]. Present in most immune cells including microglia, the NLRP3
Microglia vs macrophages
The vast majority of studies on NLRP3 inflammasome activation were performed in macrophages or other peripheral immune cells, particularly with regard to mitochondrial involvement. It is accepted by most that microglia are simply the resident macrophages in the brain and indeed the two phagocytic cell types are quite similar. For example, antigens that are commonly used to identify microglia, such as CD11b or CD68, are also present in macrophages [157]. However, although both cell types are of
M1 vs M2 activation
The majority of this review has focused on proinflammatory microglial activation. This mechanism of activation is referred to as a “classical” phenotype, or M1 activation [161]. However, microglia can also exhibit an “alternative” M2 activation [19], which serves to reduce production of proinflammatory mediators and promote brain repair. Whereas M1 polarization may be beneficial immediately after brain injury by phagocytic clearing of debris, M2 polarization may help to resolve a
Concluding remarks
Although it is generally accepted that reactive oxygen species, and in particular H2O2, play a role in M1 microglial activation, there is still disagreement as to the extent. A number of studies indicate that pro-oxidants cause microglial activation, whereas even more demonstrate that antioxidants impair activation. However, concerns exist about the methodology used to quantify and modulate ROS, particularly superoxide of mitochondrial origin. It is noteworthy that flawed approaches such as the
Acknowledgments
The authors acknowledge funding from NIH R01 NS085165 to B.M.P. In addition, the authors thank Dr. David Loane for helpful feedback during the preparation of the review.
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