Review ArticleMicroglial heterogeneity in chronic pain
Introduction
In both brain and spinal cord, microglia are ubiquitous resident immune cells that constantly surveil the parenchyma via highly dynamic processes and interactions with neurons, vasculature and other glial cells (Bernier et al., 2019, Cserép et al., 2019). Unlike other tissue-resident and non-parenchymal brain macrophages, microglia originate from erythromyeloid progenitors in the yolk sac that arise earlier than other mononuclear phagocytes (Ginhoux et al., 2010, Utz et al., 2020). However, similar to tissue-resident macrophages, microglia markedly rely on environmental cues that shape their transcriptional profile and functional properties (Gosselin et al., 2014). Through multiple interactions with surrounding cells, microglia assume diverse biological roles that aim to maintenance of homeostasis. Such roles range from securing functional development of neuronal circuits to responding and regulating neuronal excitability and clearing the CNS parenchyma from potential pathogens or dead cells (Sierra et al., 2010, Stevens et al., 2007, Umpierre and Wu, 2020). Upon CNS injury or inflammatory insult, microglia shift from a homeostatic role to a reactive state that guarantees neuroprotection but can also lead to pronounced neuroinflammation (Stratoulias et al., 2019). This divergence from homeostasis is clearly represented at cellular and subcellular levels by the occurrence of morphological changes -rapid regulation of both motility and direction of branches- and prominent transcriptional changes (Davalos et al., 2005, Nimmerjahn et al., 2005). The advent of genome-wide association studies (GWAS) has marked an important paradigm shift in our understanding of microglia in disease states as they implicate genetic variants, that are either solely expressed by microglia or regulate microglia responses, as risk factors for neurodegenerative diseases (Jansen et al., 2019) and neuropsychiatric disorders (Sekar et al., 2016). Therefore, microglia are involved in disease aetiology and actively support and modulate neuronal function.
Although most neuronal and glial cells are known to be diverse across the CNS (Bayraktar et al., 2020, Marques et al., 2016, Saunders et al., 2018), microglial heterogeneity has only recently become the centre of attention. At first, with the intention to classify microglia functional modalities, microglial heterogeneity was based on the expression of membrane markers and morphological configurations, which resulted in the generation of fixed phenotypic categories. For instance, the M1/M2 framework has been adopted from the macrophage field to describe pro-inflammatory (M1) and anti-inflammatory (M2) functions of microglia following CNS insult. However, the use of this oversimplified nomenclature is widely criticized as misleading (Ransohoff, 2016) whereas there is more consensus towards using single-cell RNA sequencing (RNA-seq) analysis to characterize microglial heterogeneity. In the healthy murine brain, RNA-seq approach has demonstrated that microglia share a conserved expression of core genes that regulate homeostatic functions (Bennett et al., 2016, Butovsky et al., 2014, Li et al., 2019). This homeostatic gene signature is associated with various roles played by microglia in a region- and time-dependent fashion (Butovsky et al., 2014, De Biase et al., 2017, Masuda et al., 2019). Furthermore, microglia are endowed with the ability to sense the environment and readily respond to external stimuli across the CNS by changing the expression of the transcriptional homeostatic signature. For instance, in mouse models of Alzheimer’s disease (AD), multiple sclerosis and neuroinflammation, microglial heterogeneity manifests through the establishment of cell subsets that alter their transcriptional profile through down-regulation of some homeostatic genes (Cx3cr1, Tmem119, P2ry12, Sall1, Hexb, Fcrls) and up-regulation of disease-specific genes (Apoe, Trem2, Ctss, Axl) (Jordão et al., 2019, Keren-Shaul et al., 2017, Krasemann et al., 2017). In support of the concept that diverse microglial profiles can be observed in response to pathology, changes in gene expression are accompanied by alteration at the molecular level (Mrdjen et al., 2018), and for instance, microglia adjacent to amyloid plaques, the pathological hallmark of AD, express specific subsets of protein markers (Kamphuis et al., 2016, Parhizkar et al., 2019).
