Lipid rafts in glial cells: role in neuroinflammation and pain processing

Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions. Activation of microglia and astrocytes secondary to inflammatory processes contributes to the development and perpetuation of pain with a neuropathic phenotype. This pain state presents as a chronic debilitating condition and affects a large population of patients with conditions like rheumatoid arthritis and diabetes, or after surgery, trauma, or chemotherapy. Here, we review the regulation of lipid rafts in glial cells and the role they play as a key component of neuroinflammatory sensitization of central pain signaling pathways. In this context, we introduce the concept of an inflammaraft (i-raft), enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as an organizing platform to initiate inflammatory signaling and the cellular response. Characteristics of the inflammaraft include increased relative abundance of lipid rafts in inflammatory cells, increased content of cholesterol per raft, and increased levels of inflammatory receptors, such as toll-like receptor (TLR)4, adaptor molecules, ion channels, and enzymes in lipid rafts. This inflammaraft motif serves an important role in the membrane assembly of protein complexes, for example, TLR4 dimerization. Operating within this framework, we demonstrate the involvement of inflammatory receptors, redox molecules, and ion channels in the inflammaraft formation and the regulation of cholesterol and sphingolipid metabolism in the inflammaraft maintenance and disruption. Strategies for targeting inflammarafts, without affecting the integrity of lipid rafts in noninflammatory cells, may lead to developing novel therapies for neuropathic pain states and other neuroinflammatory conditions. 12/15-lipoxygenase apoA-I binding protein brain-derived neurotrophic factor cluster of differentiation chemotherapy-induced peripheral neuropathy DNAX activation protein protein deglycase DJ-1 (also known as Parkinson disease protein 7) excitatory amino acid transporter extracellular vesicle monosialotetrahexosyl ganglioside interleukin inducible nitric oxide synthase Janus kinase lipopolysaccharide lipopolysaccharide from Rhodobacter sphaeroides neurokinin 1 receptor NACHT, LRR, and PYD domains-containing protein 3 NADPH oxidase P2X purinoreceptor cation channel family P2Y purinergic G protein-coupled receptor family reactive oxygen species Src homology region 2 domain-containing phosphatase signal transducer and activator of transcription toll-like receptor triggering receptor expressed on myeloid cells Tissue injury and injury to the peripheral nerve often lead to highly disruptive persistent pain states in animals and humans. In this review, we draw attention to the role played in the development of these persistent pain states by signaling organized through lipid rafts in microglia and astrocytes. The concept of lipid rafts was originally proposed based on the self-associative properties of sphingolipid and cholesterol that facilitate lateral segregation of lipids and proteins in the plasma membrane (1Simons K. Ikonen E. Functional rafts in cell membranes.Nature. 1997; 387: 569-572Crossref PubMed Scopus (7622) Google Scholar). An umbrella effect of the sphingomyelin headgroup stabilizes cholesterol near the sphingomyelin molecules by concealing the hydrophobic cholesterol core from interfacial water (2Hanashima S. Murakami K. Yura M. Yano Y. Umegawa Y. Tsuchikawa H. Matsumori N. Seo S. Shinoda W. Murata M. Cholesterol-induced conformational change in the sphingomyelin headgroup.Biophys. J. 2019; 117: 307-318Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). These and other structural characteristics of lipid rafts and the biophysical models of microdomains in the plasma membrane have been described in detail in many excellent reviews (3Lingwood D. Simons K. Lipid rafts as a membrane-organizing principle.Science. 