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The complement cascade in the regulation of neuroinflammation, nociceptive sensitization, and pain

Open AccessPublished:August 16, 2021DOI:https://doi.org/10.1016/j.jbc.2021.101085
      The complement cascade is a key component of the innate immune system that is rapidly recruited through a cascade of enzymatic reactions to enable the recognition and clearance of pathogens and promote tissue repair. Despite its well-understood role in immunology, recent studies have highlighted new and unexpected roles of the complement cascade in neuroimmune interaction and in the regulation of neuronal processes during development, aging, and in disease states. Complement signaling is particularly important in directing neuronal responses to tissue injury, neurotrauma, and nerve lesions. Under physiological conditions, complement-dependent changes in neuronal excitability, synaptic strength, and neurite remodeling promote nerve regeneration, tissue repair, and healing. However, in a variety of pathologies, dysregulation of the complement cascade leads to chronic inflammation, persistent pain, and neural dysfunction. This review describes recent advances in our understanding of the multifaceted cross-communication that takes place between the complement system and neurons. In particular, we focus on the molecular and cellular mechanisms through which complement signaling regulates neuronal excitability and synaptic plasticity in the nociceptive pathways involved in pain processing in both health and disease. Finally, we discuss the future of this rapidly growing field and what we believe to be the significant knowledge gaps that need to be addressed.

      Keywords

      Abbreviations:

      AD (Alzheimer's disease), ALS (amyotrophic lateral sclerosis), ANKA (antineutrophil cytoplasmic antibody), AS (ankylosing spondylitis), C3aR (C3a receptor), C5aR1/2 (C5a receptor 1/2), C1-INH (C1-complex inhibitor), C4BP (C4b-binding protein), CGRP (calcitonin-gene related peptide), CNS (central nervous system), CRPS (complex regional pain syndrome), DAF (decay-accelerating factor), DAMPS (danger-associated molecular patterns), DRG (dorsal root ganglion), GPCR (G-protein-coupled receptor), HAE (hereditary angioedema), IL-1β (interleukin 1β), MAC (membrane attack complex), MASP (MBL-associated serine protease), MAG (myelin-associated glycoprotein), MBL (mannose-binding lectin), MCP (membrane cofactor protein), NGF (nerve growth factor), OA (osteoarthritis), PAMPs (pathogen-associated molecular patterns), PNH (paroxysmal nocturnal hemoglobinuria), PNI (peripheral nerve injury), PNS (peripheral nervous system), SCI (spinal cord injury), TBI (traumatic brain injury), TPCC (terminal pathway complete complex), TrkA (tropomyosin-related kinase A), TRPV1 (transient receptor potential vanilloid 1)
      The complement system consists of over 40 soluble and membrane-bound proteins that are rapidly mobilized through a cascade of enzymatic reactions (Fig. 1) in response to infection or tissue injury. Activated components of the complement system participate in canonical host defenses through a range of mechanisms, including the activation and chemotaxis of immune cells, as well as opsonization (i.e., tagging) and killing of pathogens or diseased cells (
      • Monk P.N.
      • Scola A.M.
      • Madala P.
      • Fairlie D.P.
      Function, structure and therapeutic potential of complement C5a receptors.
      ,
      • Wagner E.
      • Frank M.M.
      Therapeutic potential of complement modulation.
      ,
      • Ricklin D.
      • Lambris J.D.
      Complement in immune and inflammatory disorders: Pathophysiological mechanisms.
      ,
      • Holers V.M.
      Complement and its receptors: New insights into human disease.
      ). The actions of the complement cascade are considered as a functional bridge between the two branches of the immune system, linking the intrinsic activity of the innate immune system to the lymphocytes that drive the adaptive responses (
      • Dunkelberger J.R.
      • Song W.C.
      Complement and its role in innate and adaptive immune responses.
      ). It is becoming increasingly appreciated that complement also orchestrates multiple host processes and particularly those related to the function of the nervous system in health and disease. These include complement-dependent regulation of synaptic remodeling, axonal regrowth, neuronal damage, nociceptor sensitization, and pain. Despite recent progress, significant knowledge gaps exist in our understanding of molecular and cellular mechanisms that mediate the effects of complement cascades on neurons in the central and peripheral nervous systems. Furthermore, there are numerous examples when the same component of the complement system may impose opposite effects on neurons under pathological conditions, by either promoting neuronal recovery or exacerbating neuronal damage and aberrant neuronal activity. This suggests that the same complement factors can drive distinct outcomes through state-dependent mechanisms we have yet to fully elucidate.
      Figure thumbnail gr1
      Figure 1The complement system integrates distinct stimuli into a unified immunological response. The three pathways of complement activation, Classical (blue), Lectin (yellow), and Alternative (green), are activated by distinct perturbations to organism homeostasis such as antibody-based recognition of antigens or danger-associated molecular patterns (DAMPs), to drive a cascade of enzymatic activity merging in the terminal pathway (red). The cumulative result is the production of membrane-bound fragments (e.g., C3b and C5b) and soluble factors (e.g., C3a and C5a) that activate signaling through surface receptors (purple) that are highly expressed on immune and glial cells, and to a lesser extent, on neurons. Uncontrolled activation of the terminal pathway also promotes assembly of the cell-lysing terminal pathway complete complex (TPCC), also known as the membrane attack complex (MAC), which is regulated by multiple steric inhibitors and proteases (gray). This system controls the formation of complement products, their activity once formed and proteolytic degradation. A key component of the system is the amplification loop within the alternative pathway, which provides a positive feedback loop to ensure maximal activation of complement when appropriately triggered. However, this loop can also exacerbate complement-induced pathology under the conditions when complement inhibitors are dysregulated. Linked pain states are highlighted in red square brackets and include postsurgical pain, pain associated with peripheral nerve injury (PNI), complex regional pain syndrome (CRPS), and arthritic pain, as well as pain associated with paroxysmal nocturnal hemoglobinuria (PNH), hereditary angioedema (HAE), ankylosing spondylitis (AS), and chemotherapy-induced peripheral neuropathy (CIPN). In this context, CIPN also includes a painful condition associated with the use of anti-ganglioside-GD2 antibody for treating neuroblastoma cancer (
      • Sorkin L.S.
      • Otto M.
      • Baldwin 3rd, W.M.
      • Vail E.
      • Gillies S.D.
      • Handgretinger R.
      • Barfield R.C.
      • Ming Yu H.
      • Yu A.L.
      Anti-GD(2) with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia.
      ,
      • Anghelescu D.L.
      • Goldberg J.L.
      • Faughnan L.G.
      • Wu J.
      • Mao S.
      • Furman W.L.
      • Santana V.M.
      • Navid F.
      Comparison of pain outcomes between two anti-GD2 antibodies in patients with neuroblastoma.
      ,
      • Xu J.
      • Zhang L.
      • Xie M.
      • Li Y.
      • Huang P.
      • Saunders T.L.
      • Fox D.A.
      • Rosenquist R.
      • Lin F.
      Role of complement in a rat model of paclitaxel-induced peripheral neuropathy.
      ).
      Here, we systematically discuss what is known about the complex effects of activated complement products on neurons during the normal host response to injury as well as in the development and maintenance of neurological pathologies and propose classification of these effects based on common underlying molecular and cellular mechanisms. We focus particularly on the complement-dependent changes in the function of the somatosensory system that take place in response to injury or illness and discuss how these mechanisms contribute to nociceptive sensitization and the development of acute and chronic pain. Finally, we discuss what we consider as critical open questions for future research as well as challenges and opportunities in this rapidly emerging field of complement neurobiology.

      Activation of the complement cascade

      Under normal conditions, complement proteins are continuously produced as inactive zymogens by the liver and secreted into the plasma. Other tissues can serve as local sources and provide focal enhancement of complement signaling and inflammation after injury (
      • Janeway Jr., C.A.
      • Travers P.
      • Walport M.
      • Shlomchik M.J.
      The complement system and innate immunity.
      ). Each inactive zymogen is cleaved into its active products by a factor-specific protease. For example, the C5 protein is inactive until it is cleaved into the small “a” fragment (C5a), which is soluble, and the larger “b” fragment (C5b), which is membrane-bound through a covalent bond. This a/b nomenclature is used across complement components, which are numbered according to the order of their discovery rather than their sequence of activation. When tissue becomes damaged or an autoimmune reaction occurs, the action of the two split products is complementary: the “a” fragment attracts and activates immune cells, whereas the “b” fragment opsonizes the offending cell to promote its recognition and clearance by phagocytes. The activation of the complement cascade can be driven via three major pathways: classical, lectin, and alternative.
      The classical pathway (Fig. 1, blue) is activated by binding of an antibody (IgM or IgG) to an antigen on a microbial or host cell, forming an antibody–antigen complex, also known as an immune complex. In essentially any autoimmune disorder where aberrant self-recognition occurs, the production of immune complexes drives high levels of complement activity. The primary detector of immune complexes and pathogen-associated molecular patterns (PAMPs) is the C1 complex, which consists of the pattern recognition protein, C1q, and two protease complexes formed by C1r and C1s (Fig. 1). The binding of an immune complex to the C1 complex results in autocatalytic cleavage of the latter, producing the active form of C1 complex. This in turn cleaves C4 to produce C4b, which rapidly forms a covalent bond with a membrane. The anchoring of C4b to a membrane limits all subsequent cleavages to the immediate vicinity of the site at which the immune complex was first detected, preventing the spread of activation and providing a focal source of the downstream terminal chemotactic fragments, such as C3a and C5a. Aberrant activation of the classical pathway induced by B cell overproduction of antibodies can drive immunological pain disorders such as complex regional pain syndrome (CRPS; Fig. 1) (
      • Li W.W.
      • Guo T.Z.
      • Shi X.
      • Czirr E.
      • Stan T.
      • Sahbaie P.
      • Wyss-Coray T.
      • Kingery W.S.
      • Clark J.D.
      Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome.
      ,
      • Shi X.
      • Guo T.Z.
      • Li W.W.
      • Birklein F.
      • Escolano F.L.
      • Herrnberger M.
      • Clark J.D.
      • Kingery W.S.
      C5a complement and cytokine signaling mediate the pronociceptive effects of complex regional pain syndrome patient IgM in fracture mice.
      ). Additionally, activation of the classical pathway by cytotoxic agents such as antiganglioside-GD2 antibody for treating neuroblastoma cancer, contributes to severe pain, which is a common side effect of anticancer therapeutics (
      • Sorkin L.S.
      • Otto M.
      • Baldwin 3rd, W.M.
      • Vail E.
      • Gillies S.D.
      • Handgretinger R.
      • Barfield R.C.
      • Ming Yu H.
      • Yu A.L.
      Anti-GD(2) with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia.
      ,
      • Anghelescu D.L.
      • Goldberg J.L.
      • Faughnan L.G.
      • Wu J.
      • Mao S.
      • Furman W.L.
      • Santana V.M.
      • Navid F.
      Comparison of pain outcomes between two anti-GD2 antibodies in patients with neuroblastoma.
      ,
      • Xu J.
      • Zhang L.
      • Xie M.
      • Li Y.
      • Huang P.
      • Saunders T.L.
      • Fox D.A.
      • Rosenquist R.
      • Lin F.
      Role of complement in a rat model of paclitaxel-induced peripheral neuropathy.
      ) (Fig. 1).
      Membrane-bound C4b binds C2 that is subsequently cleaved by the C1 complex, resulting in the active serine protease complex, C4b2b, termed the classical C3 convertase (Fig. 1, blue/green). Up to this point, the only “a” fragments produced are C4a and C2a, both of which have essentially no activity (
      • Barnum S.R.
      C4a: An anaphylatoxin in name only.
      ). Formation of the C3 convertase is a critical transition to terminal pathway signaling and the generation of highly active C3a and C5a components, both of which are powerful drivers of neuroinflammation and pain (Fig. 1) (
      • Shutov L.P.
      • Warwick C.A.
      • Shi X.
      • Gnanasekaran A.
      • Shepherd A.J.
      • Mohapatra D.P.
      • Woodruff T.M.
      • Clark J.D.
      • Usachev Y.M.
      The complement system component C5a produces thermal hyperalgesia via macrophage-to-nociceptor signaling that requires NGF and TRPV1.
      ,
      • Jang J.H.
      • Clark J.D.
      • Li X.
      • Yorek M.S.
      • Usachev Y.M.
      • Brennan T.J.
      Nociceptive sensitization by complement C5a and C3a in mouse.
      ,
      • Jang J.H.
      • Liang D.
      • Kido K.
      • Sun Y.
      • Clark D.J.
      • Brennan T.J.
      Increased local concentration of complement C5a contributes to incisional pain in mice.
      ,
      • Liang D.Y.
      • Li X.
      • Shi X.
      • Sun Y.
      • Sahbaie P.
      • Li W.W.
      • Clark J.D.
      The complement component C5a receptor mediates pain and inflammation in a postsurgical pain model.
      ,
      • Warwick C.A.
      • Shutov L.P.
      • Shepherd A.J.
      • Mohapatra D.P.
      • Usachev Y.M.
      Mechanisms underlying mechanical sensitization induced by complement C5a: The roles of macrophages, TRPV1, and calcitonin gene-related peptide receptors.
      ).
      The lectin pathway (Fig. 1, yellow) is activated primarily by extracellular sugar residues, most prominently mannose residues on bacterial cells. The pattern recognition complex of the lectin pathway is homologous to the C1 complex of the classical pathway at both the structural and functional levels. It consists of the mannose-binding lectin (MBL) paired with two protease complexes formed by MBL-associated serine proteases, MASP-1 and MASP-2. MASP1/2 are closely related to C1r/s and likely diverged from a common ancestor after gene duplication. Other collagenous lectins such as the ficolins and collectin-11 are also capable of activating the lectin pathway by interacting with MASPs (
      • Ricklin D.
      • Hajishengallis G.
      • Yang K.
      • Lambris J.D.
      Complement: A key system for immune surveillance and homeostasis.
      ,
      • Bajic G.
      • Degn S.E.
      • Thiel S.
      • Andersen G.R.
      Complement activation, regulation, and molecular basis for complement-related diseases.
      ). Upon recognition of a substrate, the MBL complex is converted to an active serine protease and cleaves C4 and C2 similarly to the classical pathway, producing the classical C3 convertase, C4b2b.
      The alternative pathway (Fig. 1, green) is not activated by a specific pathogen, but rather has constitutive low-level activity, which enables to continuously probe cellular surfaces for defects. The spontaneous hydrolysis of C3 allows binding of factor B that is proteolytically activated by factor D to produce the C3 convertase of the alternative pathway, C3bBb. Although this C3 convertase can form on both healthy and pathogenic surfaces, complement inhibitors present in healthy cells rapidly inactivate the convertase and its activation products. In contrast, complement activation proceeds unfettered on foreign cells and can be further amplified through various mechanisms. For example, factor P, also known as properdin, stabilizes the alternative pathway C3 convertase and localizes it to pathogenic substrates, thereby providing amplification of its activity (
      • Ricklin D.
      • Hajishengallis G.
      • Yang K.
      • Lambris J.D.
      Complement: A key system for immune surveillance and homeostasis.
      ). Furthermore, the alternative pathway can significantly amplify signaling from all three pathways through a positive feedback loop. Once a C3 convertase has formed, the cleaved C3b binds factor B to form another C3 convertase, thus amplifying the initial signal. This feedback loop is estimated to be responsible for 80–90% of all complement activity regardless of which initiation pathway was triggered (
      • Harboe M.
      • Mollnes T.E.
      The alternative complement pathway revisited.
      ).
      The terminal pathway represents the final stage of complement activation (Fig. 1, red). It begins with the addition of C3b to the C3 convertase complexes, resulting in C4bC2bC3b complex for the classical and leptin pathways and C3bBbC3b complex for the alternative pathway. These complexes function as C5 convertases that cleave C5 into two split products, C5a and C5b. A highly reactive product, C5a functions as a potent inflammatory mediator that was originally described as an anaphylatoxin due to its ability to activate mast cells and induce massive release of histamine. Its profound role in neuroinflammation and pain is discussed below. The other product, C5b, assembles with C6, C7, C8, and the pore-forming subunit C9 on the surface of a cell to put together the terminal pathway complete complex (TPCC) (
      • Kemper C.
      • Pangburn M.K.
      • Fishelson Z.
      Complement nomenclature 2014.
      ), also known as the membrane attack complex (MAC). The complex forms a large channel (up to 100 Å) in the surface membrane, which ultimately causes cell lysis. Notably, overactivation of the TPCC/MAC has been implicated in various pain states, including arthritis, neuropathic pain, and chemotherapy-induced peripheral neuropathy (CIPN; Fig. 1) (
      • Xu J.
      • Zhang L.
      • Xie M.
      • Li Y.
      • Huang P.
      • Saunders T.L.
      • Fox D.A.
      • Rosenquist R.
      • Lin F.
      Role of complement in a rat model of paclitaxel-induced peripheral neuropathy.
      ,
      • Wang Q.
      • Rozelle A.L.
      • Lepus C.M.
      • Scanzello C.R.
      • Song J.J.
      • Larsen D.M.
      • Crish J.F.
      • Bebek G.
      • Ritter S.Y.
      • Lindstrom T.M.
      • Hwang I.
      • Wong H.H.
      • Punzi L.
      • Encarnacion A.
      • Shamloo M.
      • et al.
      Identification of a central role for complement in osteoarthritis.
      ,
      • Levin M.E.
      • Jin J.G.
      • Ji R.R.
      • Tong J.
      • Pomonis J.D.
      • Lavery D.J.
      • Miller S.W.
      • Chiang L.W.
      Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain.
      ).

