Mitochondrial damage-associated molecular patterns stimulate reactive oxygen species production in human microglia
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.
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Cell culture and reagents
The human microglial HMC3 cell line was purchased from ATCC (ATCC® CRL-3304™, Lot 70016372; Manassas, VA, USA) and cultured in Eagle's Minimum Essential Medium (EMEM; from ATCC) supplemented with 10% fetal bovine serum (FBS) and antibiotics (both from Life Technologies, Carlsband, CA, USA), namely 100 IU/mL penicillin and 100 μg/mL streptomycin. Before the experiments, HMC3 cells were grown and expanded in 75 cm2 flasks maintained in 5% CO2 atmosphere at 37 °C, and were sub-cultured every 2 to
MTDs induce changes in the expression of genes involved in microglial activation pathways
To gain insights into the effects of MTDs on the expression of gene coding for proteins involved in their binding, in the signaling transduction pathways downstream of these receptors and in microglia activation, we quantified TLR9, FPR1, FPR2, CD11b, ICAM1, ERK1/2, p38, JNK, SPHK1, IL-1β and RPS18 mRNA. All evaluated transcripts were detectable in each sample, except for FPR1 and CD11b. The addition of only chloroform or DMSO, at the same concentrations used to dissolve stimuli, did not induce
Discussion
Despite the major contribution of microglial cells in neuroinflammation and the development of numerous neurodegenerative diseases, there are currently no drugs selectively targeting CNS-resident microglia directly approved for their therapy. Currently the glatiramer acetate is used in treating MS, and its neuroprotective effect indirectly reduces microglia activation (Giunti et al., 2014). Therefore, a clear understanding of the molecules and mechanisms by which microglial cell activation is
Funding
This work was supported by the Italian Multiple Sclerosis Foundation (FISM) [grant “Mitochondrial DAMPs in Multiple Sclerosis: a pilot study” to AC, code 2017/R/10].
CRediT authorship contribution statement
Milena Nasi: conceptualization, methodology, formal analysis, data curation, writing - original draft, writing - review & editing, visualization, supervision, project administration, funding acquisition;
Anna De Gaetano: investigation, resources, writing - review & editing;
Elena Bianchini: methodology, investigation, resources, writing - original draft;
Sara De Biasi: formal analysis, visualization;
Lara Gibellini: resources, investigation;
Anita Neroni: investigation, resources;
Marco Mattioli:
Declaration of competing interest
The authors have no competing interest to declare.
Acknowledgements
Elena Bianchini is a 2019 post-doctoral fellow of Fondazione Umberto Veronesi. Sara De Biasi is a Marylou Ingram Scholar for the International Society for Advancement of Cytometry. We thank Dr. Alice Prandi (Bio-techne srl, Milan, Italy) for her valuable technical support.
References (36)
- A. Andersson, R. Covacu, D. Sunnemark, A.I. Danilov, A. Dal Bianco, M. Khademi, E. Wallström, A. Lobell, L. Brundin, H....
- et al.
The Role of Mitochondrial Damage-associated Molecular Patterns in Chronic Neuroinflammation Mediators of Inflammation
(2019) - M.L. Block, L. Zecca, J.S. Hong Microglia-mediated neurotoxicity: uncovering the molecular mechanisms Nat. Rev....
- N. Cappoli, D. Mezzogori, E. Tabolacci, I. Coletta, P. Navarra, G. Pani, and C. Dello Russo The mTOR kinase inhibitor...
The role of microglial activation in disease progression
Mult. Scler.
(2014)- A. Cossarizza, M. Pinti, M. Nasi, L. Gibellini, S. Manzini, E. Roat, S. De Biasi, L. Bertoncelli, J.P. Montagna, L....
- A. Cossarizza, H.D. Chang, A. Radbruch, A. Acs, D. Adam, S. Adam-Klages, W.W. Agace, N. Aghaeepour, M. Akdis, M. Allez,...
- et al.
The human microglial HMC3 cell line: where do we stand?
J. Neuroinflammation
(2018) - C.A. Dendrou, L. Fugger, M.A. Friese Immunopathology of multiple sclerosis Nat. Rev. Immunol., 15 (2015), pp....
- D. Giunti, B. Parodi, C. Cordano, A. Uccelli, N. Kerlero de Rosbo Can we switch microglia's phenotype to foster...
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Milena Nasi and Anna De Gaetano have equally contributed to the study.