Research ArticleNEUROPATHIC PAIN

Lysozyme elicits pain during nerve injury by neuronal Toll-like receptor 4 activation and has therapeutic potential in neuropathic pain

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Science Translational Medicine  07 Aug 2019:
Vol. 11, Issue 504, eaav4176
DOI: 10.1126/scitranslmed.aav4176

Bacteriolytic pain

Neuropathic pain is a debilitating condition originating from nerve damage. The current treatments have limited efficacy or are associated with serious side effects. Toll-like receptor 4 (TLR4) activation promotes neuropathic pain; however, the molecular pathway responsible for TLR4 activation is unclear. Using rodent models and human tissue, Yadav et al. now show that the endogenous bacteriolytic enzyme lysozyme is up-regulated upon nerve injury in both rodents and humans. Lysozyme acts as TLR4 activator in sensory neurons, promoting chronic pain in rodents. Moreover, the lysozyme administration in spinal cord of healthy rats was sufficient to cause neuropathic pain by increasing neuronal excitability.

Abstract

The role of neuronal Toll-like receptor 4 (TLR4) in nerve injury is being pursued actively. However, the endogenous activation of neuronal TLR4 during neuroinflammation, in absence of the participation of glial TLR4, remains elusive. Here, we identified lysozyme as an endogenous activator of neuronal TLR4 signaling during nerve injury. Upon nerve injury, enhanced expression of lysozyme promoted neuronal hyperexcitability and neuropathic pain. Injections of lysozyme in healthy rats increased their mechanical and thermal pain sensitivity. Likewise, infusion of spinal cord slices with lysozyme increased neuronal excitability typical of neuropathic pain. Our results also showed that lysozyme activated excitability of both Aδ- and C-fibers. Thus, in addition to the discovery of lysozyme as an endogenous ligand for regulating neuronal TLR4 signaling, this study also lays the foundation of our understanding of its role in nervous system pathologies, providing multiple avenues for treating neuroinflammation.

INTRODUCTION

Neuropathic pain arises as a consequence of neuronal injury leading to altered gene expression (14), protein regulation (5), and channel expression (6). Nerve damage leads to a local neuroinflammatory response (7, 8), which contributes to the generation of heightened neuronal excitability, spontaneous neuronal firing (allodynia), and reduced threshold to noxious stimuli (hyperalgesia). Current approaches for neuropathic pain management are either ineffective (9) or are associated with undesirable side effects (10), highlighting the need for novel pain therapies.

During nerve injury, endogenous ligands associated with tissue damage and cell death [known as damage-associated molecular patterns (DAMPs) or alarmins] (11) activate Toll-like receptors (TLRs). TLRs are pattern recognition receptors, which regulate innate immune response against structurally conserved components of microbes [called as pathogen-associated molecular patterns (PAMPs)]. In this context, the role of Toll-like receptor 4 (TLR4) is pivotal in nerve injury pathologies of both central nervous system (CNS) and peripheral nervous system (PNS) (1113). TLR4 transduces its biologic effects by activation and nuclear localization of nuclear factor κB (NFκB) and enhanced production of proinflammatory cytokines (14), which activate nociceptors during nerve injury–induced neuroinflammation. Although the role of TLR4 is established in neuropathic pain (15), the identity of its endogenous ligands that activate neuronal TLR4 during sterile nerve injuries remains underexplored (16, 17).

Lysozyme is a bacteriolytic enzyme found in body secretions and in phagocytes such as macrophages, neutrophils, and dendritic cells (18, 19). Since its discovery, the antibacterial activity of lysozyme has remained the cornerstone of innate immunity. Current belief that the degradation products of lysozyme action, such as peptidoglycans, lipoproteins, and DNA, are released from the bacterial lysis, which activate nucleotide-binding oligomerization domain-containing protein (NOD1 and NOD2) receptors, TLR2 and TLR9, respectively (2023), culminating in the release of proinflammatory cytokines, does not explain all of its physiologic functions. Lysozyme, for example, can also alter the physiological properties of mammalian cells through an unknown mechanism (24, 25). Consequently, the physiological roles of lysozyme continue to remain an area of intense scientific scrutiny. Lysozyme gene expression is up-regulated during neuroinflammation associated with neurodegenerative disorders and nerve injury (1, 2, 2629). Nerve injury and neurodegenerative conditions lead to sterile neuroinflammation, where bacterial load is absent, and therefore, its bacteriolytic function is of no consequence. Hence, an enhanced expression of lysozyme in the nervous system, under these conditions, warrants an investigation of its physiological role.