      Regulators of the complement cascade

      Rapid and substantial activation of the complement system in response to foreign invaders and injury also requires equally potent and coordinated mechanisms for limiting its activity in order to prevent uncontrollable inflammation, autoimmunity, and destruction of healthy tissues (
      • Ricklin D.
      • Hajishengallis G.
      • Yang K.
      • Lambris J.D.
      Complement: A key system for immune surveillance and homeostasis.
      ,
      • Sjoberg A.P.
      • Trouw L.A.
      • Blom A.M.
      Complement activation and inhibition: A delicate balance.
      ). Multiple molecular and cellular mechanisms have evolved to regulate complement activation through various inhibitory signaling processes (Fig. 1, gray). These mechanisms help to prevent activation of the complement cascade in healthy tissues, accelerate the removal of opsins deposited on host cells, and refine the target of complement signaling by localizing convertases to the site of infection or injury. These inhibitors act through a variety of mechanisms and at all stages of complement signaling, but most target the alternative pathway amplification loop (Fig. 1). Notably, Factor H, Factor I, complement receptor 1 (CR1), decay-accelerating factor (DAF or CD55), and membrane cofactor protein (MCP or CD46) all specifically regulate C3b-containing convertases, further underscoring the importance of controlling the alternative pathway amplification loop under various conditions (
      • Ricklin D.
      • Hajishengallis G.
      • Yang K.
      • Lambris J.D.
      Complement: A key system for immune surveillance and homeostasis.
      ,
      • Bajic G.
      • Degn S.E.
      • Thiel S.
      • Andersen G.R.
      Complement activation, regulation, and molecular basis for complement-related diseases.
      ,
      • Dunn H.A.
      • Orlandi C.
      • Martemyanov K.A.
      Beyond the ligand: Extracellular and transcellular G protein-coupled receptor complexes in physiology and pharmacology.
      ).
      The initiation steps of complement activation are also controlled at multiple levels. With respect to the classical and lectin pathways, the C1 complex inhibitor (C1-INH/serpin G1) is a soluble protein that circulates throughout the body and limits the amount of protease activity “leaked” from the site of primary activation. It also limits the spread of spontaneous activation such as seen in chronic pain associated with hereditary angioedema (HAE; Fig. 1), for which the first-line treatment is to provide a plasma-derived preparation of C1-INH, Cinryze (
      • Morgan B.P.
      • Harris C.L.
      Complement, a target for therapy in inflammatory and degenerative diseases.
      ,
      • Mastellos D.C.
      • Ricklin D.
      • Lambris J.D.
      Clinical promise of next-generation complement therapeutics.
      ). Similarly, at the level of inhibiting C3 convertases, the C4b-binding protein (C4BP) displaces C2b in the classical C4b2b/C3 convertase by competitively binding with C4b to inhibit activity of the enzyme (Fig. 1). Acting together with the soluble inhibitors of C3b, Factor I and Factor H, these mechanisms reduce the levels of both alternative and classical C3 convertases within the circulatory system, while permitting their localized actions.
      The other inhibitors of complement signaling act primarily at the cell surface. These proteins vary widely in their mechanisms of action, but all prevent damage to the healthy cells that express them and protect local tissue. For example, the membrane-bound protein MCP (CD46) binds to C3b deposited on the cell surface making it susceptible to cleavage and inactivation by Factor I, which prevents formation of C3 and C5 convertases (Fig. 1). Other membrane-bound inhibitors, such as CR1 and DAF (CD55), competitively bind to C3b to prevent the formation of a convertase. They can also displace Bb from an active convertase to promote decay. These cell surface regulators are especially critical for protecting healthy tissue near an area of injury with active complement signaling, as in the case of nerve damage. DAF and other membrane-localized regulators protect healthy cells from complement deposition throughout the period of inflammation and recovery, while dysregulation of these inhibitory factors leaves healthy cells vulnerable to the actions of immune cells recruited to the site of injury in the context of neuropathic pain (
      • Levin M.E.
      • Jin J.G.
      • Ji R.R.
      • Tong J.
      • Pomonis J.D.
      • Lavery D.J.
      • Miller S.W.
      • Chiang L.W.
      Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain.
      ,
      • Renthal W.
      • Tochitsky I.
      • Yang L.
      • Cheng Y.C.
      • Li E.
      • Kawaguchi R.
      • Geschwind D.H.
      • Woolf C.J.
      Transcriptional reprogramming of distinct peripheral sensory neuron subtypes after axonal injury.
      ).
      An additional level of complement regulation occurs through various glycosaminoglycans such as heparan sulfate. These sugar molecules expressed on host and pathogen membranes interact with various complement inhibitors and activators. For example, interaction of heparan sulfate with Factor H prevents complement activation on a host cell (
      • Langford-Smith A.
      • Day A.J.
      • Bishop P.N.
      • Clark S.J.
      Complementing the sugar code: Role of GAGs and sialic acid in complement regulation.
      ). Mechanistically, heparan sulfate promotes localization of Factor H to host membrane and acts as a cofactor for Factor H to enhance localized cleavage of C3b (
      • Schmidt C.Q.
      • Herbert A.P.
      • Kavanagh D.
      • Gandy C.
      • Fenton C.J.
      • Blaum B.S.
      • Lyon M.
      • Uhrin D.
      • Barlow P.N.
      A new map of glycosaminoglycan and C3b binding sites on factor H.
      ,
      • Clark S.J.
      • Ridge L.A.
      • Herbert A.P.
      • Hakobyan S.
      • Mulloy B.
      • Lennon R.
      • Wurzner R.
      • Morgan B.P.
      • Uhrin D.
      • Bishop P.N.
      • Day A.J.
      Tissue-specific host recognition by complement factor H is mediated by differential activities of its glycosaminoglycan-binding regions.
      ,
      • Perkins S.J.
      • Fung K.W.
      • Khan S.
      Molecular interactions between complement factor H and its heparin and heparan sulfate ligands.
      ,
      • Loeven M.A.
      • Rops A.L.
      • Berden J.H.
      • Daha M.R.
      • Rabelink T.J.
      • van der Vlag J.
      The role of heparan sulfate as determining pathogenic factor in complement factor H-associated diseases.
      ). Furthermore, some glycosaminoglycans are involved in complement-mediated regulation of neurological diseases. In particular, heparan sulfate has been found to localize Factor H to Aβ plaques and also to inhibit binding of β-amyloid peptides by C1q, which likely prevents clearance of the plaques and contributes to Alzheimer's disease progression (
      • Strohmeyer R.
      • Ramirez M.
      • Cole G.J.
      • Mueller K.
      • Rogers J.
      Association of factor H of the alternative pathway of complement with agrin and complement receptor 3 in the Alzheimer's disease brain.
      ,
      • Urbanyi Z.
      • Forrai E.
      • Sarvari M.
      • Liko I.
      • Illes J.
      • Pazmany T.
      Glycosaminoglycans inhibit neurodegenerative effects of serum amyloid P component in vitro.
      ).
      The last line of defense against complement activity, whether spontaneous or pathogenic, is provided by regulators of the formation and insertion of the TPCC/MAC lytic channel (Fig. 1). Protectin, also known as CD59, prevents assembly of the TPCC by binding to transmembrane proteins C8 and C9. Similarly, vitronectin and clusterin bind to various proteins in the TPCC, preventing its assembly. These proteins are widely expressed on both healthy and apoptotic cells. Thus, formation of the TPCC is normally restricted to pathogenic cells, whereas formation of the TPCC on host cells is commonly associated with pathological conditions. A clear example of importance of TPCC inhibition is found in a rare disease called paroxysmal nocturnal hemoglobinuria (PNH). Patients with PNH experience episodes of severe pain, which is associated with complement-mediated lysis of red blood cells due to the absence of protectin and DAF on host cells (
      • Holers V.M.
      Complement and its receptors: New insights into human disease.
      • Quadros A.U.
      • Cunha T.M.
      C5a and pain development: An old molecule, a new target.
      ,
      • Risitano A.M.
      Paroxysmal nocturnal hemoglobinuria and other complement-mediated hematological disorders.
      ) (Fig. 1).

      Complement receptors

      Although many of the actions of the complement system are directed at its targets through generic PAMPs or ubiquitous tagging of lipid membranes, specific receptors present on the host cells can facilitate recruitment to the site of injury, recognition and phagocytosis of opsonized pathogens, or even stimulation of cellular replication to aid in tissue recovery (
      • Holers V.M.
      Complement and its receptors: New insights into human disease.
      ). The early components of the complement cascade (C1, C2, C4, MBL, and MASPs) have few well-characterized receptors as they are involved primarily in detection of threats and initiation of the terminal pathway. The only well-characterized receptors are those for C1q, such as C1qR, cC1qR, and gC1qR. These receptors are expressed on phagocytes and thought to be important for recognizing C1q bound to the immune complexes and promoting phagocytosis. The remaining complement receptors recognize primarily fragments of C3 and C5 (Fig. 1, purple).
      The first class of complement receptors in this group consists of those that recognize membrane-bound fragments of C3 and include CR1 (CD35), CR2 (CD21), CR3 (CD11b+CD18), and CR4 (CD11c+CD18) (Fig. 1). CR1 binds C3b with high affinity and the inactivated form of C3b (iC3b) with low affinity, whereas CR2-4 receptors universally recognize iC3b. When these receptors are expressed on phagocytes, activation promotes phagocytosis of opsonized material. Monocytes, macrophages, and neutrophils all express CR1, CR3, and CR4, whereas dendritic cells express CR4 (
      • Janeway Jr., C.A.
      • Travers P.
      • Walport M.
      • Shlomchik M.J.
      The complement system and innate immunity.
      ). As mentioned earlier, CR1 also has decay-accelerating characteristics, by competitively binding C3b and displacing Bb from the C3 convertase. B cells also express CR1 and CR2, and iC3b binding to these receptors enhances B cell activation and antibody secretion (
      • Ricklin D.
      • Hajishengallis G.
      • Yang K.
      • Lambris J.D.
      Complement: A key system for immune surveillance and homeostasis.
      ). The latter exemplifies how the complement system augments adaptive immune responses.
      The remaining receptors bind the soluble complement fragments C3a and C5a. Both C3a and C5a act through classical seven-transmembrane G-protein-coupled receptors (GPCRs) called C3aR and C5aR1 (CD88), respectively (Fig. 1). These receptors have similar expression patterns on endothelium (
      • Strunk R.C.
      • Eidlen D.M.
      • Mason R.J.
      Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways.
      ), smooth muscle (
      • Drouin S.M.
      • Kildsgaard J.
      • Haviland J.
      • Zabner J.
      • Jia H.P.
      • McCray Jr., P.B.
      • Tack B.F.
      • Wetsel R.A.
      Expression of the complement anaphylatoxin C3a and C5a receptors on bronchial epithelial and smooth muscle cells in models of sepsis and asthma.
      ), glia (
      • Gasque P.
      • Chan P.
      • Fontaine M.
      • Ischenko A.
      • Lamacz M.
      • Gotze O.
      • Morgan B.P.
      Identification and characterization of the complement C5a anaphylatoxin receptor on human astrocytes.
      ,
      • Lacy M.
      • Jones J.
      • Whittemore S.R.
      • Haviland D.L.
      • Wetsel R.A.
      • Barnum S.R.
      Expression of the receptors for the C5a anaphylatoxin, interleukin-8 and FMLP by human astrocytes and microglia.
      ), and throughout all myeloid cells including neutrophils, monocytes, macrophages, and restricted populations of dendritic cells (
      • Karsten C.M.
      • Laumonnier Y.
      • Eurich B.
      • Ender F.
      • Broker K.
      • Roy S.
      • Czabanska A.
      • Vollbrandt T.
      • Figge J.
      • Kohl J.
      Monitoring and cell-specific deletion of C5aR1 using a novel floxed GFP-C5aR1 reporter knock-in mouse.
      • Dunkelberger J.
      • Zhou L.
      • Miwa T.
      • Song W.C.
      C5aR expression in a novel GFP reporter gene knockin mouse: Implications for the mechanism of action of C5aR signaling in T cell immunity.
      ). Activation of these receptors produces a variety of effects, including increase in vascular permeability, chemotaxis, production and release of inflammatory factors, and stimulation of phagocytosis. C3aR- and C5aR1-mediated effects are particularly prominent in various pain states, including postsurgical pain, neuropathic pain, CRPS, and arthritis (Fig. 1) (
      • Shi X.
      • Guo T.Z.
      • Li W.W.
      • Birklein F.
      • Escolano F.L.
      • Herrnberger M.
      • Clark J.D.
      • Kingery W.S.
      C5a complement and cytokine signaling mediate the pronociceptive effects of complex regional pain syndrome patient IgM in fracture mice.
      ,
      • Jang J.H.
      • Clark J.D.
      • Li X.
      • Yorek M.S.
      • Usachev Y.M.
      • Brennan T.J.
      Nociceptive sensitization by complement C5a and C3a in mouse.
      ,
      • Quadros A.U.
      • Cunha T.M.
      C5a and pain development: An old molecule, a new target.
      ,
      • Clark J.D.
      • Qiao Y.
      • Li X.
      • Shi X.
      • Angst M.S.
      • Yeomans D.C.
      Blockade of the complement C5a receptor reduces incisional allodynia, edema, and cytokine expression.
      ,
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ,
      • Doolen S.
      • Cook J.
      • Riedl M.
      • Kitto K.
      • Kohsaka S.
      • Honda C.N.
      • Fairbanks C.A.
      • Taylor B.K.
      • Vulchanova L.
      Complement 3a receptor in dorsal horn microglia mediates pronociceptive neuropeptide signaling.
      ,
      • Giorgio C.
      • Zippoli M.
      • Cocchiaro P.
      • Castelli V.
      • Varrassi G.
      • Aramini A.
      • Allegretti M.
      • Brandolini L.
      • Cesta M.C.
      Emerging role of C5 complement pathway in peripheral neuropathies: Current treatments and future perspectives.
      ). To limit systemic activation, both C3a and C5a are rapidly “des-arginated” by serum carboxypeptidases. However, while this entirely removes the activity of C3a at C3aR, C5a-desArg retains some, albeit reduced, activity at C5aR1, thus making C5a highly potent in biological tissues (
      • Monk P.N.
      • Scola A.M.
      • Madala P.
      • Fairlie D.P.
      Function, structure and therapeutic potential of complement C5a receptors.
      ,
      • Klos A.
      • Wende E.
      • Wareham K.J.
      • Monk P.N.
      International union of basic and clinical pharmacology. [corrected]. LXXXVII. Complement peptide C5a, C4a, and C3a receptors.
      ).
      The other C5a receptor, C5aR2 (C5L2; Fig. 1), is less fully characterized and has more nuanced function. Although C5aR2 has structural homology to C5aR1, it is uncoupled from intracellular heterotrimeric G-proteins due to mutations in the G-protein recognition sequence (
      • Okinaga S.
      • Slattery D.
      • Humbles A.
      • Zsengeller Z.
      • Morteau O.
      • Kinrade M.B.
      • Brodbeck R.M.
      • Krause J.E.
      • Choe H.R.
      • Gerard N.P.
      • Gerard C.
      C5L2, a nonsignaling C5A binding protein.
      ). As such, C5a-C5aR2 engagement does not induce classical GPCR signaling events, initially promoting the concept of C5aR2 as a decoy receptor (
      • Okinaga S.
      • Slattery D.
      • Humbles A.
      • Zsengeller Z.
      • Morteau O.
      • Kinrade M.B.
      • Brodbeck R.M.
      • Krause J.E.
      • Choe H.R.
      • Gerard N.P.
      • Gerard C.
      C5L2, a nonsignaling C5A binding protein.
      ,
      • Cain S.A.
      • Monk P.N.
      The orphan receptor C5L2 has high affinity binding sites for complement fragments C5a and C5a des-Arg(74).
      ). Indeed, deletion of C5aR2 appears to enhance the C5aR1-mediated proinflammatory activity of C5a (
      • Gerard N.P.
      • Lu B.
      • Liu P.
      • Craig S.
      • Fujiwara Y.
      • Okinaga S.
      • Gerard C.
      An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2.
      ,
      • Wu M.C.L.
      • Lee J.D.
      • Ruitenberg M.J.
      • Woodruff T.M.
      Absence of the C5a receptor C5aR2 worsens ischemic tissue injury by increasing C5aR1-mediated neutrophil infiltration.
      ). However, further studies demonstrated that C5aR2 can signal through β-arrestins independent of G-proteins, which may mediate some functional activities including pro- and anti-inflammatory responses (
      • Klos A.
      • Wende E.
      • Wareham K.J.
      • Monk P.N.
      International union of basic and clinical pharmacology. [corrected]. LXXXVII. Complement peptide C5a, C4a, and C3a receptors.
      ,
      • Gerard N.P.
      • Lu B.
      • Liu P.
      • Craig S.
      • Fujiwara Y.
      • Okinaga S.
      • Gerard C.
      An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2.
      ,
      • Chen N.J.
      • Mirtsos C.
      • Suh D.
      • Lu Y.C.
      • Lin W.J.
      • McKerlie C.
      • Lee T.
      • Baribault H.
      • Tian H.
      • Yeh W.C.
      C5L2 is critical for the biological activities of the anaphylatoxins C5a and C3a.
      ,
      • Li X.X.
      • Clark R.J.
      • Woodruff T.M.
      C5aR2 activation broadly modulates the signaling and function of primary human macrophages.
      ).
      The interactions of the complement receptors and their targets on various cellular populations promote significant cross-talk among immune cells, allowing complement to integrate and enhance a wide variety of responses throughout the peripheral and central nervous systems (PNS and CNS, respectively; Fig. 2), as well as in other tissues (
      • Quadros A.U.
      • Cunha T.M.
      C5a and pain development: An old molecule, a new target.
      ,
      • Klos A.
      • Wende E.
      • Wareham K.J.
      • Monk P.N.
      International union of basic and clinical pharmacology. [corrected]. LXXXVII. Complement peptide C5a, C4a, and C3a receptors.
      ,
      • Lee J.D.
      • Coulthard L.G.
      • Woodruff T.M.
      Complement dysregulation in the central nervous system during development and disease.
      ). Although much is known about intrinsic mechanisms that drive complement activation, we know comparatively less about the myriad of effects that complement effectors have on downstream targets. In the next sections, we discuss the current literature regarding the roles of complement in the nervous system and outline the key knowledge gaps that need to be filled by additional studies.
      Figure thumbnail gr2
      Figure 2Complement-mediated mechanisms in the regulation of neuronal functions in health and disease. Where labeled, physiological mechanisms are shown in green and pathological mechanisms in red. Some mechanisms, such as synaptic pruning (gray), can contribute to both physiological and pathological processes, depending on developmental state and disease conditions.