Several comprehensive reviews discuss microglial heterogeneity in the context of regional diversity, sexual dimorphism, healthy development, ageing and nervous system diseases (Masuda et al., 2020, Stratoulias et al., 2019, Tan et al., 2019). We discuss microglial heterogeneity in the context of chronic pain. In the last two decades, it has become apparent that under neuropathic pain conditions following peripheral damage, dorsal horn microglia in the spinal cord play a causal role in the establishment and maintenance of nociceptive mechanisms via well-defined pathways for communication with neurons (Malcangio, 2019). Typical examples of neuroimmune interactions include 1) neuron-derived ATP activation of microglial P2X4/BDNF pathway which results in a TrkB-mediated reduction of GABAergic inhibition of dorsal horn neurons and an overall increase in excitability (Coull et al., 2005, Masuda et al., 2016) 2) microglial P2X7 receptor activation by neuronal ATP that promotes release of the cysteine protease Cathepsin S which liberates neuronal fractalkine (CX3CL1) chemokine domain that further stimulates microglia to secrete pro-nociceptive cytokines via activation of CX3CR1 receptor (Clark et al., 2007a, Malcangio, 2019) and 3) de novo expression of colony-stimulating factor 1 (CSF1) in injured nociceptors which drives the expression of neuropathic pain-associated genes in dorsal horn microglia through binding to CSF1 receptor/DAP12 (Guan et al., 2016). Despite significant progress in our understanding of microglial pathways that facilitate nociceptive transmission, so far very few studies investigated microglial heterogeneous transcriptional changes in chronic pain states. Hence, this review provides an overview on the contemporary framework under which microglial heterogeneity is being studied in the CNS in the context of disease, sex, and intraregional differences. Then against this background, we discuss microglial heterogeneity in chronic pain. Our prediction is that the advent of novel technologies will facilitate a better characterization of functions, transcriptome and structure of microglia and their dynamic role in the establishment and maintenance of chronic pain. This type of approach may lead to the identification of innovative microglial targets for chronic pain treatments.
Section snippets
Membrane markers and morphology
With the aim to understand microglial functions in healthy murine CNS, early studies examined microglial distribution, morphology and membrane marker expression and suggested that CNS regional density accounts for microglia’s ability to adapt and respond to local cues from neuronal environments. Specifically, immunostaining of the membrane marker F4/80 indicates that microglia density in thalamus (101 cells/mm2), hippocampus (120 cells/mm2) and substantia nigra (134 cells/mm2) is higher than in
Chronic pain
In contrast to acute pain which is an adaptive process and a defensive mechanism from harmful stimuli, chronic pain is maladaptive, outlasts noxious stimuli and poorly controlled by current analgesics. Harmful stimuli are detected in the periphery of the body by specialized sensory neurons, namely the nociceptors, which transmit electrical impulses to the dorsal horn of the spinal cord. From this first relay station, nociceptive signalling continues to the higher centres in the brain where pain
Sensory discriminative
In addition to microglia heterogeneous immune responses across the CNS, there is clinical and preclinical evidence that alteration in microglia properties occurs in pain-related areas that are closely linked with both the sensory-discriminative and the affective-motivational component of chronic pain (Austin and Fiore, 2019). Preclinical data in neuropathic pain models indicates that microglia influence nociceptive mechanisms in a variety of brain regions (Fig. 3), as both Iba-1 gene expression
Conclusions
Microglia can assume diverse states to integrate inward and outward signals within microenvironments and under homeostasis, microglia exert multiple functions. Upon stimulation by a pathogen or in disease states, some microglia diverge from homeostatic profiles and acquire distinct modes of activation: cells alter morphology and membrane marker expression, as well as core transcriptional profiles (Mathys et al., 2019, Sala Frigerio et al., 2019). Thus, the association of a microglia
Acknowledgements
The authors acknowledge funds from the European Union’s Horizon 2020 research and innovation programme “TOBeATPAIN” under the Marie Skłodowska-Curie grant agreement No 764860.
We specially thank Ana Rita Alves da Silva for providing the image showing microgliosis in the dorsal horn of the spinal cord in animals with nerve injury (Fig. 2B).
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