2010; 327: 46-50Crossref PubMed Scopus (2785) Google Scholar, 4Sezgin E. Levental I. Mayor S. Eggeling C. The mystery of membrane organization: composition, regulation and roles of lipid rafts.Nat. Rev. Mol. Cell Biol. 2017; 18: 361-374Crossref PubMed Scopus (617) Google Scholar, 5Robinson C.V. Rohacs T. Hansen S.B. Tools for understanding nanoscale lipid regulation of ion channels.Trends Biochem. Sci. 2019; 44: 795-806Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). The purpose of this article is different, it focuses on the biology and changes in glial cell lipid rafts underlying neuroinflammation and nociceptive signaling. The nidus around which this review on lipid rafts, glia, and pain processing is organized results from three convergent observations. They focus on toll-like receptor (TLR)4 signaling but have much broader implications. First, we and others observed that spinal TLR4 signaling has little effect upon the acute pain state (as after a brief application of a high-intensity thermal stimulus or a mechanical compression), but appears to mediate development of the persistent pain phenotype secondary to local inflammation and to peripheral nerve injury (6Tanga F.Y. Nutile-McMenemy N. DeLeo J.A. The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy.Proc. Natl. Acad. Sci. USA. 2005; 102: 5856-5861Crossref PubMed Scopus (489) Google Scholar, 7Sorge R.E. LaCroix-Fralish M.L. Tuttle A.H. Sotocinal S.G. Austin J.S. Ritchie J. Chanda M.L. Graham A.C. Topham L. Beggs S. et al.Spinal cord Toll-like receptor 4 mediates inflammatory and neuropathic hypersensitivity in male but not female mice.J. Neurosci. 2011; 31: 15450-15454Crossref PubMed Scopus (241) Google Scholar, 8Christianson C.A. Dumlao D.S. Stokes J.A. Dennis E.A. Svensson C.I. Corr M. Yaksh T.L. Spinal TLR4 mediates the transition to a persistent mechanical hypersensitivity after the resolution of inflammation in serum-transferred arthritis.Pain. 2011; 152: 2881-2891Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 9Woller S.A. Ravula S.B. Tucci F.C. Beaton G. Corr M. Isseroff R.R. Soulika A.M. Chigbrow M. Eddinger K.A. Yaksh T.L. Systemic TAK-242 prevents intrathecal LPS evoked hyperalgesia in male, but not female mice and prevents delayed allodynia following intraplantar formalin in both male and female mice: the role of TLR4 in the evolution of a persistent pain state.Brain Behav. Immun. 2016; 56: 271-280Crossref PubMed Scopus (37) Google Scholar, 10Xu Q. Yaksh T.L. A brief comparison of the pathophysiology of inflammatory versus neuropathic pain.Curr. Opin. Anaesthesiol. 2011; 24: 400-407Crossref PubMed Scopus (0) Google Scholar, 11Woller S.A. Ocheltree C. Wong S.Y. Bui A. Fujita Y. Goncalves Dos Santos G. Yaksh T.L. Corr M. Neuraxial TNF and IFN-beta co-modulate persistent allodynia in arthritic mice.Brain Behav. Immun. 2019; 76: 151-158Crossref PubMed Scopus (4) Google Scholar, 12Ramachandran R. Wang Z. Saavedra C. DiNardo A. Corr M. Powell S.B. Yaksh T.L. Role of Toll-like receptor 4 signaling in mast cell-mediated migraine pain pathway.Mol. Pain. 2019; 15 (4806919867842): 174Crossref Scopus (3) Google Scholar, 13Woller S.A. Corr M. Yaksh T.L. Differences in cisplatin-induced mechanical allodynia in male and female mice.Eur. J. Pain. 2015; 19: 1476-1485Crossref PubMed Scopus (17) Google Scholar). This transition of pain from an acute stimulus-linked condition to a chronic state has been shown to be a surprisingly common event in both animals and humans (14Woller S.A. Eddinger K.A. Corr M. Yaksh T.L. An overview of pathways encoding nociception.Clin. Exp. Rheumatol. 2017; 35 (Suppl. 107): 40-46PubMed Google Scholar). For example, in rheumatoid arthritis, joint pain can persist despite resolution of swelling (15Wolfe F. Michaud K. Assessment of pain in rheumatoid arthritis: minimal clinically significant difference, predictors, and the effect of anti-tumor necrosis factor therapy.J. Rheumatol. 