      Complement cascade in the nervous system: mechanisms and functions

      In addition to protecting the nervous system from pathogens, the complement system plays major roles in controlling many fundamental processes within the PNS and CNS during development and aging, as well as in response to injury, disease, ischemic damage, or toxic stress. Figure 2 describes four prominent generalizable mechanisms through which complement acts in the nervous system in both development and physiological homeostasis as well as in neuropathology. Circulating complement components are not traditionally thought to cross the blood–brain or blood–spinal cord barriers in any meaningful concentrations in the healthy state. Rather, the complement proteins present in the CNS are thought to be produced locally within the CNS, predominantly by glia (
      • Brennan F.H.
      • Anderson A.J.
      • Taylor S.M.
      • Woodruff T.M.
      • Ruitenberg M.J.
      Complement activation in the injured central nervous system: Another dual-edged sword?.
      • Morgan B.P.
      Complement in the pathogenesis of Alzheimer's disease.
      ). CNS-derived complement components can act on neurons, glia, and immune cells to mediate both physiological and pathological effects (
      • Brennan F.H.
      • Anderson A.J.
      • Taylor S.M.
      • Woodruff T.M.
      • Ruitenberg M.J.
      Complement activation in the injured central nervous system: Another dual-edged sword?.
      ). During development of the nervous system, complement proteins regulate the proliferation of neural progenitor cells and neuronal migration, as well sculpting developing synaptic networks (
      • Stephan A.H.
      • Barres B.A.
      • Stevens B.
      The complement system: An unexpected role in synaptic pruning during development and disease.
      ,
      • Coulthard L.G.
      • Hawksworth O.A.
      • Woodruff T.M.
      Complement: The emerging architect of the developing brain.
      ). In addition, deregulation of the complement cascade has been implicated in synaptic loss and neuronal damage in aging and various neurodegenerative diseases including Alzheimer's disease (AD) and amyotrophic lateral sclerosis (ALS) (
      • Lee J.D.
      • Coulthard L.G.
      • Woodruff T.M.
      Complement dysregulation in the central nervous system during development and disease.
      ,
      • Tenner A.J.
      Complement-mediated events in Alzheimer's disease: Mechanisms and potential therapeutic targets.
      ). Following stroke, the complement system is rapidly activated, promoting inflammation and exacerbating oxidative tissue damage in the first few days after stroke, while facilitating synaptogenesis, neuronal plasticity, and poststroke recovery at later stages (
      • Pavlovski D.
      • Thundyil J.
      • Monk P.N.
      • Wetsel R.A.
      • Taylor S.M.
      • Woodruff T.M.
      Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis.
      ,
      • Arumugam T.V.
      • Tang S.C.
      • Lathia J.D.
      • Cheng A.
      • Mughal M.R.
      • Chigurupati S.
      • Magnus T.
      • Chan S.L.
      • Jo D.G.
      • Ouyang X.
      • Fairlie D.P.
      • Granger D.N.
      • Vortmeyer A.
      • Basta M.
      • Mattson M.P.
      Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing complement-mediated neuronal cell death.
      ,
      • Rahpeymai Y.
      • Hietala M.A.
      • Wilhelmsson U.
      • Fotheringham A.
      • Davies I.
      • Nilsson A.K.
      • Zwirner J.
      • Wetsel R.A.
      • Gerard C.
      • Pekny M.
      • Pekna M.
      Complement: A novel factor in basal and ischemia-induced neurogenesis.
      ,
      • Stokowska A.
      • Atkins A.L.
      • Moran J.
      • Pekny T.
      • Bulmer L.
      • Pascoe M.C.
      • Barnum S.R.
      • Wetsel R.A.
      • Nilsson J.A.
      • Dragunow M.
      • Pekna M.
      Complement peptide C3a stimulates neural plasticity after experimental brain ischaemia.
      ). Similarly, the complement system plays a dual role following spinal cord injury (SCI), contributing to the inflammatory response and clearance of cell debris and damaged tissues during an acute phase, while facilitating tissue repair during a later recovery phase (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.
      ,
      • Brennan F.H.
      • Gordon R.
      • Lao H.W.
      • Biggins P.J.
      • Taylor S.M.
      • Franklin R.J.
      • Woodruff T.M.
      • Ruitenberg M.J.
      The complement receptor C5aR controls acute inflammation and astrogliosis following spinal cord injury.
      ). Another important function of the complement system following tissue or nerve injury is its contribution to sensitization of nociceptive neurons and amplification of pain signaling, which serves to guard and protect injured tissues and support normal healing. However, aberrant activation of the complement cascade can promote the development of conditions characterized by chronic pain, such as complex regional pain syndrome, arthritic pain, and neuropathic pain (
      • Li W.W.
      • Guo T.Z.
      • Shi X.
      • Czirr E.
      • Stan T.
      • Sahbaie P.
      • Wyss-Coray T.
      • Kingery W.S.
      • Clark J.D.
      Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome.
      ,
      • Wang Q.
      • Rozelle A.L.
      • Lepus C.M.
      • Scanzello C.R.
      • Song J.J.
      • Larsen D.M.
      • Crish J.F.
      • Bebek G.
      • Ritter S.Y.
      • Lindstrom T.M.
      • Hwang I.
      • Wong H.H.
      • Punzi L.
      • Encarnacion A.
      • Shamloo M.
      • et al.
      Identification of a central role for complement in osteoarthritis.
      ,
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ).
      The complement system can regulate various aspects of neuronal life and death both directly, through specific complement receptors expressed on the neuronal plasma membrane, as well as indirectly, by recruiting glial and immune cells that convey complement signaling to neurons through various mechanisms (
      • Lee J.D.
      • Coulthard L.G.
      • Woodruff T.M.
      Complement dysregulation in the central nervous system during development and disease.
      ,
      • Stephan A.H.
      • Barres B.A.
      • Stevens B.
      The complement system: An unexpected role in synaptic pruning during development and disease.
      ,
      • Tenner A.J.
      Complement-mediated events in Alzheimer's disease: Mechanisms and potential therapeutic targets.
      ). The complex effects of the complement system on neuronal excitability, plasticity, survival, and demise involve intricate molecular, cellular, and intercellular mechanisms that can be broadly divided into four groups: (1) synaptic pruning; (2) regulation of axonal growth; (3) regulation of neuroinflammation; and (4) TPCC/MAC-mediated neuronal toxicity (Fig. 2).

      Synaptic pruning

      Activity-dependent elimination of synapses, also known as synaptic pruning, is a fundamental mechanism that shapes neuronal wiring during development (
      • Stephan A.H.
      • Barres B.A.
      • Stevens B.
      The complement system: An unexpected role in synaptic pruning during development and disease.
      ,
      • Katz L.C.
      • Shatz C.J.
      Synaptic activity and the construction of cortical circuits.
      ). Recent studies have showed that synaptic pruning in the developing visual system is guided by components of the classical complement cascade (
      • Stevens B.
      • Allen N.J.
      • Vazquez L.E.
      • Howell G.R.
      • Christopherson K.S.
      • Nouri N.
      • Micheva K.D.
      • Mehalow A.K.
      • Huberman A.D.
      • Stafford B.
      • Sher A.
      • Litke A.M.
      • Lambris J.D.
      • Smith S.J.
      • John S.W.
      • et al.
      The classical complement cascade mediates CNS synapse elimination.
      ,
      • Schafer D.P.
      • Lehrman E.K.
      • Kautzman A.G.
      • Koyama R.
      • Mardinly A.R.
      • Yamasaki R.
      • Ransohoff R.M.
      • Greenberg M.E.
      • Barres B.A.
      • Stevens B.
      Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.
      ). This process involves opsonization of synapses by the complement fragment C3b and its interaction with complement receptor CR3 expressed on microglia, to direct phagocytosis of C3b-expressing synapses (Fig. 2) (
      • Stevens B.
      • Allen N.J.
      • Vazquez L.E.
      • Howell G.R.
      • Christopherson K.S.
      • Nouri N.
      • Micheva K.D.
      • Mehalow A.K.
      • Huberman A.D.
      • Stafford B.
      • Sher A.
      • Litke A.M.
      • Lambris J.D.
      • Smith S.J.
      • John S.W.
      • et al.
      The classical complement cascade mediates CNS synapse elimination.
      ,
      • Schafer D.P.
      • Lehrman E.K.
      • Kautzman A.G.
      • Koyama R.
      • Mardinly A.R.
      • Yamasaki R.
      • Ransohoff R.M.
      • Greenberg M.E.
      • Barres B.A.
      • Stevens B.
      Microglia sculpt postnatal neural circuits in an activity and complement-dependent manner.
      ,
      • Anderson S.R.
      • Zhang J.
      • Steele M.R.
      • Romero C.O.
      • Kautzman A.G.
      • Schafer D.P.
      • Vetter M.L.
      Complement targets newborn retinal ganglion cells for phagocytic elimination by microglia.
      ). The presence of C1q at synapses suggests that C3b is produced locally at these sites as a result of activation of the classical cascade. Similar mechanisms are involved during the development in other brain regions, and they also contribute to synaptic plasticity and learning in the adult brain (
      • Chu Y.
      • Jin X.
      • Parada I.
      • Pesic A.
      • Stevens B.
      • Barres B.
      • Prince D.A.
      Enhanced synaptic connectivity and epilepsy in C1q knockout mice.
      ,
      • Kakegawa W.
      • Mitakidis N.
      • Miura E.
      • Abe M.
      • Matsuda K.
      • Takeo Y.H.
      • Kohda K.
      • Motohashi J.
      • Takahashi A.
      • Nagao S.
      • Muramatsu S.
      • Watanabe M.
      • Sakimura K.
      • Aricescu A.R.
      • Yuzaki M.
      Anterograde c1ql1 signaling is required in order to determine and maintain a single-winner climbing fiber in the mouse cerebellum.
      ,
      • Martinelli D.C.
      • Chew K.S.
      • Rohlmann A.
      • Lum M.Y.
      • Ressl S.
      • Hattar S.
      • Brunger A.T.
      • Missler M.
      • Sudhof T.C.
      Expression of C1ql3 in discrete neuronal populations controls efferent synapse numbers and diverse behaviors.
      ). However, excessive synaptic pruning during development and aging has been implicated in psychiatric and neurodegenerative diseases (
      • Stephan A.H.
      • Barres B.A.
      • Stevens B.
      The complement system: An unexpected role in synaptic pruning during development and disease.
      ,
      • Tenner A.J.
      • Stevens B.
      • Woodruff T.M.
      New tricks for an ancient system: Physiological and pathological roles of complement in the CNS.
      ). For example, increased expression of the complement protein C4 has been linked to schizophrenia (
      • Sekar A.
      • Bialas A.R.
      • de Rivera H.
      • Davis A.
      • Hammond T.R.
      • Kamitaki N.
      • Tooley K.
      • Presumey J.
      • Baum M.
      • Van Doren V.
      • Genovese G.
      • Rose S.A.
      • Handsaker R.E.
      • Daly M.J.
      • et al.
      Schizophrenia Working Group of the Psychiatric Genomics Consortium
      Schizophrenia risk from complex variation of complement component 4.
      ). Mechanistically, it has been proposed that elevations in levels of C4 lead to excessive downstream activation of C3 and complement/microglia-mediated elimination of synapses, ultimately leading to impaired synaptic wiring in the prefrontal cortex and other brain regions implicated in schizophrenia (
      • Sekar A.
      • Bialas A.R.
      • de Rivera H.
      • Davis A.
      • Hammond T.R.
      • Kamitaki N.
      • Tooley K.
      • Presumey J.
      • Baum M.
      • Van Doren V.
      • Genovese G.
      • Rose S.A.
      • Handsaker R.E.
      • Daly M.J.
      • et al.
      Schizophrenia Working Group of the Psychiatric Genomics Consortium
      Schizophrenia risk from complex variation of complement component 4.
      ,
      • Sellgren C.M.
      • Gracias J.
      • Watmuff B.
      • Biag J.D.
      • Thanos J.M.
      • Whittredge P.B.
      • Fu T.
      • Worringer K.
      • Brown H.E.
      • Wang J.
      • Kaykas A.
      • Karmacharya R.
      • Goold C.P.
      • Sheridan S.D.
      • Perlis R.H.
      Increased synapse elimination by microglia in schizophrenia patient-derived models of synaptic pruning.
      ). Another example of the pathological role of complement in excessive synaptic pruning relates to its involvement in synaptic loss in AD. Indeed, increased expression of the components of the classical complement cascade (e.g., C1q, C3 and C4) has been reported in the hippocampus and frontal cortex of AD patients, as well as in AD mouse models (
      • Lee J.D.
      • Coulthard L.G.
      • Woodruff T.M.
      Complement dysregulation in the central nervous system during development and disease.
      ,
      • Dejanovic B.
      • Huntley M.A.
      • De Maziere A.
      • Meilandt W.J.
      • Wu T.
      • Srinivasan K.
      • Jiang Z.
      • Gandham V.
      • Friedman B.A.
      • Ngu H.
      • Foreman O.
      • Carano R.A.D.
      • Chih B.
      • Klumperman J.
      • Bakalarski C.
      • et al.
      Changes in the synaptic proteome in tauopathy and rescue of tau-induced synapse loss by C1q antibodies.
      ). Elevated levels of C1q were specifically associated with synapses in two different Aβ mouse models of AD, whereas inhibition of C1q or deletion of C3 or CR3 in these mice led to reductions in the number of phagocytic microglia and synaptic loss, as well improvements in learning and memory tasks (
      • Hong S.
      • Beja-Glasser V.F.
      • Nfonoyim B.M.
      • Frouin A.
      • Li S.
      • Ramakrishnan S.
      • Merry K.M.
      • Shi Q.
      • Rosenthal A.
      • Barres B.A.
      • Lemere C.A.
      • Selkoe D.J.
      • Stevens B.
      Complement and microglia mediate early synapse loss in Alzheimer mouse models.
      • Shi Q.
      • Chowdhury S.
      • Ma R.
      • Le K.X.
      • Hong S.
      • Caldarone B.J.
      • Stevens B.
      • Lemere C.A.
      Complement C3 deficiency protects against neurodegeneration in aged plaque-rich APP/PS1 mice.
      ). Similarly, in Tau-P301S mice, C1q was found at the synapses in association with phospho-Tau and phagocyting microglia, and a C1q-scavenging antibody rescued synaptic loss in these mice (
      • Dejanovic B.
      • Huntley M.A.
      • De Maziere A.
      • Meilandt W.J.
      • Wu T.
      • Srinivasan K.
      • Jiang Z.
      • Gandham V.
      • Friedman B.A.
      • Ngu H.
      • Foreman O.
      • Carano R.A.D.
      • Chih B.
      • Klumperman J.
      • Bakalarski C.
      • et al.
      Changes in the synaptic proteome in tauopathy and rescue of tau-induced synapse loss by C1q antibodies.
      ).
      Thus, complement-mediated synaptic pruning likely represents a general mechanism that plays critical roles in both physiological processes (e.g., synaptic refinement during development and structural remodeling during synaptic plasticity and memory formation in mature brain) and the pathogenesis of various psychiatric and neurological diseases (e.g., schizophrenia, AD, glaucoma).