Here, we examined the role of lysozyme during nerve injury in murine models of neuropathic pain. Using chemical and genetic inhibition of lysozyme, we uncovered a correlation between lysozyme overexpression and neuropathic pain. In addition, bath application of lysozyme triggered neuronal hyperexcitability typical of neuropathic pain. Pharmacological inhibition of TLR4 in healthy rats, as well as studies on C3H/HeJ (TLR4 mutant) and B6.129-Tlr2tm1Kir/J (TLR2 knockout) mice, established lysozyme as a specific endogenous activator of neuronal TLR4, responsible for modulation of neuronal excitability and pain during nerve injury. Our studies with human spinal cord injury samples are consistent with the above findings. Thus, the study uncovered a physiological function of lysozyme in the nervous system during nerve injury–induced neuroinflammation and suggests that lysozyme might be targeted for obtaining potentially nonaddictive pharmacological interventions for treating neuropathic pain.

RESULTS

Endogenous up-regulation of lysozyme contributes to neuropathic pain

Subsequent to a partial sciatic ligation (PSL), neuropathic pain symptoms such as mechanical allodynia and thermal hyperalgesia develop at day 1, which were sustained for a number of weeks thereafter (fig. S1A). To check the expression of lysozyme in the neuronal tissue upon nerve injury, we performed Western blotting of the lysates from dorsal root ganglia (L4 and L5 DRGs) of PSL rats, isolated a day before surgery and 1, 7, and 14 days after surgery. Lysozyme expression was substantially increased after nerve injury [~4.87-fold (P < 0.001), ~5.53-fold (P < 0.001), and ~4.27-fold (P < 0.005) at days 1, 7, and 14, respectively] (n = 4 Western blots from different tissue samples; Fig. 1A). To confirm the specificity of lysozyme expression with neuronal injury, we examined the expression of lysozyme in the DRG lysates of the PSL animals with respect to sham-operated animals (at day 3 after surgery) by Western blotting. Lysozyme expression increased in the DRGs of PSL rats (~5.4-fold) and not in the sham-operated animals (n = 4 Western blots from different tissue samples, P < 0.001; Fig. 1B). Subsequent to nerve injury, infiltrating macrophages may also contribute to lysozyme concentration buildup in DRGs, as well as in the spinal cord. To test the contribution of infiltrating macrophages in the appearance of lysozyme concentrations in the DRGs, we inhibited the activation and proliferation of macrophages and glial cells using minocycline, in both PSL and sham-operated rats. Intrathecal injections of minocycline (300 μg per animal) did not reduce the lysozyme expression (~4.1-fold) in the DRGs of PSL rats (n = 4 Western blots from different tissue samples, P < 0.001; Fig. 1C) suggesting a neuronal origin of lysozyme during nerve injury. We next sought to identify the cellular source of lysozyme by examining the expression of lysozyme in both neuronal and glial cells isolated from the L4 and L5 DRGs of both sham-operated and PSL rats (3 days after surgery). Western blots from the isolated cellular lysates showed increased expression of lysozyme (~4.0-fold, P < 0.001) in neurons but not in the glial cells from the PSL rats (P = 0.71) (n = 3 Western blots from cell extracts of animal DRGs; Fig. 1D), confirming a neuronal overexpression of lysozyme during nerve injury.

Fig. 1 Endogenous up-regulation of lysozyme induces neuropathic pain in rat models.

