Cytotoxic Immunity in Peripheral Nerve Injury and Pain

Abstract

Cytotoxicity and consequent cell death pathways are a critical component of the immune response to infection, disease or injury. While numerous examples of inflammation causing neuronal sensitization and pain have been described, there is a growing appreciation of the role of cytotoxic immunity in response to painful nerve injury. In this review we highlight the functions of cytotoxic immune effector cells, focusing in particular on natural killer (NK) cells, and describe the consequent action of these cells in the injured nerve as well as other chronic pain conditions and peripheral neuropathies. We describe how targeted delivery of cytotoxic factors via the immune synapse operates alongside Wallerian degeneration to allow local axon degeneration in the absence of cell death and is well-placed to support the restoration of homeostasis within the nerve. We also summarize the evidence for the expression of endogenous ligands and receptors on injured nerve targets and infiltrating immune cells that facilitate direct neuro-immune interactions, as well as modulation of the surrounding immune milieu. A number of chronic pain and peripheral neuropathies appear comorbid with a loss of function of cellular cytotoxicity suggesting such mechanisms may actually help to resolve neuropathic pain. Thus while the immune response to peripheral nerve injury is a major driver of maladaptive pain, it is simultaneously capable of directing resolution of injury in part through the pathways of cellular cytotoxicity. Our growing knowledge in tuning immune function away from inflammation toward recovery from nerve injury therefore holds promise for interventions aimed at preventing the transition from acute to chronic pain.

Keywords: cellular cytotoxicity; innate immunity; natural killer cells; nerve injury; neuro-immunology; neuron-glia crosstalk; neuropathic pain; peripheral neuropathy.

FIGURE 1

The cytotoxic neuro-immune synapse: Potential downstream intracellular pathways. Cytotoxic natural killer cells form an immunological synapse upon recognition and adhesion to a target, in this case the axon of a stressed sensory neuron. (1) Cytotoxic/lytic granules are held by the microtubule organizing center (MTOC) of the NK cell. Immune synapse formation triggers reorientation and polarization of the granules by the MTOC which directs the fusion of the granules to the synaptic membrane. This mechanism ensures the precision release of the cytotoxic contents directly through the targeted axonal membrane. (2) The assembly of perforin pore complexes in the axonal membrane from individual perforin subunits released to the extracellular space allows the flow of ions including Ca²⁺, and larger molecules such as the serine protease granzyme B, into the intracellular environment of the target neuron. (3) Ca²⁺ flux leads to rapid axon microtubule destabilization and axon degeneration. The full mechanism of cytotoxicity in the axon is unclear. However, in the case of neuron-autonomous Wallerian degeneration, the late-phase Ca²⁺ elevation and subsequent calpain activation required for microtubule destabilization is gated by a metabolic signaling cascade involving depletion of nicotinamide adenine dinucleotide (NAD⁺), activation of Sterile-alpha and armadillo motif containing protein (SARM) and C-Jun N-terminal kinase (JNK) (Llobet Rosell and Neukomm, 2019). Although caspase 3 is a major target of granzyme B protease activity (Adrain et al., 2005), its role in cytotoxic axon degeneration remains unclear. Pathways shown in gray italics are yet to be determined specifically in cytotoxicity-induced axonal degeneration.

FIGURE 2

The receptors and ligands of cellular cytotoxicity. There are several key cellular effector mechanisms capable of evoking intracellular cell death pathways. In the case of nerve injury this could be a targeted sensory axon. Natural killer (NK) cells are specialist cytotoxic effector cells expressing multiple receptors and ligands for executing cellular cytotoxicity. (1) CD95 (Fas) is a transmembrane protein that belongs to the tumor necrosis factor (TNF) family. Engagement with its ligand CD95L (FasL) expressed on NK cells (as well as activated CD8⁺ T cells and plasma cells) triggers apoptotic signaling in the target cell. Apoptosis may also be induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) via its receptor on target axons. (2) Cross-linking of an antigen-antibody complex with the low affinity Fc receptor FcγRIIIa (CD16a) on human NK cells (FcγRIII/CD16 in mice) can trigger cytotoxic granule release (Rosales, 2017). (3) NK cells express a host of activating receptors such as NKG2D which recognize ligands expressed at times of neural stress, tumorigenesis or infection. Cytotoxic activity by either Fc or activator receptor function is kept in balance by inhibitory signaling from major histocompatibility complex (MHC) class I molecules or human leukocyte antigens (HLA), present on nearly all somatic cells; these molecules signal through inhibitory receptors expressed on NK cells, e.g., Ly49 family in mice, and killer immunoglobulin-like receptors (KIR) in humans. (4) Macrophages may elicit a form of antibody-dependent cellular phagocytosis (ADCP) via engagement with another low affinity Fc receptor FcγRIIa (CD32a) in humans. Macrophages/monocytes may also express natural killer receptors and ligands allowing them to recognize stressed target cells as well as interact with NK cells via natural killer receptor ligands (e.g., RAE1) while protecting themselves from killing by inhibitory MHC I molecules such as Qa1b (in mice) (Zhou et al., 2012). (5) Sensitized CD8⁺ T cells recognize MHC I-presented antigens at the target membrane surface via corresponding T cell receptors (TCR), which together with co-stimulation via receptors such as NKG2D, triggers cytotoxic granule release in a manner similar to NK cells. CD8⁺ T cells may also trigger apoptosis in target cells via FasL. (6) Neutrophils can recognize opsonized targets, such as antibody-antigen complexes, via complement receptors (e.g., C3R) or Fc receptors including FcγRIIa (Rosales, 2017). Complement or antibody Fc receptor signaling neutrophils can lead to respiratory burst and the release of reactive oxygen species (ROS), or an active mechanical disruption of the target cell membrane known as ‘trogoptosis’ (Matlung et al., 2018). Signaling via death receptor 6 (DR6) is thought to be involved in axon degeneration in the peripheral nervous system as well as neurodegeneration in the brain but the extrinsic signal for the receptor remains unknown (Gamage et al., 2017). ‘+’ and ‘−’ within the immune cell denotes stimulatory and inhibitory signaling, respectively.

FIGURE 3

Inhibition of apoptosis in the cell bodies of sensory neurons. Adult sensory neurons are protected from cell death by expression of Bcl2 (B-cell lymphoma 2). The gene product BCL-2 suppresses the function of the pro-apoptotic protein BAX (BCL2 Associated X) which can be induced by cell death signaling. The expression of further anti-apoptotic genes in the cell bodies of sensory neurons prevent axon degeneration intracellular signaling cascades from causing neuronal death. For example, expression of Hspb1 (Heat shock protein family B1), also known as HSP27, prevents sensory neuron death after nerve injury. X-linked inhibitor of apoptosis protein (XIAP) prevents cell death during developmental axon pruning by inhibiting caspases in the cell body. Thus axonal activation of caspases 3, 6 and 9, which is sufficient for certain forms of axon-restricted degeneration, occurs in the absence of cell apoptosis (Simon et al., 2012; Cusack et al., 2013).