      Regulation of axonal growth

      The complement system can both promote and suppress axonal growth, depending on the specific complement factors involved (Fig. 2). The initiating factor of the classical cascade, C1q, was shown to stimulate axonal growth both in vitro and in vivo through noncanonical mechanisms, independent of classical pathway activation (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.
      ,
      • Benoit M.E.
      • Tenner A.J.
      Complement protein C1q-mediated neuroprotection is correlated with regulation of neuronal gene and microRNA expression.
      ). For example, C1q was shown to promote neurite outgrowth in rat primary cortical neurons by upregulating expression of nerve growth factor (NGF) and neurotrophin 3 (NT3) (
      • Benoit M.E.
      • Tenner A.J.
      Complement protein C1q-mediated neuroprotection is correlated with regulation of neuronal gene and microRNA expression.
      ) (Fig. 2). In addition, C1q can promote axonal growth through its direct interaction with myelin-associated glycoprotein (MAG), which belongs to a class of proteins known as myelin-associated inhibitors of axonal growth (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.
      ,
      • Chaudhry N.
      • Filbin M.T.
      Myelin-associated inhibitory signaling and strategies to overcome inhibition.
      ). This interaction blocks the growth inhibitory signaling of MAG, thereby promoting cytoskeletal rearrangement and neurite outgrowth (Fig. 2). The described C1q-dependent mechanism is especially important for supporting axonal regeneration following spinal cord injury (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.
      ) (Fig. 3).
      Figure thumbnail gr3
      Figure 3The complement system orchestrates cellular responses to injury in the peripheral nervous system. After injury, inflammation, or infection, activation of the complement system contributes to nociceptor sensitization and wound healing. In the case of peripheral nerve damage, activated components of the complement system (red) stimulate immune cells and promote nerve regrowth. Damaged nerves also release myelin-associated glycoprotein (MAG), which can inhibit regrowth of sensory axons. C5a/C3a-dependent recruitment of macrophages helps to clear MAG and to inhibit its action through binding of C1q, which promotes axonal regrowth. C3b, when deposited on nerves, can inhibit regrowth, but is typically inactivated by DAF (CD55) under physiological conditions. The soluble complement factors C3a and C5a act through their receptors, C3aR and C5aR1, respectively, to recruit and activate immune cells. This causes Ca2+-dependent release of numerous inflammatory mediators (e.g., NGF, PGE2, CGRP), which can sensitize nociceptive neurons by modulating TRPV1, tetrodotoxin-resistant (TTX-R) voltage-gated Na+ channels, and other ion channels and receptors through various mechanisms (
      • Zhang X.
      • Huang J.
      • McNaughton P.A.
      NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels.
      ,
      • Chuang H.H.
      • Prescott E.D.
      • Kong H.
      • Shields S.
      • Jordt S.E.
      • Basbaum A.I.
      • Chao M.V.
      • Julius D.
      Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition.
      ,
      • Zhu W.
      • Oxford G.S.
      Phosphoinositide-3-kinase and mitogen activated protein kinase signaling pathways mediate acute NGF sensitization of TRPV1.
      ,
      • Natura G.
      • von Banchet G.S.
      • Schaible H.G.
      Calcitonin gene-related peptide enhances TTX-resistant sodium currents in cultured dorsal root ganglion neurons from adult rats.
      ).
      In contrast, C3 was shown to inhibit axonal growth, as revealed by examining the effects of C3 deletion on axonal regeneration (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C3 suppresses axon growth and promotes neuron loss.
      ). In this study, the regeneration of sensory axons after spinal cord injury was 2-fold higher in mice lacking C3 than in wild-type counterparts. Furthermore, in vitro examination suggested that this effect was mediated by the C3 split product, C3b, by either causing neuronal toxicity or inhibiting neural adhesion (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C3 suppresses axon growth and promotes neuron loss.
      ). Notably, the other split product of C3, C3a, mildly promoted, rather than inhibited, neurite outgrowth in cultured cortical neurons (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C3 suppresses axon growth and promotes neuron loss.
      ) (Fig. 2). As discussed below, preferential activation of a growth-promoting versus growth-inhibiting mechanism can profoundly affect healing processes and normal pain resolution following injury (Fig. 3).

      Regulation of neuroinflammation: the roles of C3a and C5a

      Within the CNS, complement activation is commonly found alongside neuropathology and inflammation (
      • Lee J.D.
      • Coulthard L.G.
      • Woodruff T.M.
      Complement dysregulation in the central nervous system during development and disease.
      ), and is accompanied by robust generation of the inflammatory peptides C3a and C5a (Fig. 2). Through their conjugate receptors, C3aR and C5aR1/2, these peptides can drive both glial and neuronal dysregulation in a number of diseases including stroke (
      • Lee J.D.
      • Coulthard L.G.
      • Woodruff T.M.
      Complement dysregulation in the central nervous system during development and disease.
      ,
      • Pavlovski D.
      • Thundyil J.
      • Monk P.N.
      • Wetsel R.A.
      • Taylor S.M.
      • Woodruff T.M.
      Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis.
      ,
      • Arumugam T.V.
      • Tang S.C.
      • Lathia J.D.
      • Cheng A.
      • Mughal M.R.
      • Chigurupati S.
      • Magnus T.
      • Chan S.L.
      • Jo D.G.
      • Ouyang X.
      • Fairlie D.P.
      • Granger D.N.
      • Vortmeyer A.
      • Basta M.
      • Mattson M.P.
      Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing complement-mediated neuronal cell death.
      ,
      • Rahpeymai Y.
      • Hietala M.A.
      • Wilhelmsson U.
      • Fotheringham A.
      • Davies I.
      • Nilsson A.K.
      • Zwirner J.
      • Wetsel R.A.
      • Gerard C.
      • Pekny M.
      • Pekna M.
      Complement: A novel factor in basal and ischemia-induced neurogenesis.
      ,
      • Stokowska A.
      • Atkins A.L.
      • Moran J.
      • Pekny T.
      • Bulmer L.
      • Pascoe M.C.
      • Barnum S.R.
      • Wetsel R.A.
      • Nilsson J.A.
      • Dragunow M.
      • Pekna M.
      Complement peptide C3a stimulates neural plasticity after experimental brain ischaemia.
      ), spinal cord injury (
      • Brennan F.H.
      • Gordon R.
      • Lao H.W.
      • Biggins P.J.
      • Taylor S.M.
      • Franklin R.J.
      • Woodruff T.M.
      • Ruitenberg M.J.
      The complement receptor C5aR controls acute inflammation and astrogliosis following spinal cord injury.
      ,
      • Biggins P.J.C.
      • Brennan F.H.
      • Taylor S.M.
      • Woodruff T.M.
      • Ruitenberg M.J.
      The alternative receptor for complement component 5a, C5aR2, conveys neuroprotection in traumatic spinal cord injury.
      ), AD (
      • Tenner A.J.
      Complement-mediated events in Alzheimer's disease: Mechanisms and potential therapeutic targets.
      ,
      • Maier M.
      • Peng Y.
      • Jiang L.
      • Seabrook T.J.
      • Carroll M.C.
      • Lemere C.A.
      Complement C3 deficiency leads to accelerated amyloid beta plaque deposition and neurodegeneration and modulation of the microglia/macrophage phenotype in amyloid precursor protein transgenic mice.
      ,
      • Wyss-Coray T.
      • Yan F.
      • Lin A.H.
      • Lambris J.D.
      • Alexander J.J.
      • Quigg R.J.
      • Masliah E.
      Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice.
      ), and ALS (
      • Woodruff T.M.
      • Costantini K.J.
      • Crane J.W.
      • Atkin J.D.
      • Monk P.N.
      • Taylor S.M.
      • Noakes P.G.
      The complement factor C5a contributes to pathology in a rat model of amyotrophic lateral sclerosis.
      ,
      • Woodruff T.M.
      • Lee J.D.
      • Noakes P.G.
      Role for terminal complement activation in amyotrophic lateral sclerosis disease progression.
      ,
      • Mantovani S.
      • Gordon R.
      • Macmaw J.K.
      • Pfluger C.M.
      • Henderson R.D.
      • Noakes P.G.
      • McCombe P.A.
      • Woodruff T.M.
      Elevation of the terminal complement activation products C5a and C5b-9 in ALS patient blood.
      ,
      • Lee J.D.
      • Kumar V.
      • Fung J.N.
      • Ruitenberg M.J.
      • Noakes P.G.
      • Woodruff T.M.
      Pharmacological inhibition of complement C5a-C5a(1) receptor signalling ameliorates disease pathology in the hSOD1(G93A) mouse model of amyotrophic lateral sclerosis.
      ). In most cases, elevated levels of the inactive zymogens C3 and C5 are largely attributed to their production by glia, which subsequently leads to the formation of the small soluble fragments, C3a and C5a by the respective convertases (
      • Brennan F.H.
      • Anderson A.J.
      • Taylor S.M.
      • Woodruff T.M.
      • Ruitenberg M.J.
      Complement activation in the injured central nervous system: Another dual-edged sword?.
      ,
      • Morgan B.P.
      Complement in the pathogenesis of Alzheimer's disease.
      ). Once formed, C3a and C5a activate their cognate receptors, which are expressed on both glia and neurons, though generally at higher levels on glia. Activation of C3aR and C5aR1 initiates a variety of effects, including chemotaxis of immune and glial cells, production and release of inflammatory factors in a Ca2+-dependent manner, and stimulation of phagocytosis (
      • Ricklin D.
      • Hajishengallis G.
      • Yang K.
      • Lambris J.D.
      Complement: A key system for immune surveillance and homeostasis.
      ,
      • Quadros A.U.
      • Cunha T.M.
      C5a and pain development: An old molecule, a new target.
      ,
      • Klos A.
      • Wende E.
      • Wareham K.J.
      • Monk P.N.
      International union of basic and clinical pharmacology. [corrected]. LXXXVII. Complement peptide C5a, C4a, and C3a receptors.
      ,
      • Tenner A.J.
      Complement-mediated events in Alzheimer's disease: Mechanisms and potential therapeutic targets.
      ). Levels of C3a and C5a are significantly elevated in the plasma after stroke (
      • Mocco J.
      • Wilson D.A.
      • Komotar R.J.
      • Sughrue M.E.
      • Coates K.
      • Sacco R.L.
      • Elkind M.S.
      • Connolly Jr., E.S.
      Alterations in plasma complement levels after human ischemic stroke.
      ), and inhibition or genetic ablation of C5a/C5aR1 signaling significantly reduced the infarct volume and functional deficits after stroke (
      • Pavlovski D.
      • Thundyil J.
      • Monk P.N.
      • Wetsel R.A.
      • Taylor S.M.
      • Woodruff T.M.
      Generation of complement component C5a by ischemic neurons promotes neuronal apoptosis.
      ,
      • Arumugam T.V.
      • Tang S.C.
      • Lathia J.D.
      • Cheng A.
      • Mughal M.R.
      • Chigurupati S.
      • Magnus T.
      • Chan S.L.
      • Jo D.G.
      • Ouyang X.
      • Fairlie D.P.
      • Granger D.N.
      • Vortmeyer A.
      • Basta M.
      • Mattson M.P.
      Intravenous immunoglobulin (IVIG) protects the brain against experimental stroke by preventing complement-mediated neuronal cell death.
      ). Similarly in ALS patients, plasma and leukocyte levels of C5a are increased relative to healthy individuals (
      • Mantovani S.
      • Gordon R.
      • Macmaw J.K.
      • Pfluger C.M.
      • Henderson R.D.
      • Noakes P.G.
      • McCombe P.A.
      • Woodruff T.M.
      Elevation of the terminal complement activation products C5a and C5b-9 in ALS patient blood.
      ). In addition, levels of complement inhibitors DAF (CD55) and protectin (CD59) were altered at the endplates of motor neurons in ALS patients (
      • Bahia El Idrissi N.
      • Bosch S.
      • Ramaglia V.
      • Aronica E.
      • Baas F.
      • Troost D.
      Complement activation at the motor end-plates in amyotrophic lateral sclerosis.
      ). The role of complement in ALS was also supported by the findings that in mouse models of ALS, genetic or pharmacologic inhibition of C5aR1 delayed disease progression (
      • Woodruff T.M.
      • Costantini K.J.
      • Crane J.W.
      • Atkin J.D.
      • Monk P.N.
      • Taylor S.M.
      • Noakes P.G.
      The complement factor C5a contributes to pathology in a rat model of amyotrophic lateral sclerosis.
      ,
      • Woodruff T.M.
      • Lee J.D.
      • Noakes P.G.
      Role for terminal complement activation in amyotrophic lateral sclerosis disease progression.
      ,
      • Lee J.D.
      • Kumar V.
      • Fung J.N.
      • Ruitenberg M.J.
      • Noakes P.G.
      • Woodruff T.M.
      Pharmacological inhibition of complement C5a-C5a(1) receptor signalling ameliorates disease pathology in the hSOD1(G93A) mouse model of amyotrophic lateral sclerosis.
      ) and that complement inhibitors DAF (CD55) and protectin (CD59) were downregulated in ALS mouse models (
      • Lee J.D.
      • Kamaruzaman N.A.
      • Fung J.N.T.
      • Taylor S.M.
      • Turner B.J.
      • Atkin J.D.
      • Woodruff T.M.
      • Noakes P.G.
      Dysregulation of the complement cascade in the hSOD1G93Atransgenic mouse model of amyotrophic lateral sclerosis.
      ,
      • Lee J.D.
      • Levin S.C.
      • Willis E.F.
      • Li R.
      • Woodruff T.M.
      • Noakes P.G.
      Complement components are upregulated and correlate with disease progression in the TDP-43(Q331K) mouse model of amyotrophic lateral sclerosis.
      ). C3 and C3aR are also increased in the brain of AD patients as well as in the mouse brain of an AD model (
      • Litvinchuk A.
      • Wan Y.W.
      • Swartzlander D.B.
      • Chen F.
      • Cole A.
      • Propson N.E.
      • Wang Q.
      • Zhang B.
      • Liu Z.
      • Zheng H.
      Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer's disease.
      ). Mechanistically, C3a/C3aR signaling was shown to induce microglia activation via STAT3-dependent transcriptional program, which in turn drives tau pathogenesis in AD mice (
      • Litvinchuk A.
      • Wan Y.W.
      • Swartzlander D.B.
      • Chen F.
      • Cole A.
      • Propson N.E.
      • Wang Q.
      • Zhang B.
      • Liu Z.
      • Zheng H.
      Complement C3aR inactivation attenuates tau pathology and reverses an immune network deregulated in tauopathy models and Alzheimer's disease.
      ). Intriguingly, it was also noted by the same group that C3aR expression and activation on neurons were also capable of driving Aβ production (
      • Lian H.
      • Litvinchuk A.
      • Chiang A.C.
      • Aithmitti N.
      • Jankowsky J.L.
      • Zheng H.
      Astrocyte-microglia cross talk through complement activation modulates amyloid pathology in mouse models of Alzheimer's disease.
      ), underscoring the functional importance of C3aR on both neurons and microglia to the development of AD.
      However, inhibition of C3aR or C5aR1 signaling is not always disease modifying or palliative, as both C3a and C5a can play roles in tissue repair. For example, intranasal administration of C3a drives neurogenesis during recovery from stroke (
      • Rahpeymai Y.
      • Hietala M.A.
      • Wilhelmsson U.
      • Fotheringham A.
      • Davies I.
      • Nilsson A.K.
      • Zwirner J.
      • Wetsel R.A.
      • Gerard C.
      • Pekny M.
      • Pekna M.
      Complement: A novel factor in basal and ischemia-induced neurogenesis.
      ,
      • Stokowska A.
      • Atkins A.L.
      • Moran J.
      • Pekny T.
      • Bulmer L.
      • Pascoe M.C.
      • Barnum S.R.
      • Wetsel R.A.
      • Nilsson J.A.
      • Dragunow M.
      • Pekna M.
      Complement peptide C3a stimulates neural plasticity after experimental brain ischaemia.
      ), and a higher C3a:C3 ratio in the serum (indicating enhanced convertase activity) correlates with more positive outcomes following cardioembolic strokes (
      • Stokowska A.
      • Olsson S.
      • Holmegaard L.
      • Jood K.
      • Blomstrand C.
      • Jern C.
      • Pekna M.
      Cardioembolic and small vessel disease stroke show differences in associations between systemic C3 levels and outcome.
      ). Similarly, in murine models of AD, inhibition or genetic knockout of C3 worsens AD pathology, which is characterized by higher levels of Aβ deposition and neuronal death that in complement sufficient animals (
      • Maier M.
      • Peng Y.
      • Jiang L.
      • Seabrook T.J.
      • Carroll M.C.
      • Lemere C.A.
      Complement C3 deficiency leads to accelerated amyloid beta plaque deposition and neurodegeneration and modulation of the microglia/macrophage phenotype in amyloid precursor protein transgenic mice.
      • Wyss-Coray T.
      • Yan F.
      • Lin A.H.
      • Lambris J.D.
      • Alexander J.J.
      • Quigg R.J.
      • Masliah E.
      Prominent neurodegeneration and increased plaque formation in complement-inhibited Alzheimer's mice.
      ). Finally, in models of spinal cord injury, genetic or pharmacological blockage of C5aR1 signaling enhanced recovery during the acute phase (<7 days) by reducing microglia-mediated inflammation and cytokine release. However, chronic or later blockade of C5aR1, or C5aR1 genetic deletion, appears to be detrimental to full recovery after injury (
      • Brennan F.H.
      • Gordon R.
      • Lao H.W.
      • Biggins P.J.
      • Taylor S.M.
      • Franklin R.J.
      • Woodruff T.M.
      • Ruitenberg M.J.
      The complement receptor C5aR controls acute inflammation and astrogliosis following spinal cord injury.
      ,
      • Beck K.D.
      • Nguyen H.X.
      • Galvan M.D.
      • Salazar D.L.
      • Woodruff T.M.
      • Anderson A.J.
      Quantitative analysis of cellular inflammation after traumatic spinal cord injury: Evidence for a multiphasic inflammatory response in the acute to chronic environment.
      ). These apparently contrasting effects for C3a and C5a signaling illustrate the overall dual roles of the complement system in neurological disorders and CNS/PNS injury, where the same complement factor can either exacerbate neuronal damage or stimulate the recovery process, depending on both the type and stage of a particular neuropathological condition.