2007; 34: 1674-1683PubMed Google Scholar, 16Taylor P. Manger B. Alvaro-Gracia J. Johnstone R. Gomez-Reino J. Eberhardt E. Wolfe F. Schwartzman S. Furfaro N. Kavanaugh A. Patient perceptions concerning pain management in the treatment of rheumatoid arthritis.J. Int. Med. Res. 2010; 38: 1213-1224Crossref PubMed Google Scholar). Following surgeries, up to 30% of the population report pain that lasts greater than 3 months (17Katz J. Seltzer Z. Transition from acute to chronic postsurgical pain: risk factors and protective factors.Expert Rev. Neurother. 2009; 9: 723-744Crossref PubMed Scopus (426) Google Scholar, 18Kehlet H. Rathmell J.P. Persistent postsurgical pain: the path forward through better design of clinical studies.Anesthesiology. 2010; 112: 514-515Crossref PubMed Scopus (100) Google Scholar). Current work emphasizes that these persistent changes may result from neuroinflammatory processes in the spinal cord resembling those arising from physical or chemical injury to the peripheral nerve, with peripheral and central (spinal cord and dorsal root ganglion) immune and glial cells being activated, resulting in a facilitated input/output function of the dorsal horn secondary to neuronal sensitization (10Xu Q. Yaksh T.L. A brief comparison of the pathophysiology of inflammatory versus neuropathic pain.Curr. Opin. Anaesthesiol. 2011; 24: 400-407Crossref PubMed Scopus (0) Google Scholar, 19Yaksh T.L. Woller S.A. Ramachandran R. Sorkin L.S. The search for novel analgesics: targets and mechanisms.F1000Prime Rep. 2015; 7: 56Crossref PubMed Google Scholar). Next came the realization that these effects of Tlr4 knockout or TLR4 inhibition on pain processing are mediated, at least in part, by an action upon spinal glial cells. Intrathecal delivery of agents that are thought to maintain microglia in a quiescent state, such as miR-124 (20Willemen H.L. Huo X.J. Mao-Ying Q.L. Zijlstra J. Heijnen C.J. Kavelaars A. MicroRNA-124 as a novel treatment for persistent hyperalgesia.J. Neuroinflammation. 2012; 9: 143Crossref PubMed Scopus (0) Google Scholar) or minocycline (21Mei X.P. Xu H. Xie C. Ren J. Zhou Y. Zhang H. Xu L.X. Post-injury administration of minocycline: an effective treatment for nerve-injury induced neuropathic pain.Neurosci. Res. 2011; 70: 305-312Crossref PubMed Scopus (0) Google Scholar, 22Hua X.Y. Svensson C.I. Matsui T. Fitzsimmons B. Yaksh T.L. Webb M. Intrathecal minocycline attenuates peripheral inflammation-induced hyperalgesia by inhibiting p38 MAPK in spinal microglia.Eur. J. Neurosci. 2005; 22: 2431-2440Crossref PubMed Scopus (206) Google Scholar), have been reported to attenuate hyperalgesia (abnormally increased sensitivity to high intensity "painful" stimuli) in models of tissue and nerve injury, albeit the specificity of microglial "inhibitors" is not absolute (23Möller T. Bard F. Bhattacharya A. Biber K. Campbell B. Dale E. Eder C. Gan L. Garden G.A. Hughes Z.A. et al.Critical data-based re-evaluation of minocycline as a putative specific microglia inhibitor.Glia. 2016; 64: 1788-1794Crossref PubMed Scopus (82) Google Scholar). Spinal microglia express TLR4 in abundance and when activated, it mediates a robust secretion of cytokines, chemokines, and lipids (10Xu Q. Yaksh T.L. A brief comparison of the pathophysiology of inflammatory versus neuropathic pain.Curr. Opin. Anaesthesiol. 2011; 24: 400-407Crossref PubMed Scopus (0) Google Scholar, 24Zhao H. Alam A. Chen Q. Eusman M.A. Pal A. Eguchi S. Wu L. Ma D. The role of microglia in the pathobiology of neuropathic pain development: what do we know?.Br. J. Anaesth. 