Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia

Highlights

• Chronic microglial activation underlies inflammation in neurodegenerative diseases.

• MTDs cause changes in the expression of genes involved in pro-inflammatory activation.

• MTDs do not alter the expression of HLA-DR and the release of TNF-α and IL-6.

• mtDNA and CL increase cellular oxidative stress, but not mtO₂⁻ production.

• MTDs cause a failure in microglia activation toward a pro-inflammatory phenotype.

Abstract

Microglia are the resident innate immune cells of the central nervous system and exert functions of host defense and maintenance of normal tissue homeostasis, along with support of neuronal processes in the healthy brain. Chronic and dysregulated microglial cell activation has increasingly been linked to the status of neuroinflammation underlying many neurodegenerative diseases, including multiple sclerosis (MS). However, the stimulus (or stimuli) and mechanisms by which microglial activation is initiated and maintained MS are still debated. The purpose of our research was to investigate whether the endogenous mitochondrial (mt)-derived damage-associated molecular patterns (MTDs) mtDNA, N-formyl peptides and cardiolipin (CL) contribute to these phenomena. We characterized the effects of the abovementioned MTDs on microglia activation in vitro (i.e. using HMC3 cells) by evaluating the expression of gene coding for proteins involved in their binding and coupled to downstream signaling pathways, the up-regulation of markers of activation on the cell surface and the production of pro-inflammatory cytokines and reactive oxygen species. At the transcriptional level, significant variations in the mRNA relative expression of five of eleven selected genes were observed in response to stimulation. No changes in activation of antigenic profile or functional properties of HMC3 cells were observed; there was no up-regulation of HLA-DR expression or increased secretion of tumor necrosis factor-α and interleukin-6. However, after stimulation with mtDNA and CL, an increase in cellular oxidative stress, but not in the mt ROS O₂⁻, compared to control cells, were observed. There were no effects on cell viability. Overall, our data suggest that MTDs could cause a failure in microglial activation toward a pro-inflammatory phenotype, possibly triggering an endogenous regulatory mechanism for the resolution of neuroinflammation. This could open a door for the development of drugs selectively targeting microglia and modulating its functionality to treat MS and/or other neurodegenerative conditions in which MTDs have a pathogenic relevance.

Introduction

Microglia are highly specialized macrophages residing in the central nervous system (CNS) parenchyma that originate from myeloid progenitors in the yolk sac and enter the CNS during early embryogenesis (Saijo and Glass, 2011). As resident innate immune cells within the mature brain and spinal cord, they constantly monitor the CNS environment and act as local sentinels for pathogen infection and tissue injury (Dello Russo et al., 2018). In addition to their functions of immune surveillance and maintenance of normal tissue homeostasis, microglial cells also support cerebral processes such as the regulation of synaptic architecture and neurogenesis (Dello Russo et al., 2018).

Microglia are professional phagocytes which express various receptors, including pattern recognition receptors (PRRs), that sense pathogen-associated molecular patterns in the CNS, along with endogenous damage-associated molecular patterns (DAMPs) released from damaged or dead/dying cells (Block et al., 2007). Following activation in response to noxious stimuli, microglial cells up-regulate major histocompatibility complex (MHC) class II expression to present antigens to naïve T cells and trigger signaling pathways that mediate the production of numerous molecules, among which antimicrobial peptides, cytokines, chemokines, reactive oxygen species (ROS) and nitric oxide (Saijo and Glass, 2011).

Although microglial activation is essential for clearance of pathogen infections and neuronal survival, an excessive activation of microglia exerts deleterious neurotoxic effects, and chronic and dysregulated microglial cell activation has been associated to the pathological forms of inflammation underlying several neurodegenerative disorders and their pathology (Block et al., 2007). Parkinson's disease (PD), Alzheimer's disease (AD) and multiple sclerosis (MS) provide examples of neurodegenerative conditions in which chronically activated microglia might drive persistent CNS inflammation and contribute to disease progression, as extensively reviewed elsewhere (Block et al., 2007; Hickman et al., 2018; Saijo et al., 2010; Saijo and Glass, 2011). As has been well established for MS, in neurodegenerative diseases, it is important to note that microglial activation is featured by dual, divergent roles of pro-inflammatory and neurotoxic exertion as well as anti-inflammatory and neuroprotective effects, fuelling and/or dampening down neuroinflammation (Correale, 2014; Wang et al., 2019).

MS is a heterogeneous, chronic, inflammatory demyelinating disease which affects the CNS, pathologic hallmark consists of multiple focal demyelinated areas disseminated throughout both the white and gray matter within the brain and spinal cord, referred to as plaques or lesions (Dendrou et al., 2015). Autoreactive T and B lymphocytes mounting aberrant responses against CNS autoantigens have major pathological roles (Dendrou et al., 2015), but also innate immunity is deeply implicated in the course of the disease (Mayo et al., 2012). In particular, CNS-resident microglia are known to be among the most prominent innate contributors to pathological changes in MS; diffuse activation either in lesions or normal-appearing white matter of patients has been reported (Zrzavy et al., 2017).

Although for some neurodegenerative disorders plausible chronic inducers of microglial cell activation through PRRs have been identified – such as the neuron-derived α-synuclein for PD (Roodveldt et al., 2008) and amyloid-β for AD (Halle et al., 2008) – for others, including MS, the pathogenic stimulus (or stimuli) and mechanisms by which microglia activation is established and maintained are still unknown. Therefore, we focused on DAMPs arising from mitochondrial (mt) components (MTDs), whose relevance in neurodegenerative diseases, including MS, has increasingly been demonstrated (Bajwa et al., 2019; Leurs et al., 2018; Lowes et al., 2019; Nasi et al., 2019; Wilkins et al., 2015), as possible contributors to these phenomena. There is increasing evidence that DAMPs play a role in the high mobility group box protein 1 (Andersson et al., 2008) and cell-free mtDNA in MS (Leurs et al., 2018; Lowes et al., 2019; Nasi et al., 2019). In particular, our group recently reported that patients affected by secondary progressive MS had higher levels of circulating mtDNA compared to patients with a primary, progressive form (Nasi et al., 2019).

Physiologically, mtDNA is the circular, coding DNA within mitochondria, N-formyl peptides are chemoattractant peptides participating in the clearance of damaged cells and in host defense against bacteria, and CL is a lipid dimer that plays an important role in several mt functions such as mt respiration and biogenesis (Grazioli and Pugin, 2018). Therefore, we characterized the effects of MTDs on microglia activation in vitro using a microglial cell line of human origin as an experimental model. Additionally, we analyzed the effects of the N-term portion of proteins synthesized within mitochondria (i.e. N-formyl peptides) and of the inner mt membrane lipid cardiolipin (CL) on the expression of genes involved in microglia activation and the up-regulation of markers of activation on the cell surface, together with the production of pro-inflammatory cytokines and ROS.