      TPCC/MAC-mediated neuronal toxicity

      Unlike many typical signaling mechanisms including those of C3a and C5a, the activity of the TPCC/MAC (
      • Kemper C.
      • Pangburn M.K.
      • Fishelson Z.
      Complement nomenclature 2014.
      ) can occur in the absence of a conjugate receptor on the affected cell (Fig. 2). This unusual property enables the TPCC to form a large lytic pore on any lipid structure of a host cell or pathogen, where it can cause cytolytic destruction (
      • Hess C.
      • Kemper C.
      Complement-mediated regulation of metabolism and basic cellular processes.
      ,
      • Xie C.B.
      • Jane-Wit D.
      • Pober J.S.
      Complement membrane attack complex: New roles, mechanisms of action, and therapeutic targets.
      ). In addition, the TPCC can play a signaling role when conditions are sublytic (i.e., a pore is formed but the cell has not lysed), causing an increase in the release of proinflammatory cytokines (
      • Kilgore K.S.
      • Schmid E.
      • Shanley T.P.
      • Flory C.M.
      • Maheswari V.
      • Tramontini N.L.
      • Cohen H.
      • Ward P.A.
      • Friedl H.P.
      • Warren J.S.
      Sublytic concentrations of the membrane attack complex of complement induce endothelial interleukin-8 and monocyte chemoattractant protein-1 through nuclear factor-kappa B activation.
      ,
      • Jane-Wit D.
      • Manes T.D.
      • Yi T.
      • Qin L.
      • Clark P.
      • Kirkiles-Smith N.C.
      • Abrahimi P.
      • Devalliere J.
      • Moeckel G.
      • Kulkarni S.
      • Tellides G.
      • Pober J.S.
      Alloantibody and complement promote T cell-mediated cardiac allograft vasculopathy through noncanonical nuclear factor-κB signaling in endothelial cells.
      ) through a variety of mechanisms, both Ca2+-dependent and independent (
      • Xie C.B.
      • Jane-Wit D.
      • Pober J.S.
      Complement membrane attack complex: New roles, mechanisms of action, and therapeutic targets.
      ). However, in the context of neuronal toxicity, the lytic function of the TPCC is suspected to be the primary cause of neuronal cell death. This is the case in a variety of neurological disorders including stroke (
      • Széplaki G.
      • Szegedi R.
      • Hirschberg K.
      • Gombos T.
      • Varga L.
      • Karádi I.
      • Entz L.
      • Széplaki Z.
      • Garred P.
      • Prohászka Z.
      • Füst G.
      Strong complement activation after acute ischemic stroke is associated with unfavorable outcomes.
      ,
      • Pedersen E.D.
      • Løberg E.M.
      • Vege E.
      • Daha M.R.
      • Maehlen J.
      • Mollnes T.E.
      In situ deposition of complement in human acute brain ischaemia.
      ,
      • Pedersen E.D.
      • Waje-Andreassen U.
      • Vedeler C.A.
      • Aamodt G.
      • Mollnes T.E.
      Systemic complement activation following human acute ischaemic stroke.
      ,
      • Harhausen D.
      • Khojasteh U.
      • Stahel P.F.
      • Morgan B.P.
      • Nietfeld W.
      • Dirnagl U.
      • Trendelenburg G.
      Membrane attack complex inhibitor CD59a protects against focal cerebral ischemia in mice.
      ), traumatic brain injury (TBI) (
      • Bellander B.M.
      • Singhrao S.K.
      • Ohlsson M.
      • Mattsson P.
      • Svensson M.
      Complement activation in the human brain after traumatic head injury.
      ,
      • Kossmann T.
      • Stahel P.F.
      • Morganti-Kossmann M.C.
      • Jones J.L.
      • Barnum S.R.
      Elevated levels of the complement components C3 and factor B in ventricular cerebrospinal fluid of patients with traumatic brain injury.
      ,
      • Stahel P.F.
      • Morganti-Kossmann M.C.
      • Perez D.
      • Redaelli C.
      • Gloor B.
      • Trentz O.
      • Kossmann T.
      Intrathecal levels of complement-derived soluble membrane attack complex (sC5b-9) correlate with blood-brain barrier dysfunction in patients with traumatic brain injury.
      ), spinal cord injury (
      • Liu L.
      • Tornqvist E.
      • Mattsson P.
      • Eriksson N.P.
      • Persson J.K.
      • Morgan B.P.
      • Aldskogius H.
      • Svensson M.
      Complement and clusterin in the spinal cord dorsal horn and gracile nucleus following sciatic nerve injury in the adult rat.
      ,
      • Qiao F.
      • Atkinson C.
      • Kindy M.S.
      • Shunmugavel A.
      • Morgan B.P.
      • Song H.
      • Tomlinson S.
      The alternative and terminal pathways of complement mediate post-traumatic spinal cord inflammation and injury.
      ,
      • Anderson A.J.
      • Robert S.
      • Huang W.
      • Young W.
      • Cotman C.W.
      Activation of complement pathways after contusion-induced spinal cord injury.
      ), ALS (
      • Woodruff T.M.
      • Lee J.D.
      • Noakes P.G.
      Role for terminal complement activation in amyotrophic lateral sclerosis disease progression.
      ,
      • Lee J.D.
      • Kumar V.
      • Fung J.N.
      • Ruitenberg M.J.
      • Noakes P.G.
      • Woodruff T.M.
      Pharmacological inhibition of complement C5a-C5a(1) receptor signalling ameliorates disease pathology in the hSOD1(G93A) mouse model of amyotrophic lateral sclerosis.
      ,
      • Bahia El Idrissi N.
      • Bosch S.
      • Ramaglia V.
      • Aronica E.
      • Baas F.
      • Troost D.
      Complement activation at the motor end-plates in amyotrophic lateral sclerosis.
      ,
      • Lee J.D.
      • Kamaruzaman N.A.
      • Fung J.N.T.
      • Taylor S.M.
      • Turner B.J.
      • Atkin J.D.
      • Woodruff T.M.
      • Noakes P.G.
      Dysregulation of the complement cascade in the hSOD1G93Atransgenic mouse model of amyotrophic lateral sclerosis.
      ,
      • Lee J.D.
      • Levin S.C.
      • Willis E.F.
      • Li R.
      • Woodruff T.M.
      • Noakes P.G.
      Complement components are upregulated and correlate with disease progression in the TDP-43(Q331K) mouse model of amyotrophic lateral sclerosis.
      ,
      • Bahia El Idrissi N.
      • Fluiter K.
      • Vieira F.
      • Baas F.
      Complement component C6 inhibition decreases neurological disability in female transgenic SOD1G93A mouse model of Amyotrophic Lateral Sclerosis.
      ), and neuropathic pain (
      • Xu J.
      • Zhang L.
      • Xie M.
      • Li Y.
      • Huang P.
      • Saunders T.L.
      • Fox D.A.
      • Rosenquist R.
      • Lin F.
      Role of complement in a rat model of paclitaxel-induced peripheral neuropathy.
      ).
      Many pathologies are characterized by elevated deposition of the TPCC in affected tissues. For example, in ALS, TPCC levels are increased at the end plate of motor neurons (
      • Sta M.
      • Sylva-Steenland R.M.
      • Casula M.
      • de Jong J.M.
      • Troost D.
      • Aronica E.
      • Baas F.
      Innate and adaptive immunity in amyotrophic lateral sclerosis: Evidence of complement activation.
      ,
      • Humayun S.
      • Gohar M.
      • Volkening K.
      • Moisse K.
      • Leystra-Lantz C.
      • Mepham J.
      • McLean J.
      • Strong M.J.
      The complement factor C5a receptor is upregulated in NFL-/- mouse motor neurons.
      ). In spinal cord injury, TPCCs surround the primary site of injury (
      • Liu L.
      • Tornqvist E.
      • Mattsson P.
      • Eriksson N.P.
      • Persson J.K.
      • Morgan B.P.
      • Aldskogius H.
      • Svensson M.
      Complement and clusterin in the spinal cord dorsal horn and gracile nucleus following sciatic nerve injury in the adult rat.
      ,
      • Anderson A.J.
      • Robert S.
      • Huang W.
      • Young W.
      • Cotman C.W.
      Activation of complement pathways after contusion-induced spinal cord injury.
      ). In TBI, TPCC insertion is positively correlated with the disruption of blood–brain barrier after the injury (
      • Bellander B.M.
      • Singhrao S.K.
      • Ohlsson M.
      • Mattsson P.
      • Svensson M.
      Complement activation in the human brain after traumatic head injury.
      ,
      • Kossmann T.
      • Stahel P.F.
      • Morganti-Kossmann M.C.
      • Jones J.L.
      • Barnum S.R.
      Elevated levels of the complement components C3 and factor B in ventricular cerebrospinal fluid of patients with traumatic brain injury.
      ,
      • Stahel P.F.
      • Morganti-Kossmann M.C.
      • Perez D.
      • Redaelli C.
      • Gloor B.
      • Trentz O.
      • Kossmann T.
      Intrathecal levels of complement-derived soluble membrane attack complex (sC5b-9) correlate with blood-brain barrier dysfunction in patients with traumatic brain injury.
      ). This increased deposition of TPCC in various neuropathologies is likely driven by poor regulation of the formation and insertion of the complex. Indeed, the ubiquitously expressed inhibitor of TPCC formation, protectin (CD59), is downregulated in ALS patients (
      • Lee J.D.
      • Kamaruzaman N.A.
      • Fung J.N.T.
      • Taylor S.M.
      • Turner B.J.
      • Atkin J.D.
      • Woodruff T.M.
      • Noakes P.G.
      Dysregulation of the complement cascade in the hSOD1G93Atransgenic mouse model of amyotrophic lateral sclerosis.
      ,
      • Lee J.D.
      • Levin S.C.
      • Willis E.F.
      • Li R.
      • Woodruff T.M.
      • Noakes P.G.
      Complement components are upregulated and correlate with disease progression in the TDP-43(Q331K) mouse model of amyotrophic lateral sclerosis.
      ), and its genetic knockout impairs recovery in rodent models of stroke (
      • Harhausen D.
      • Khojasteh U.
      • Stahel P.F.
      • Morgan B.P.
      • Nietfeld W.
      • Dirnagl U.
      • Trendelenburg G.
      Membrane attack complex inhibitor CD59a protects against focal cerebral ischemia in mice.
      ) and spinal cord injury (
      • Qiao F.
      • Atkinson C.
      • Kindy M.S.
      • Shunmugavel A.
      • Morgan B.P.
      • Song H.
      • Tomlinson S.
      The alternative and terminal pathways of complement mediate post-traumatic spinal cord inflammation and injury.
      ). Although these various pathologies are defined by different types of neurological trauma or neurodegeneration, a common hallmark of these diseases is the contribution of excessive TPCC activity to neuronal death, due to either excessive activation of the TPCC or dysregulation of the inhibitory mechanisms (Fig. 2).