2017; 118: 504-516Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 25Gregus A.M. Buczynski M.W. Dumlao D.S. Norris P.C. Rai G. Simeonov A. Maloney D.J. Jadhav A. Xu Q. Wei S.C. et al.Inhibition of spinal 15-LOX-1 attenuates TLR4-dependent, nonsteroidal anti-inflammatory drug-unresponsive hyperalgesia in male rats.Pain. 2018; 159: 2620-2629Crossref PubMed Scopus (4) Google Scholar, 26Bruno K. Woller S.A. Miller Y.I. Yaksh T.L. Wallace M. Beaton G. Chakravarthy K. Targeting toll-like receptor-4 (TLR4)-an emerging therapeutic target for persistent pain states.Pain. 2018; 159: 1908-1915Crossref PubMed Scopus (19) Google Scholar). These products acting upon neuraxial receptors and targets (dorsal horn neurons) can lead to a pronounced facilitation of neuraxial afferent processing, yielding an enhanced pain signal driven by an otherwise innocuous or moderately noxious stimulus, i.e., allodynia or hyperalgesia. We note here that while there is a consensus that spinal glial cells are activated and induce sensitization of nociceptive neuronal pathways, disagreement exists regarding the specific cell types that play a major role in various pain conditions. As an example, a pivotal role of microglial activation is suggested for the pain state generated by injuries to nerve trunks, i.e., mononeuropathies (6Tanga F.Y. Nutile-McMenemy N. DeLeo J.A. The CNS role of Toll-like receptor 4 in innate neuroimmunity and painful neuropathy.Proc. Natl. Acad. Sci. USA. 2005; 102: 5856-5861Crossref PubMed Scopus (489) Google Scholar). Conversely, activation of astrocytes is seen in pain states associated with polyneuropathies induced by chemotherapeutics. Treatment with several agents (oxaliplatin, paclitaxel, or bortezomib) induces increases in spinal astrocytes (27Robinson C.R. Zhang H. Dougherty P.M. Astrocytes, but not microglia, are activated in oxaliplatin and bortezomib-induced peripheral neuropathy in the rat.Neuroscience. 2014; 274: 308-317Crossref PubMed Scopus (79) Google Scholar, 28Makker P.G. Duffy S.S. Lees J.G. Perera C.J. Tonkin R.S. Butovsky O. Park S.B. Goldstein D. Moalem-Taylor G. Characterisation of immune and neuroinflammatory changes associated with chemotherapy-induced peripheral neuropathy.PLoS One. 2017; 12: e0170814Crossref PubMed Scopus (88) Google Scholar). Other investigators show changes in spinal microglia associated with chemotherapy-induced peripheral neuropathy (CIPN) (29Beh S.T. Kuo Y.M. Chang W.W. Wilder-Smith E. Tsao C.H. Tsai C.H. Chen L.T. Liao L.D. Preventive hypothermia as a neuroprotective strategy for paclitaxel-induced peripheral neuropathy.Pain. 2019; 160: 1505-1521Crossref PubMed Scopus (3) Google Scholar, 30Hu L.Y. Zhou Y. Cui W.Q. Hu X.M. Du L.X. Mi W.L. Chu Y.X. Wu G.C. Wang Y.Q. Mao-Ying Q.L. Triggering receptor expressed on myeloid cells 2 (TREM2) dependent microglial activation promotes cisplatin-induced peripheral neuropathy in mice.Brain Behav. Immun. 2018; 68: 132-145Crossref PubMed Scopus (3) Google Scholar). Finally, the pivot from TLR4 activation in glial cells to lipid rafts comes from the recognition that the first step in TLR4 signaling, receptor dimerization, requires the ordered membrane microenvironment of lipid rafts (31Fessler M.B. Parks J.S. Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.J. Immunol. 2011; 187: 1529-1535Crossref PubMed Scopus (163) Google Scholar, 32Tall A.R. Yvan-Charvet L. Cholesterol, inflammation and innate immunity.Nat. Rev. Immunol. 2015; 15: 104-116Crossref PubMed Scopus (537) Google Scholar). The requirement for lipid raft localization is important to activation not only of TLR4 but also of a large number of other inflammatory receptors, enzymes, and ion channels, as will be considered in this review. In this context, the TLR4 dimerization, which can be easily assessed with a flow cytometry method, reflects not only a ligand-induced TLR4 receptor activation event but also the permissive membrane microenvironment that supports receptor dimerization. Thus, depletion of cholesterol inhibits lipopolysaccharide (LPS)-induced TLR4 dimerization (33Woller S.A. Choi S.H. An E.J. Low H. Schneider D.A. Ramachandran R. Kim J. Bae Y.S. Sviridov D. Corr M. et al.Inhibition of neuroinflammation by AIBP: spinal effects upon facilitated pain states.Cell Reports. 2018; 23: 2667-2677Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 34Powers K.A. Szaszi K. Khadaroo R.G. Tawadros P.S. Marshall J.C. Kapus A. Rotstein O.D. Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages.J. Exp. Med. 2006; 203: 1951-1961Crossref PubMed Scopus (136) Google Scholar). apoA-I binding protein (AIBP), which augments cholesterol efflux and reduces lipid rafts, also inhibits TLR4 dimerization and alleviates neuropathic pain in mouse models (33Woller S.A. Choi S.H. An E.J. Low H. Schneider D.A. Ramachandran R. Kim J. Bae Y.S. Sviridov D. Corr M. et al.Inhibition of neuroinflammation by AIBP: spinal effects upon facilitated pain states.Cell Reports. 2018; 23: 2667-2677Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Conversely, increased cholesterol and lipid raft content in the plasma membrane, secondary to knockout of the cholesterol transporters ABCA1 and ABCG1, augments TLR4 dimerization (35Yvan-Charvet L. Welch C. Pagler T.A. Ranalletta M. Lamkanfi M. Han S. Ishibashi M. Li R. Wang N. Tall A.R. Increased inflammatory gene expression in ABC transporter-deficient macrophages: free cholesterol accumulation, increased signaling via toll-like receptors, and neutrophil infiltration of atherosclerotic lesions.Circulation. 2008; 118: 1837-1847Crossref PubMed Scopus (284) Google Scholar, 36Zhu X. Owen J.S. Wilson M.D. Li H. Griffiths G.L. Thomas M.J. Hiltbold E.M. Fessler M.B. Parks J.S. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.J. Lipid Res. 2010; 51: 3196-3206Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar). Based on the three sets of observations outlined above and current literature, we concluded that cholesterol-rich membrane rafts in glial cells serve as local organizing matrices for membrane receptors and channels involved in neuroinflammation and pain processing in the spinal cord. In this article, we summarize the current knowledge in support of this hypothesis. We also suggest a new framework that we believe can be useful for operating in the space of lipid rafts and neuroinflammation, the inflammaraft or, for brevity, the i-raft. In the course of many conversations on the topic of lipid rafts and their role in inflammatory processes, particularly in spinal microglia, the authors of this article often employed an "inflammasome of the lipid raft" phraseology. It conveyed the connotation of the role played by membrane lipid rafts as a focal point for the organization of a cascade defining the cellular inflammatory response. The notion, while useful, was conversationally cumbersome and not entirely correct. The term "NLRP3 inflammasome" has been reserved for an intracellular molecular complex, which triggers the release of the cytokines interleukin (IL)-1β and IL-18 (37Martinon F. Burns K. Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-β.Mol. Cell. 2002; 10: 417-426Abstract Full Text Full Text PDF PubMed Scopus (3422) Google Scholar). Thus, a new term was coined, "inflammaraft," defined, for the purposes of this article, as enlarged lipid rafts harboring activated receptors and adaptor molecules and serving as a scaffold to organize the cellular inflammatory response (Fig. 1). In our own work, we observed that in response to inflammatory stimuli there was increased cellular binding of cholera toxin B to a raft-localized ganglioside, monosialotetrahexosyl ganglioside (GM)1, indicating enlarged lipid rafts, increased cholesterol levels in isolated lipid rafts, increased TLR4 occupancy in lipid rafts, and most important in our view, increased TLR4 dimerization (33Woller S.A. Choi S.H. An E.J. Low H. Schneider D.A. Ramachandran R. Kim J. Bae Y.S. Sviridov D. Corr M. et al.Inhibition of neuroinflammation by AIBP: spinal effects upon facilitated pain states.Cell Reports. 