      Mechanisms underlying complement action in tissue injury and pain

      Tissue injury is commonly associated with an amplified pain sensation in response to a noxious stimulation (hyperalgesia) and/or an innocuous stimulation (allodynia) at the affected site. This sensitized reaction represents a fundamental adaptive mechanism that helps to protect injured tissue and assists in wound healing. However, chronic sensitization is deleterious and serves no physiological function. Peripherally, after an injury there is a complex interplay between the immune cells and nociceptive neurons (Fig. 3) to promote healing and facilitate behavioral guarding of the wound. Complement assists in both these aspects by recruiting and activating immune cells (e.g., macrophages) to fight infection and clear debris, while at the same time stimulating these cells to release inflammatory factors that sensitize neurons to promote behavioral guarding until the wound has healed. However, the persistent or unbalanced signaling of complement factors during chronic pain points to a potential role for complement in the maladaptive mechanisms of peripherally driven chronic pain. Numerous studies have highlighted the critical roles of the complement cascade in various chronic pain conditions. Indeed, elevated levels of many key complement factors (e.g., C3a, C5, and C5a) have been reported in patients with various pathological conditions associated with pain, including osteoarthritis, rheumatoid arthritis, pancreatitis, burns, and surgical trauma (
      • Wang Q.
      • Rozelle A.L.
      • Lepus C.M.
      • Scanzello C.R.
      • Song J.J.
      • Larsen D.M.
      • Crish J.F.
      • Bebek G.
      • Ritter S.Y.
      • Lindstrom T.M.
      • Hwang I.
      • Wong H.H.
      • Punzi L.
      • Encarnacion A.
      • Shamloo M.
      • et al.
      Identification of a central role for complement in osteoarthritis.
      ,
      • Jose P.J.
      • Moss I.K.
      • Maini R.N.
      • Williams T.J.
      Measurement of the chemotactic complement fragment C5a in rheumatoid synovial fluids by radioimmunoassay: Role of C5a in the acute inflammatory phase.
      ,
      • Roxvall L.
      • Bengtson A.
      • Heideman M.
      Anaphylatoxin generation in acute pancreatitis.
      ,
      • Bengtsson A.
      • Bengtson J.P.
      • Rydenhag A.
      • Roxvall L.
      • Heideman M.
      Accumulation of anaphylatoxins and terminal complement complexes in inflammatory fluids.
      ,
      • Fosse E.
      • Mollnes T.E.
      • Ingvaldsen B.
      Complement activation during major operations with or without cardiopulmonary bypass.
      ,
      • Kiener H.P.
      • Baghestanian M.
      • Dominkus M.
      • Walchshofer S.
      • Ghannadan M.
      • Willheim M.
      • Sillaber C.
      • Graninger W.B.
      • Smolen J.S.
      • Valent P.
      Expression of the C5a receptor (CD88) on synovial mast cells in patients with rheumatoid arthritis.
      ,
      • Chello M.
      • Mastroroberto P.
      • Romano R.
      • Ascione R.
      • Pantaleo D.
      • De Amicis V.
      Complement and neutrophil activation during cardiopulmonary bypass: A randomized comparison of hypothermic and normothermic circulation.
      ). Similarly, increased production of complement factors C3, C5, and C5a was reported in several rodent models of acute and chronic pain, including models of postsurgical pain, cancer pain, and neuropathic pain (
      • Jang J.H.
      • Liang D.
      • Kido K.
      • Sun Y.
      • Clark D.J.
      • Brennan T.J.
      Increased local concentration of complement C5a contributes to incisional pain in mice.
      ,
      • Levin M.E.
      • Jin J.G.
      • Ji R.R.
      • Tong J.
      • Pomonis J.D.
      • Lavery D.J.
      • Miller S.W.
      • Chiang L.W.
      Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain.
      ,
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ,
      • Huang X.
      • Li J.
      • Xie J.
      • Li Y.
      • Gao Y.
      • Li X.
      • Xu X.
      • Shi R.
      • Yao W.
      • Ke C.
      Neuronal complement cascade drives bone cancer pain via C3R mediated microglial activation.
      ), while genetic deletion of C3 and C5 as well as pharmacological inhibition of C3aR and C5aR1 produced significant analgesic effects in these models (
      • Xu J.
      • Zhang L.
      • Xie M.
      • Li Y.
      • Huang P.
      • Saunders T.L.
      • Fox D.A.
      • Rosenquist R.
      • Lin F.
      Role of complement in a rat model of paclitaxel-induced peripheral neuropathy.
      ,
      • Liang D.Y.
      • Li X.
      • Shi X.
      • Sun Y.
      • Sahbaie P.
      • Li W.W.
      • Clark J.D.
      The complement component C5a receptor mediates pain and inflammation in a postsurgical pain model.
      ,
      • Clark J.D.
      • Qiao Y.
      • Li X.
      • Shi X.
      • Angst M.S.
      • Yeomans D.C.
      Blockade of the complement C5a receptor reduces incisional allodynia, edema, and cytokine expression.
      ,
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ,
      • Doolen S.
      • Cook J.
      • Riedl M.
      • Kitto K.
      • Kohsaka S.
      • Honda C.N.
      • Fairbanks C.A.
      • Taylor B.K.
      • Vulchanova L.
      Complement 3a receptor in dorsal horn microglia mediates pronociceptive neuropeptide signaling.
      ,
      • Moriconi A.
      • Cunha T.M.
      • Souza G.R.
      • Lopes A.H.
      • Cunha F.Q.
      • Carneiro V.L.
      • Pinto L.G.
      • Brandolini L.
      • Aramini A.
      • Bizzarri C.
      • Bianchini G.
      • Beccari A.R.
      • Fanton M.
      • Bruno A.
      • Costantino G.
      • et al.
      Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief.
      ). Furthermore, a meta-analysis of 20 microarray studies that examined changes in gene expression in various chronic pain models in rodents found that complement was one of the most commonly and highest regulated categories of genes upregulated after induction of both neuropathic (i.e., induced by peripheral nerve injury) and inflammatory (i.e., induced by tissue injury) pain (
      • LaCroix-Fralish M.L.
      • Austin J.S.
      • Zheng F.Y.
      • Levitin D.J.
      • Mogil J.S.
      Patterns of pain: meta-analysis of microarray studies of pain.
      ). Below, we discuss the major mechanisms that underlie the contribution of the complement system to tissue injury and pain.

      Sensitization of nociceptive neurons

      Activation of the complement system in peripheral tissues triggers the production and release of numerous inflammatory factors that can significantly modify neuronal excitability. The main soluble effectors of the complement cascade that drive inflammatory responses are C3a and C5a. These peptides have profound effects on a wide array of physiological systems that promote inflammatory responses to infection or injury. Indeed, direct application of C5a to an ex vivo skin–nerve preparation sensitized primary afferent fibers to thermal stimulation and also caused spontaneous firing of action potentials (
      • Jang J.H.
      • Clark J.D.
      • Li X.
      • Yorek M.S.
      • Usachev Y.M.
      • Brennan T.J.
      Nociceptive sensitization by complement C5a and C3a in mouse.
      ,
      • Jang J.H.
      • Liang D.
      • Kido K.
      • Sun Y.
      • Clark D.J.
      • Brennan T.J.
      Increased local concentration of complement C5a contributes to incisional pain in mice.
      ). Similar effects were induced by C3a, indicating that complement activity is capable of heightening sensitivity of peripheral neurons to cutaneous stimulation.
      Specific factors responsible for complement-induced sensitization have only recently begun to be unraveled and include a variety of local inflammatory mediators, which are released primarily from immune cells but also from peripheral neuronal terminals (Fig. 3). One particularly well-characterized inflammatory mediator is nerve growth factor (NGF). The levels of NGF are prominently elevated after peripheral injury (
      • Li W.W.
      • Guo T.Z.
      • Shi X.
      • Czirr E.
      • Stan T.
      • Sahbaie P.
      • Wyss-Coray T.
      • Kingery W.S.
      • Clark J.D.
      Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome.
      ,
      • Shutov L.P.
      • Warwick C.A.
      • Shi X.
      • Gnanasekaran A.
      • Shepherd A.J.
      • Mohapatra D.P.
      • Woodruff T.M.
      • Clark J.D.
      • Usachev Y.M.
      The complement system component C5a produces thermal hyperalgesia via macrophage-to-nociceptor signaling that requires NGF and TRPV1.
      ,
      • Liang D.Y.
      • Li X.
      • Shi X.
      • Sun Y.
      • Sahbaie P.
      • Li W.W.
      • Clark J.D.
      The complement component C5a receptor mediates pain and inflammation in a postsurgical pain model.
      ), concomitant with the expression of complement factors (
      • Jang J.H.
      • Liang D.
      • Kido K.
      • Sun Y.
      • Clark D.J.
      • Brennan T.J.
      Increased local concentration of complement C5a contributes to incisional pain in mice.
      ). Subcutaneous NGF can also be increased by injecting activated complement fragments such as C5a (
      • Shutov L.P.
      • Warwick C.A.
      • Shi X.
      • Gnanasekaran A.
      • Shepherd A.J.
      • Mohapatra D.P.
      • Woodruff T.M.
      • Clark J.D.
      • Usachev Y.M.
      The complement system component C5a produces thermal hyperalgesia via macrophage-to-nociceptor signaling that requires NGF and TRPV1.
      ). NGF promotes sensitization of nociceptive fibers primarily through signaling of the tropomyosin-related kinase A (TrkA) receptor expressed on the peripheral terminals of the afferents. In particular, NGF/TrkA-mediated signaling stimulates plasma membrane insertion of the polymodal nociceptive ion channel TRPV1 and also sensitizes the channel to noxious heat, acidic pH, and some endogenous lipid-derived ligands (
      • Zhang X.
      • Huang J.
      • McNaughton P.A.
      NGF rapidly increases membrane expression of TRPV1 heat-gated ion channels.
      ,
      • Chuang H.H.
      • Prescott E.D.
      • Kong H.
      • Shields S.
      • Jordt S.E.
      • Basbaum A.I.
      • Chao M.V.
      • Julius D.
      Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition.
      ,
      • Zhu W.
      • Oxford G.S.
      Phosphoinositide-3-kinase and mitogen activated protein kinase signaling pathways mediate acute NGF sensitization of TRPV1.
      ). In cases when acute injury is not readily resolved, NGF can promote a transition to chronic pain through various mechanisms, including changes in the expression of nociceptive ion channels, the production of reactive oxygen species, and stimulation of peripheral axonal sprouting (
      • Eskander M.A.
      • Ruparel S.
      • Green D.P.
      • Chen P.B.
      • Por E.D.
      • Jeske N.A.
      • Gao X.
      • Flores E.R.
      • Hargreaves K.M.
      Persistent nociception triggered by nerve growth factor (NGF) is mediated by TRPV1 and oxidative mechanisms.
      ,
      • Mantyh P.W.
      • Koltzenburg M.
      • Mendell L.M.
      • Tive L.
      • Shelton D.L.
      Antagonism of nerve growth factor-TrkA signaling and the relief of pain.
      ).
      Although the primary cellular source of the NGF that is released in the periphery in response to complement activation is likely macrophages (
      • Shutov L.P.
      • Warwick C.A.
      • Shi X.
      • Gnanasekaran A.
      • Shepherd A.J.
      • Mohapatra D.P.
      • Woodruff T.M.
      • Clark J.D.
      • Usachev Y.M.
      The complement system component C5a produces thermal hyperalgesia via macrophage-to-nociceptor signaling that requires NGF and TRPV1.
      ,
      • Marcinkiewicz M.
      • Marcinkiewicz J.
      • Chen A.
      • Leclaire F.
      • Chretien M.
      • Richardson P.
      Nerve growth factor and proprotein convertases furin and PC7 in transected sciatic nerves and in nerve segments cultured in conditioned media: Their presence in Schwann cells, macrophages, and smooth muscle cells.
      ) (Fig. 3), another distinct source of complement-induced nociceptive sensitization lies within the afferent fibers themselves. For example, the pronociceptive mediator calcitonin gene-related peptide (CGRP), which is synthesized and released by peptidergic C-fibers in the setting of neurogenic inflammation (
      • Usoskin D.
      • Furlan A.
      • Islam S.
      • Abdo H.
      • Lonnerberg P.
      • Lou D.
      • Hjerling-Leffler J.
      • Haeggstrom J.
      • Kharchenko O.
      • Kharchenko P.V.
      • Linnarsson S.
      • Ernfors P.
      Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.
      ,
      • Richardson J.D.
      • Vasko M.R.
      Cellular mechanisms of neurogenic inflammation.
      ,
      • Cavanaugh D.J.
      • Chesler A.T.
      • Braz J.M.
      • Shah N.M.
      • Julius D.
      • Basbaum A.I.
      Restriction of transient receptor potential vanilloid-1 to the peptidergic subset of primary afferent neurons follows its developmental downregulation in nonpeptidergic neurons.
      ,
      • Planells-Cases R.
      • Garcìa-Sanz N.
      • Morenilla-Palao C.
      • Ferrer-Montiel A.
      Functional aspects and mechanisms of TRPV1 involvement in neurogenic inflammation that leads to thermal hyperalgesia.
      ), is responsible for the mechanical sensitization caused by the actions of C5a in the dermis (
      • Warwick C.A.
      • Shutov L.P.
      • Shepherd A.J.
      • Mohapatra D.P.
      • Usachev Y.M.
      Mechanisms underlying mechanical sensitization induced by complement C5a: The roles of macrophages, TRPV1, and calcitonin gene-related peptide receptors.
      ), and this effect may involve facilitation of tetrodotoxin-resistant voltage-gated Na+ currents (
      • Natura G.
      • von Banchet G.S.
      • Schaible H.G.
      Calcitonin gene-related peptide enhances TTX-resistant sodium currents in cultured dorsal root ganglion neurons from adult rats.
      ) (Fig. 3). In addition, levels of a pronociceptive cytokine, interleukin 1β (IL-1β), increase after injury or downstream of C5aR1 activation (
      • Li W.W.
      • Guo T.Z.
      • Shi X.
      • Czirr E.
      • Stan T.
      • Sahbaie P.
      • Wyss-Coray T.
      • Kingery W.S.
      • Clark J.D.
      Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome.
      ,
      • Liang D.Y.
      • Li X.
      • Shi X.
      • Sun Y.
      • Sahbaie P.
      • Li W.W.
      • Clark J.D.
      The complement component C5a receptor mediates pain and inflammation in a postsurgical pain model.
      ,
      • Sahbaie P.
      • Li X.
      • Shi X.
      • Clark J.D.
      Roles of Gr-1+ leukocytes in postincisional nociceptive sensitization and inflammation.
      ). IL-1β likely acts through p38 protein kinase-dependent enhancement of voltage-gated Na+ currents, to increase excitability of nociceptive neurons in various pain states (
      • Binshtok A.M.
      • Wang H.
      • Zimmermann K.
      • Amaya F.
      • Vardeh D.
      • Shi L.
      • Brenner G.J.
      • Ji R.R.
      • Bean B.P.
      • Woolf C.J.
      • Samad T.A.
      Nociceptors are interleukin-1beta sensors.
      ,
      • Dib-Hajj S.D.
      • Cummins T.R.
      • Black J.A.
      • Waxman S.G.
      Sodium channels in normal and pathological pain.
      ). Notably, a blockade of IL-1β receptor produced analgesic effects in a pain model that relies on activation of the alternative pathway (
      • Twining C.M.
      • Sloane E.M.
      • Milligan E.D.
      • Chacur M.
      • Martin D.
      • Poole S.
      • Marsh H.
      • Maier S.F.
      • Watkins L.R.
      Peri-sciatic proinflammatory cytokines, reactive oxygen species, and complement induce mirror-image neuropathic pain in rats.
      ). Similarly, inhibition of complement signaling reduces pronociceptive signaling and inflammatory factor release in various rodent pain models, including the models of inflammatory and postsurgical pain (
      • Shutov L.P.
      • Warwick C.A.
      • Shi X.
      • Gnanasekaran A.
      • Shepherd A.J.
      • Mohapatra D.P.
      • Woodruff T.M.
      • Clark J.D.
      • Usachev Y.M.
      The complement system component C5a produces thermal hyperalgesia via macrophage-to-nociceptor signaling that requires NGF and TRPV1.
      ,
      • Jang J.H.
      • Liang D.
      • Kido K.
      • Sun Y.
      • Clark D.J.
      • Brennan T.J.
      Increased local concentration of complement C5a contributes to incisional pain in mice.
      ,
      • Clark J.D.
      • Qiao Y.
      • Li X.
      • Shi X.
      • Angst M.S.
      • Yeomans D.C.
      Blockade of the complement C5a receptor reduces incisional allodynia, edema, and cytokine expression.
      ,
      • Ting E.
      • Guerrero A.T.
      • Cunha T.M.
      • Verri Jr., W.A.
      • Taylor S.M.
      • Woodruff T.M.
      • Cunha F.Q.
      • Ferreira S.H.
      Role of complement C5a in mechanical inflammatory hypernociception: Potential use of C5a receptor antagonists to control inflammatory pain.
      ). Overall, these findings suggest that the mechanisms responsible for complement-induced pronociceptive effects usually involve the release of potent inflammatory factors (e.g., NGF, CGRP, or IL-1β) that act on their respective receptors on nociceptive neurons to increase their excitability and sensitize them to noxious and innocuous stimuli (Fig. 3).