2018; 23: 2667-2677Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Thus, we suggest that characteristics of the inflammaraft should include (but are not be limited to) the following: i) increased relative abundance of lipid rafts in inflammatory cells; ii) increased content of cholesterol per raft; iii) increased levels of inflammatory receptors, adaptor molecules, ion channels, and enzymes in lipid rafts; together with iv) the evidence of protein complex assemblies in the plasma membrane. The data supporting this characterization of the inflammaraft are ample but scattered throughout literature. Other characteristics may emerge as equally important from work in other laboratories. The purpose of this article is to review the current evidence emphasizing the functionality of inflammarafts, specifically in glial cells, providing their relevance to systems functioning in nociceptive processing. It is well appreciated that the function of many ion channels and synaptic transmission require lipid rafts (38Aureli M. Grassi S. Prioni S. Sonnino S. Prinetti A. Lipid membrane domains in the brain.Biochim. Biophys. Acta. 2015; 1851: 1006-1016Crossref PubMed Scopus (65) Google Scholar, 39Zaidi A. Adewale M. McLean L. Ramlow P. The plasma membrane calcium pumps-the old and the new.Neurosci. Lett. 2018; 663: 12-17Crossref PubMed Scopus (5) Google Scholar), and an extensive literature exists to highlight roles of lipid rafts in the regulation of neuronal activity, including in pain processing (40Sághy E. Szoke E. Payrits M. Helyes Z. Borzsei R. Erostyak J. Janosi T.Z. Setalo Jr., G. Szolcsanyi J. Evidence for the role of lipid rafts and sphingomyelin in Ca2+-gating of transient receptor potential channels in trigeminal sensory neurons and peripheral nerve terminals.Pharmacol. Res. 2015; 100: 101-116Crossref PubMed Scopus (49) Google Scholar, 41Schrattenholz A. Soskic V. NMDA receptors are not alone: dynamic regulation of NMDA receptor structure and function by neuregulins and transient cholesterol-rich membrane domains leads to disease-specific nuances of glutamate-signalling.Curr. Top. Med. Chem. 2006; 6: 663-686Crossref PubMed Scopus (0) Google Scholar, 42Gnanasekaran A. Sundukova M. van den Maagdenberg A.M. Fabbretti E. Nistri A. Lipid rafts control P2X3 receptor distribution and function in trigeminal sensory neurons of a transgenic migraine mouse model.Mol. Pain. 2011; 7: 77Crossref PubMed Scopus (30) Google Scholar, 43Startek J.B. Boonen B. Lopez-Requena A. Talavera A. Alpizar Y.A. Ghosh D. Van Ranst N. Nilius B. Voets T. Talavera K. Mouse TRPA1 function and membrane localization are modulated by direct interactions with cholesterol.eLife. 2019; 8: e46084Crossref PubMed Scopus (15) Google Scholar). Yet, as we discussed above, glial cells are considered to play an important role as a component of neuroinflammatory sensitization of neuraxial signaling pathways. We will demonstrate that the concept of inflammaraft helps to formalize the lipid raft-centric view of neuroinflammation in the context of neuropathic pain and its control by cholesterol and sphingolipid metabolism, as well as processing of pro- and anti-inflammatory signals at the cell surface. The self-assembly property of cholesterol and sphingolipids is in the core of lipid raft formation (1Simons K. Ikonen E. Functional rafts in cell membranes.Nature. 1997; 387: 569-572Crossref PubMed Scopus (7622) Google Scholar). Because cholesterol concentrates in lipid rafts, proteins that have specific cholesterol binding motifs tend to localize to the rafts: examples of such protein assemblies being TLRs, several gated ion channels, and G protein-coupled receptors (44Ruysschaert J.M. Lonez C. Role of lipid microdomains in TLR-mediated signalling.Biochim. Biophys. Acta. 2015; 1848: 1860-1867Crossref PubMed Scopus (48) Google Scholar, 45Katritch V. Cherezov V. Stevens R.C. Structure-function of the G protein-coupled receptor superfamily.Annu. Rev. Pharmacol. Toxicol. 2013; 53: 531-556Crossref PubMed Scopus (684) Google Scholar, 46Fantini J. Epand R.M. Barrantes F.J. Cholesterol-recognition motifs in membrane proteins.Adv. Exp. Med. Biol. 2019; 1135: 3-25Crossref PubMed Scopus (11) Google Scholar). Binding to gangliosides guides amyloid proteins to lipid rafts (47Yahi N. Fantini J. Deciphering the glycolipid code of Alzheimer's and Parkinson's amyloid proteins allowed the creation of a universal ganglioside-binding peptide.PLoS One. 2014; 9: e104751Crossref PubMed Scopus (20) Google Scholar). In addition, post-translational modification of proteins by saturated lipids, e.g., glycosylphosphatidylinositol anchors, N-terminal myristic acid tails, cysteine acylation, isoprenylation, and C-terminal sterol moieties, recruits them to rafts (48Levental I. Grzybek M. Simons K. Greasing their way: lipid modifications determine protein association with membrane rafts.Biochemistry. 2010; 49: 6305-6316Crossref PubMed Scopus (276) Google Scholar). These data suggest that in addition to being a structural backbone of lipid rafts responsible for forming ordered membrane domains, cholesterol and sphingolipids are often required for targeting specific proteins to lipid rafts. Further, inflammarafts form as a result of inflammatory signal-dependent oligomerization of resident raft proteins, which then pulls together isolated lipid rafts to form larger assemblies, with a longer lifetime and higher occupancy of raft-associated lipids and proteins, many of which are recruited from non-raft regions of the membrane or from the cytosol (4Sezgin E. Levental I. Mayor S. Eggeling C. The mystery of membrane organization: composition, regulation and roles of lipid rafts.Nat. Rev. Mol. Cell Biol. 2017; 18: 361-374Crossref PubMed Scopus (617) Google Scholar). Thus, protein-dependent signals constitute the front end of inflammaraft formation, driving lipid raft clustering and enlargement. Conversely, because the integrity of lipid rafts depends on their lipid composition, depletion of cholesterol and/or sphingolipids disrupts lipid rafts and serves to inhibit inflammaraft-associated cellular processes. In the next two sections, we will summarize current knowledge of protein-driven inflammaraft formation and lipid-dependent mechanisms of its disassembly, specifically related to activation of microglia and astrocytes in neuropathic pain. It is well documented that TLR4 is activated in lipid rafts (31Fessler M.B. Parks J.S. Intracellular lipid flux and membrane microdomains as organizing principles in inflammatory cell signaling.J. Immunol. 2011; 187: 1529-1535Crossref PubMed Scopus (163) Google Scholar, 32Tall A.R. Yvan-Charvet L. Cholesterol, inflammation and innate immunity.Nat. Rev. Immunol. 2015; 15: 104-116Crossref PubMed Scopus (537) Google Scholar, 33Woller S.A. Choi S.H. An E.J. Low H. Schneider D.A. Ramachandran R. Kim J. Bae Y.S. Sviridov D. Corr M. et al.Inhibition of neuroinflammation by AIBP: spinal effects upon facilitated pain states.Cell Reports. 2018; 23: 2667-2677Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 34Powers K.A. Szaszi K. Khadaroo R.G. Tawadros P.S. Marshall J.C. Kapus A. Rotstein O.D. Oxidative stress generated by hemorrhagic shock recruits Toll-like receptor 4 to the plasma membrane in macrophages.J. Exp. Med. 2006; 203: 1951-1961Crossref PubMed Scopus (136) Google Scholar, 36Zhu X. Owen J.S. Wilson M.D. Li H. Griffiths G.L. Thomas M.J. Hiltbold E.M. Fessler M.B. Parks J.S. Macrophage ABCA1 reduces MyD88-dependent Toll-like receptor trafficking to lipid rafts by reduction of lipid raft cholesterol.J. Lipid Res. 2010; 51: 3196-3206Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 49Fernandez-Lizarbe S. Pascual M. Gascon M.S. Blanco A. Guerri C. Lipid rafts regulate ethanol-induced activation of TLR4 signaling in murine macrophages.Mol. Immunol. 2008; 45: 2007-2016Crossref PubMed Scopus (67) Google Scholar, 50Wong S.W. Kwon M.J. Choi A.M.K. Kim H.P. Nakahira K. Hwang D.H. Fatty acids modulate Toll-like receptor 4 activation through regulation of receptor dimerization and recruitment into lipid rafts in a reactive oxygen species-dependent manner.J. Biol. Chem. 2009; 284: 27384-27392Abstract Full Text Full Text PDF PubMed Scopus (352) Google Scholar, 51Shridas P. Bailey W.M. Talbott K.R. Oslund R.C. Gelb M.H. Webb N.R. Group X secretory phospholipase A2 enhances TLR4 signaling in macrophages.J. Immu