      Recruitment and activation of immune cells

      B cells, neutrophils, and macrophages are rapidly recruited to the site of injury to aid in fighting infections, killing pathogens, and clearing myelin and debris. These are critical steps for promoting tissue recovery because the detritus from injury inhibits the regrowth of sensory afferents, while necrotic death of pathogens and infected cells causes massive release of inflammatory mediators that can increase pain and prevent healing (
      • Taylor R.C.
      • Cullen S.P.
      • Martin S.J.
      Apoptosis: Controlled demolition at the cellular level.
      ,
      • Oppenheim J.J.
      • Yang D.
      Alarmins: Chemotactic activators of immune responses.
      ,
      • Chen C.J.
      • Kono H.
      • Golenbock D.
      • Reed G.
      • Akira S.
      • Rock K.L.
      Identification of a key pathway required for the sterile inflammatory response triggered by dying cells.
      ). When immune cells are stimulated by complement acutely after injury, they produce and release numerous inflammatory factors such as CCL2, NGF, ATP, TNF-α, PGE2, or IL-1β. These factors recruit additional immune cells and promote sensitization of the sensory neurons to guard and protect the injury site during recovery (Fig. 3). Chronically, however, the activation of these immune cells can prove deleterious for both the local tissue and the neurons that innervate it. For example, after surgery or infection, activation of the complement system and infiltration of immune cells are transient events that start with the injury to support the healing process and gradually disappear along with the recovery process. However, during injuries such as a peripheral nerve trauma (
      • Gaudet A.D.
      • Popovich P.G.
      • Ramer M.S.
      Wallerian degeneration: Gaining perspective on inflammatory events after peripheral nerve injury.
      ) or autoimmune disorders (
      • Li W.W.
      • Guo T.Z.
      • Shi X.
      • Czirr E.
      • Stan T.
      • Sahbaie P.
      • Wyss-Coray T.
      • Kingery W.S.
      • Clark J.D.
      Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome.
      ), where pain sensitization persists well past the local tissue recovery, prolonged activity of the complement cascade and the continuous presence of macrophages, neutrophils, T cells, and B cells are key factors contributing to pain chronification.
      Numerous studies support a key detrimental role of overactivated complement cascade in various chronic pain conditions. For example, in a model of rheumatoid arthritis (RA), knockout of C5aR1 (but not of C3aR) protects against histological pathology, inflammatory factors, and infiltration of immune cells such as neutrophils, T cells, and macrophages (
      • Grant E.P.
      • Picarella D.
      • Burwell T.
      • Delaney T.
      • Croci A.
      • Avitahl N.
      • Humbles A.A.
      • Gutierrez-Ramos J.C.
      • Briskin M.
      • Gerard C.
      • Coyle A.J.
      Essential role for the C5a receptor in regulating the effector phase of synovial infiltration and joint destruction in experimental arthritis.
      ). In contrast to RA and osteoarthritis (OA), which are associated with joint pain, ankylosing spondylitis (AS) is a form of arthritis whose primary symptom is lower back pain. In a mouse model of AS, there is substantial activation of the complement along with macrophage and neutrophil activation. Notably, administration of the C3-binding complement inhibitor, Efb-C (C-terminal of extracellular fibrinogen-binding protein), to AS mice significantly attenuates these pathological processes and reduces the disease progression (
      • Yang C.
      • Ding P.
      • Wang Q.
      • Zhang L.
      • Zhang X.
      • Zhao J.
      • Xu E.
      • Wang N.
      • Chen J.
      • Yang G.
      • Hu W.
      • Zhou X.
      Inhibition of complement retards ankylosing spondylitis progression.
      ). Another example of a posttraumatic chronic pain disorder associated with autoimmunity and complement action is complex regional pain syndrome (CRPS). Indeed, in a mouse model of CRPS, the sensitization of afferents and complement activation/deposition in the skin and on nearby afferent fibers is dependent on antibody production by B cells (
      • Li W.W.
      • Guo T.Z.
      • Shi X.
      • Czirr E.
      • Stan T.
      • Sahbaie P.
      • Wyss-Coray T.
      • Kingery W.S.
      • Clark J.D.
      Autoimmunity contributes to nociceptive sensitization in a mouse model of complex regional pain syndrome.
      ). Similarly, rodent models of peripheral nerve injury (PNI) are characterized by prominent tissue infiltration by immune cells (
      • Shepherd A.J.
      • Mickle A.D.
      • Golden J.P.
      • Mack M.R.
      • Halabi C.M.
      • de Kloet A.D.
      • Samineni V.K.
      • Kim B.S.
      • Krause E.G.
      • Gereau R.W.t.
      • Mohapatra D.P.
      Macrophage angiotensin II type 2 receptor triggers neuropathic pain.
      ) and complement deposition on damaged nerves (
      • Li M.
      • Peake P.W.
      • Charlesworth J.A.
      • Tracey D.J.
      • Moalem-Taylor G.
      Complement activation contributes to leukocyte recruitment and neuropathic pain following peripheral nerve injury in rats.
      ,
      • Hu P.
      • Bembrick A.L.
      • Keay K.A.
      • McLachlan E.M.
      Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve.
      ). Generalized pharmacologic inhibition of immune cells (
      • Mika J.
      • Rojewska E.
      • Makuch W.
      • Przewlocka B.
      Minocycline reduces the injury-induced expression of prodynorphin and pronociceptin in the dorsal root ganglion in a rat model of neuropathic pain.
      ) and more specific chemogenetic ablation of macrophages are each sufficient to reverse the PNI-induced sensitization of peripheral afferents (
      • Shepherd A.J.
      • Mickle A.D.
      • Golden J.P.
      • Mack M.R.
      • Halabi C.M.
      • de Kloet A.D.
      • Samineni V.K.
      • Kim B.S.
      • Krause E.G.
      • Gereau R.W.t.
      • Mohapatra D.P.
      Macrophage angiotensin II type 2 receptor triggers neuropathic pain.
      ,
      • Yu X.
      • Liu H.
      • Hamel K.A.
      • Morvan M.G.
      • Yu S.
      • Leff J.
      • Guan Z.
      • Braz J.M.
      • Basbaum A.I.
      Dorsal root ganglion macrophages contribute to both the initiation and persistence of neuropathic pain.
      ). Thus, complement-dependent recruitment of immune cells is an essential component of tissue responses to injury, which can affect the function of nociceptive neurons through release of inflammatory factors, deposition of complement on nerves that promote cell death/clearance through the TPCC, or clearance of myelin to promote axonal regeneration and tissue repair (Figs. 2 and 3).

      Axonal regeneration and recovery after injury

      An important property of the dorsal root ganglion (DRG) sensory neurons is that after peripheral nerve injury, these neurons are able to repair and regrow the damaged axons and reinnervate target tissues. Complement signaling appears to contribute to the regulation of this process directly at the site of injury (Fig. 3). After nerve injury there is a significant amount of myelin from the damage, which strongly inhibits DRG axonal outgrowth mainly through myelin-associated glycoprotein, known as MAG (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.
      ). This glycoprotein dose-dependently reduced the growth of damaged DRG axons, and the addition of C1q blocked the effects of MAG, leading to unencumbered regeneration of DRG axons (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.
      ). Although the presence of C3a similarly led to an increase in average neurite length in DRG cultures, the addition of C3b decreased the viability of DRG neurons and the number of neurons with neurite outgrowth (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C3 suppresses axon growth and promotes neuron loss.
      ). One potential explanation for the cytotoxic effects of C3b deposits on DRG neurons would be its promotion of formation of the TPCC resulting in cell lysis. However, C3b impaired neurite outgrowth even in cultures free of immune cells and serum, suggesting that its inhibitory effects on axonal regeneration were primarily due to the inhibition of cell adhesion (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C3 suppresses axon growth and promotes neuron loss.
      ). While this might initially appear as a net-zero push–pull action of the C3 split products, the described observations are consistent with the proposed role of complement as a “dual-edged sword” (
      • Brennan F.H.
      • Anderson A.J.
      • Taylor S.M.
      • Woodruff T.M.
      • Ruitenberg M.J.
      Complement activation in the injured central nervous system: Another dual-edged sword?.
      ), where depending on the precise levels of complement factors, receptors, and inhibitors, the complement system can drive seemingly opposing processes. Nevertheless, the observed effects suggest that an appropriate amount of complement activity can promote the recovery of normal neuronal function after injury and that the levels of specific complement factors must be closely regulated both spatially and temporally.
      A recent study (
      • Renthal W.
      • Tochitsky I.
      • Yang L.
      • Cheng Y.C.
      • Li E.
      • Kawaguchi R.
      • Geschwind D.H.
      • Woolf C.J.
      Transcriptional reprogramming of distinct peripheral sensory neuron subtypes after axonal injury.
      ) examining single-nucleus RNA sequencing data from DRG after different types of nerve injury, including some that recover normal sensitivity (sciatic nerve crush) and others that do not (spinal nerve transection), provides an illuminating example of the temporal role and activity of complement inhibitors in recovery after nerve injury. Specifically, in the crush model, the cell surface convertase inhibitor DAF (CD55) was significantly downregulated immediately after injury and then recovered concomitantly with healing of the injury and amelioration of the behavioral deficits. This is consistent with the role of acute complement activity driving tissue remodeling, beneficial inflammation, and overall recovery. In contrast to a transient crush injury, the chronic transection model led to reduced levels of DAF (CD55), which never returned to basal levels indicating that the injury still had not resolved and that the complement cascade was chronically disinhibited. Consistent with this single-cell sequencing study (
      • Renthal W.
      • Tochitsky I.
      • Yang L.
      • Cheng Y.C.
      • Li E.
      • Kawaguchi R.
      • Geschwind D.H.
      • Woolf C.J.
      Transcriptional reprogramming of distinct peripheral sensory neuron subtypes after axonal injury.
      ), other groups have confirmed similar decreases in DAF/CD55 after spinal nerve transection and that complement depletion prevents the development of pain behaviors (
      • Levin M.E.
      • Jin J.G.
      • Ji R.R.
      • Tong J.
      • Pomonis J.D.
      • Lavery D.J.
      • Miller S.W.
      • Chiang L.W.
      Complement activation in the peripheral nervous system following the spinal nerve ligation model of neuropathic pain.
      ,
      • Nie F.
      • Wang J.
      • Su D.
      • Shi Y.
      • Chen J.
      • Wang H.
      • Qin W.
      • Shi L.
      Abnormal activation of complement C3 in the spinal dorsal horn is closely associated with progression of neuropathic pain.
      ) further supporting the idea that chronic complement activity can be detrimental to the recovery from nerve injury. Additionally, in transection models, the levels of cell death are generally higher than in crush models due to the axotomy (
      • Groves M.J.
      • Ng Y.W.
      • Ciardi A.
      • Scaravilli F.
      Sciatic nerve injury in the adult rat: Comparison of effects on oligosaccharide, CGRP and GAP43 immunoreactivity in primary afferents following two types of trauma.
      ,
      • Groves M.J.
      • Christopherson T.
      • Giometto B.
      • Scaravilli F.
      Axotomy-induced apoptosis in adult rat primary sensory neurons.
      ,
      • Tandrup T.
      • Woolf C.J.
      • Coggeshall R.E.
      Delayed loss of small dorsal root ganglion cells after transection of the rat sciatic nerve.
      ). Consistent with this, the TPCC inhibitor protectin (CD59) remains highly expressed on all surviving cells after injury, unlike the transient elevation observed in the crush model (
      • Renthal W.
      • Tochitsky I.
      • Yang L.
      • Cheng Y.C.
      • Li E.
      • Kawaguchi R.
      • Geschwind D.H.
      • Woolf C.J.
      Transcriptional reprogramming of distinct peripheral sensory neuron subtypes after axonal injury.
      ). These findings suggest that the survival of primary afferent neurons after injury is highly dependent on the expression of terminal complement inhibitors as there is a lack of convertase inhibition due to the loss of DAF expression. Finally, global genetic knockout of C6, which inhibits formation of the TPCC, prevented the development of persistent pain in a model of anti-GD2 immunotherapy (
      • Sorkin L.S.
      • Otto M.
      • Baldwin 3rd, W.M.
      • Vail E.
      • Gillies S.D.
      • Handgretinger R.
      • Barfield R.C.
      • Ming Yu H.
      • Yu A.L.
      Anti-GD(2) with an FC point mutation reduces complement fixation and decreases antibody-induced allodynia.
      ), and global knockout of C5 alleviated neuropathic pain (
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ).
      Thus, various components of the complement system play distinct roles in regulating axonal damage and recovery after nerve injury. The complement factors, such as C1q and C3a, promote axonal regeneration and recovery by facilitating the recruitment and activation of immune cells to clear debris and by interacting with myelin-associated glycoproteins that inhibit axonal growth. In contrast, the components of the TPCC/MAC pathway, C5b-C9, promote axonal degeneration and contribute to the development of pathological pain state. Accordingly, selective therapeutic targeting of specific elements of the complement cascade has the potential of promoting axonal regeneration and effective recovery after injury, as well accelerating resolution of injury-induced pain.

      Spinal mechanisms of pain processing

      In addition to the peripheral roles of the complement system in regulating tissue repair, nociceptor sensitization, and pain, recent work has highlighted multifaceted roles of complement in controlling central mechanisms of pain processing in the spinal cord (Fig. 4). Indeed, gene microarray studies comparing changes in gene expression in several different models of neuropathic pain showed a marked enrichment of genes related to the complement system within the spinal cord, including C1q, C3, and C4 (
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ,
      • Mika J.
      • Rojewska E.
      • Makuch W.
      • Korostynski M.
      • Luvisetto S.
      • Marinelli S.
      • Pavone F.
      • Przewlocka B.
      The effect of botulinum neurotoxin A on sciatic nerve injury-induced neuroimmunological changes in rat dorsal root ganglia and spinal cord.
      ). These complement transcripts were also the most strongly upregulated of all genes examined and were expressed primarily in microglia (
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ), clearly indicating a common role for the complement system after most types of injury. An increased expression of C1q and C3 in the spinal cord could potentially contribute to synaptic remodeling via localized production of C3b and C3b-dependent synaptic pruning, as described in Figure 2. In support of this idea, treatment with recombinant C1q was shown to reduce dendritic spine density in spinal cord neurons both in vitro and in vivo (
      • Simonetti M.
      • Hagenston A.M.
      • Vardeh D.
      • Freitag H.E.
      • Mauceri D.
      • Lu J.
      • Satagopam V.P.
      • Schneider R.
      • Costigan M.
      • Bading H.
      • Kuner R.
      Nuclear calcium signaling in spinal neurons drives a genomic program required for persistent inflammatory pain.
      ). The same study reported that C1q expression in the spinal cord was upregulated following peripheral nerve injury, but was downregulated after peripheral inflammation. In the latter case, intrathecal administration of C1q partially reversed an increase in the dendritic spine density associated with peripheral inflammation and also reduced inflammatory pain (
      • Simonetti M.
      • Hagenston A.M.
      • Vardeh D.
      • Freitag H.E.
      • Mauceri D.
      • Lu J.
      • Satagopam V.P.
      • Schneider R.
      • Costigan M.
      • Bading H.
      • Kuner R.
      Nuclear calcium signaling in spinal neurons drives a genomic program required for persistent inflammatory pain.
      ). Although the significance of spinal C1q upregulation in the context of neuropathic pain has not been specifically tested in this report, the described analgesic effects of intrathecal C1q administration suggest that an increase in C1q expression is an adaptive response serving to promote tissue healing and recovery following nerve injury. This is also consistent with the described noncanonical roles of C1q in regulating axonal growth and neuronal survival (
      • Peterson S.L.
      • Nguyen H.X.
      • Mendez O.A.
      • Anderson A.J.
      Complement protein C1q modulates neurite outgrowth in vitro and spinal cord axon regeneration in vivo.
      ,
      • Benoit M.E.
      • Tenner A.J.
      Complement protein C1q-mediated neuroprotection is correlated with regulation of neuronal gene and microRNA expression.
      ,
      • Thielens N.M.
      • Tedesco F.
      • Bohlson S.S.
      • Gaboriaud C.
      • Tenner A.J.
      C1q: A fresh look upon an old molecule.
      ).
      Figure thumbnail gr4
      Figure 4Model describing the regulation of nociceptive signaling via complement in the spinal dorsal horn. The activity of complement in the spinal cord has distinct roles following nerve injury. C1q has been shown to contribute to the clearance of damaged tissue, synaptic remodeling, and neurite outgrowth. In contrast, C3a and C5a are thought to amplify nociceptive signaling through microglia and possibly astrocytic activation. This activation releases inflammatory mediators and growth factors that regulate neuronal excitability and synaptic plasticity in the spinal dorsal horn circuit, which includes local interneurons, projection neurons, and the central terminals of primary afferent fibers (C-fibers: slow-conducting unmyelinated primary afferents that transduce pain and thermal stimuli; Aδ-fibers: intermediate-conduction velocity thinly myelinated fibers that transduce pain and thermal stimuli; Aβ-fibers: fast-conducting myelinated fibers that transduce innocuous mechanical stimuli). These primary afferent inputs are processed by the interneurons and are integrated by projection neurons that transmit sensory information to the brain. The specific mechanisms through which activated complement factors modulate neuronal excitability and synaptic transmission in the spinal cord remain to be determined.
      The components of the terminal complement cascade, C5 and C5aR1, are also highly upregulated in spinal cord microglia after peripheral nerve injury, and activation of C5aR1 through intrathecal administration of C5a produces cold allodynia, whereas intrathecal administration of a potent and selective antagonist of C5aR1, PMX53, alleviated neuropathic pain (
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ). These latter findings suggest that C5a-mediated activation of microglia is a key event in the spinal cord in the context of neuropathic pain (Fig. 4). This notion is further supported by numerous studies showing that after peripheral nerve injury, microglia are strongly activated in the dorsal horn of the spinal cord (
      • Griffin R.S.
      • Costigan M.
      • Brenner G.J.
      • Ma C.H.
      • Scholz J.
      • Moss A.
      • Allchorne A.J.
      • Stahl G.L.
      • Woolf C.J.
      Complement induction in spinal cord microglia results in anaphylatoxin C5a-mediated pain hypersensitivity.
      ,
      • Hu P.
      • Bembrick A.L.
      • Keay K.A.
      • McLachlan E.M.
      Immune cell involvement in dorsal root ganglia and spinal cord after chronic constriction or transection of the rat sciatic nerve.
      ,
      • Mika J.
      • Rojewska E.
      • Makuch W.
      • Przewlocka B.
      Minocycline reduces the injury-induced expression of prodynorphin and pronociceptin in the dorsal root ganglion in a rat model of neuropathic pain.
      ,
      • Li Z.
      • Wei H.
      • Piirainen S.
      • Chen Z.
      • Kalso E.
      • Pertovaara A.
      • Tian L.
      Spinal versus brain microglial and macrophage activation traits determine the differential neuroinflammatory responses and analgesic effect of minocycline in chronic neuropathic pain.
      ,
      • Mika J.
      • Osikowicz M.
      • Rojewska E.
      • Korostynski M.
      • Wawrzczak-Bargiela A.
      • Przewlocki R.
      • Przewlocka B.
      Differential activation of spinal microglial and astroglial cells in a mouse model of peripheral neuropathic pain.
      ) and that pharmacological inactivation of microglia by intrathecal administration of minocycline, a tetracycline antibiotic known to also inhibit microglia activation (
      • Yrjanheikki J.
      • Keinanen R.
      • Pellikka M.
      • Hokfelt T.
      • Koistinaho J.
      Tetracyclines inhibit microglial activation and are neuroprotective in global brain ischemia.
      ,
      • Tikka T.
      • Fiebich B.L.
      • Goldsteins G.
      • Keinanen R.
      • Koistinaho J.
      Minocycline, a tetracycline derivative, is neuroprotective against excitotoxicity by inhibiting activation and proliferation of microglia.
      ), prevents the development of allodynia and elevation of proinflammatory markers (
      • Mika J.
      • Rojewska E.
      • Makuch W.
      • Przewlocka B.
      Minocycline reduces the injury-induced expression of prodynorphin and pronociceptin in the dorsal root ganglion in a rat model of neuropathic pain.
      ,
      • Li Z.
      • Wei H.
      • Piirainen S.
      • Chen Z.
      • Kalso E.
      • Pertovaara A.
      • Tian L.
      Spinal versus brain microglial and macrophage activation traits determine the differential neuroinflammatory responses and analgesic effect of minocycline in chronic neuropathic pain.
      ).
      Besides the products of the complement cascade, other factors, such as VGF-derived neuropeptide TLQP-21, have been reported to activate the complement receptors C3aR and gC1qR (also known as C1qBP) in rodents (
      • Elmadany N.
      • de Almeida Sassi F.
      • Wendt S.
      • Logiacco F.
      • Visser J.
      • Haage V.
      • Hernandez D.P.
      • Mertins P.
      • Hambardzumyan D.
      • Wolf S.
      • Kettenmann H.
      • Semtner M.
      The VGF-derived peptide TLQP21 impairs purinergic control of chemotaxis and phagocytosis in mouse microglia.
      ) (Fig. 4). Notably, TLQP-21 has been implicated in the spinal mechanisms that underlie both neuropathic and inflammatory pain (
      • Fairbanks C.A.
      • Peterson C.D.
      • Speltz R.H.
      • Riedl M.S.
      • Kitto K.F.
      • Dykstra J.A.
      • Braun P.D.
      • Sadahiro M.
      • Salton S.R.
      • Vulchanova L.
      The VGF-derived peptide TLQP-21 contributes to inflammatory and nerve injury-induced hypersensitivity.
      ). Inhibition of TLQP-21 signaling in the spinal cord attenuated mechanical hypersensitivity in rodent models of inflammatory and neuropathic pain, whereas its activation promoted mechanical sensitization (
      • Doolen S.
      • Cook J.
      • Riedl M.
      • Kitto K.
      • Kohsaka S.
      • Honda C.N.
      • Fairbanks C.A.
      • Taylor B.K.
      • Vulchanova L.
      Complement 3a receptor in dorsal horn microglia mediates pronociceptive neuropeptide signaling.
      ,
      • Fairbanks C.A.
      • Peterson C.D.
      • Speltz R.H.
      • Riedl M.S.
      • Kitto K.F.
      • Dykstra J.A.
      • Braun P.D.
      • Sadahiro M.
      • Salton S.R.
      • Vulchanova L.
      The VGF-derived peptide TLQP-21 contributes to inflammatory and nerve injury-induced hypersensitivity.
      ). The pronociceptive action of TLQP-21 involves microglial activation via C3aR, with the receptor being significantly upregulated after nerve injury (
      • Doolen S.
      • Cook J.
      • Riedl M.
      • Kitto K.
      • Kohsaka S.
      • Honda C.N.
      • Fairbanks C.A.
      • Taylor B.K.
      • Vulchanova L.
      Complement 3a receptor in dorsal horn microglia mediates pronociceptive neuropeptide signaling.
      ).
      How activation of complement receptors on microglia translates to the amplification of nociceptive signaling in the spinal cord following peripheral nerve injury remains unclear. Mechanistically, central sensitization caused by enhanced synaptic activity in the dorsal horn of spinal cord is a hallmark of neuropathic pain, and many aspects of central sensitization are controlled by microglia, including profound structural and functional changes within the nociceptive synaptic network (
      • Liu Y.
      • Zhou L.J.
      • Wang J.
      • Li D.
      • Ren W.J.
      • Peng J.
      • Wei X.
      • Xu T.
      • Xin W.J.
      • Pang R.P.
      • Li Y.Y.
      • Qin Z.H.
      • Murugan M.
      • Mattson M.P.
      • Wu L.J.
      • et al.
      TNF-α differentially regulates synaptic plasticity in the Hippocampus and spinal cord by microglia-dependent mechanisms after peripheral nerve injury.
      ,
      • Zhou L.J.
      • Peng J.
      • Xu Y.N.
      • Zeng W.J.
      • Zhang J.
      • Wei X.
      • Mai C.L.
      • Lin Z.J.
      • Liu Y.
      • Murugan M.
      • Eyo U.B.
      • Umpierre A.D.
      • Xin W.J.
      • Chen T.
      • Li M.
      • et al.
      Microglia are indispensable for synaptic plasticity in the spinal dorsal horn and chronic pain.
      ,
      • Kuner R.
      • Flor H.
      Structural plasticity and reorganisation in chronic pain.
      ,
      • Inoue K.
      • Tsuda M.
      Microglia in neuropathic pain: Cellular and molecular mechanisms and therapeutic potential.
      ). Thus, it is plausible that complement-dependent activation of microglia (and possibly other glial cells, e.g., astrocytes) and the release of inflammatory factors contribute to synaptic remodeling within the spinal cord through molecular and structural modifications, as well as through changes in neuronal excitability and synaptic transmission, ultimately leading to central sensitization and the amplification of pain processing (Fig. 4). Future studies will help to identify specific molecular and cellular mechanisms that link complement activation with amplified nociceptive signaling within the spinal cord.

      Concluding remarks and perspectives

      Accumulating evidence suggests that the complement cascade is strongly activated and upregulated in a variety of neuropathological states, although the mechanisms underlying the complement-dependent modulation of neuronal activity are still being unraveled. Preclinical data show that inhibition of particular aspects of complement signaling can ameliorate or even prevent conditions such as inflammatory or neuropathic chronic pain, identifying exciting new alternatives to opioids for the treatment of these conditions (
      • Moriconi A.
      • Cunha T.M.
      • Souza G.R.
      • Lopes A.H.
      • Cunha F.Q.
      • Carneiro V.L.
      • Pinto L.G.
      • Brandolini L.
      • Aramini A.
      • Bizzarri C.
      • Bianchini G.
      • Beccari A.R.
      • Fanton M.
      • Bruno A.
      • Costantino G.
      • et al.
      Targeting the minor pocket of C5aR for the rational design of an oral allosteric inhibitor for inflammatory and neuropathic pain relief.
      ). However, the homeostatic role of the complement system also critically depends on the time after injury, as modulating a single specific component could have either deleterious or healing effects, as in the case of C5aR1 inhibition in a model of spinal cord injury (
      • Brennan F.H.
      • Gordon R.
      • Lao H.W.
      • Biggins P.J.
      • Taylor S.M.
      • Franklin R.J.
      • Woodruff T.M.
      • Ruitenberg M.J.
      The complement receptor C5aR controls acute inflammation and astrogliosis following spinal cord injury.
      ). This highlights one of the major questions in the field that is important to address: How does the role of specific components of the complement system change during the progression of injury or disease, and what are the underlying mechanisms and consequences of these changes?
      Indeed, we now have evidence that complement plays multifaceted roles in both disease progression and recovery. The specific roles and timing can be substantially different for each pathology. For example, in the context of acute neurological trauma and stroke, activation of the complement system during early stages exacerbates inflammation and tissue damage, whereas at later stages, the activity of complement factors (e.g., C3a and C5a) is key to axonal regeneration and the restoration of synaptic connectivity (
      • Mika J.
      • Rojewska E.
      • Makuch W.
      • Przewlocka B.
      Minocycline reduces the injury-induced expression of prodynorphin and pronociceptin in the dorsal root ganglion in a rat model of neuropathic pain.
      ,
      • Li Z.
      • Wei H.
      • Piirainen S.
      • Chen Z.
      • Kalso E.
      • Pertovaara A.
      • Tian L.
      Spinal versus brain microglial and macrophage activation traits determine the differential neuroinflammatory responses and analgesic effect of minocycline in chronic neuropathic pain.
      ). On the other hand, in chronic neurodegenerative diseases such as AD and ALS, complement activation during early stages may help to clear toxic protein aggregates and damaged cells. As these chronic diseases progress, the complement cascade becomes persistently activated, overwhelming complement-regulating inhibitory mechanisms and leading to uncontrolled inflammation, synaptic loss, and neurotoxicity (
      • Lee J.D.
      • Coulthard L.G.
      • Woodruff T.M.
      Complement dysregulation in the central nervous system during development and disease.