(A to C) Western blot analyses showing expression of lysozyme (Lyz) in (A) PSL rat models at day −1 (***P < 0.001), day 7 (***P < 0.001), and day 14 (**P < 0.005) after nerve injury (n = 4, one way ANOVA followed by Tukey post hoc test multiple comparison test), (B) PSL rats with respect to sham-operated control rats [n = 4, unpaired (two-tailed) t test, ***P < 0.001], and (C) PSL rat DRGs after minocycline treatment [n = 4, unpaired (two-tailed) t test, ***P < 0.001]. (D) Western blot analysis showing lysozyme expression in neurons (***P < 0.001) and in glial cells [n = 3 Western blots from cells isolated from PSL rat DRGs, unpaired (two-tailed) t test]. n.s., not significant. (E and F) (E) Mechanical allodynia (von Frey threshold; n = 18 animals, ***P < 0.001 versus aCSF at 30 min) and (F) thermal hyperalgesia (n = 16 animals, ***P < 0.001 versus aCSF at 30 min) in PSL and sham rats after single injection of chitobiose or aCSF. (G) Mechanical allodynia in PSL or sham rats after continuous infusion of chitobiose or aCSF (n = 18 animals, ***P < 0.001 versus aCSF). (H) Mechanical allodynia in PSL or sham rats after single injection of bromophenol blue (n = 18 animals, ***P < 0.001 versus aCSF at 30 min). (I and J) Effect of repeated intrathecal injections of siRNA against lysozyme from day 1 on (I) mechanical allodynia and (J) thermal hyperalgesia in PSL rats (n = 18 animals, ***P < 0.001). Corresponding Western blots show lysozyme expression in the lysozyme-siRNA–treated PSL rats. (K) Expression of lysozyme in CCI and SNL rats compared to sham-operated controls [n = 4, unpaired (two-tailed) t test, **P < 0.005]. (L and M) Effect of repeated intrathecal injections of siRNA against lysozyme from days 1 to 6 after surgery on mechanical allodynia in both (L) CCI and (M) SNL rats (n = 8 animals, ***P < 0.001). Corresponding Western blots show lysozyme expression upon lysozyme-siRNA treatment. Dotted lines represent time points of injections, and day 0 is defined as the day of first siRNA treatment after 1 day of nerve ligation surgery in (I), (J), (L), and (M). All Western blot repeats were performed with different tissue samples. All behavioral data were analyzed using two-way ANOVA, followed by Bonferroni’s test.

Subsequently, we explored the role of endogenously up-regulated lysozyme in the pathophysiology of neuropathic pain. We intrathecally injected N,N′-diacetylchitobiose (chitobiose), an inhibitor of lysozyme activity (30), into PSL rats to attain a cerebrospinal fluid (CSF) concentration of 10 μg/ml. Treatment with chitobiose reduced mechanical allodynia (n = 18 animals, P < 0.001 at 30 min; Fig. 1E) and thermal hyperalgesia (n = 16 animals, P < 0.001; Fig. 1F) in PSL rats, as compared with the artificial CSF (aCSF)–treated littermates. The pain mitigatory effects of chitobiose treatment were short-lived, leading to sharp peaks for both mechanical allodynia and thermal hyperalgesia. This is likely to be due to high turnover of lysozyme, which is of the order of minutes (75 min) (31). In addition, the clearance of chitobiose (molecular weight, 424.4) in vivo is expected to be equally rapid. Accordingly, continuous infusion of chitobiose in the spinal cord reduced the mechanical allodynia in PSL rats for longer durations [n = 18 animals, P < 0.001 versus aCSF, two-way analysis of variance (ANOVA) followed by Bonferroni’s test; Fig. 1G]. To further affirm the role of lysozyme in neuropathic pain, we injected another inhibitor of lysozyme, bromophenol blue (32), at a CSF concentration of 20 μg/ml. Intrathecal injection relieved the mechanical allodynia in PSL rats (n = 18 animals, P < 0.001 versus aCSF at 30 min, two-way ANOVA followed by Bonferroni’s test; Fig. 1H). To ascertain that pain relief achieved by chitobiose and bromophenol blue injections was mediated by lysozyme inhibition and not the independent effects of these compounds themselves, we intrathecally injected small interfering RNA (siRNA) against lysozyme gene into PSL rats. Lysozyme-siRNA treatment ameliorated mechanical allodynia and thermal hyperalgesia and increased motility in rats, whereas scrambled siRNA treatment showed no relief (Fig. 1, I and J, fig. S1B, and movies S1 to S3) attesting to the direct physiologic effect of endogenous lysozyme in modulation of neuropathic pain (n = 18 animals, P < 0.001). To investigate whether these findings could be a common feature in neuropathic pain, we generated chronic constriction injury (CCI) and spinal nerve ligation (SNL) rat models of neuropathic pain and tested the expression of lysozyme (at day 3 after surgery) in the DRGs of these rats with respect to sham-operated rats. Lysozyme expression was significantly increased after nerve injury in CCI (~3.9-fold) and SNI rats (~4.3-fold) (n = 4 Western blots from different tissue samples, P < 0.005; Fig. 1K). Next, we intrathecally injected siRNA against lysozyme gene into CCI and SNL models. Lysozyme-siRNA treatment ameliorated mechanical allodynia in both CCI and SNL rats, as compared with the scrambled siRNA–treated rat models, respectively (n = 8 animals, P < 0.001; Fig. 1, L and M). Collectively, these results confirm that endogenous up-regulation of lysozyme in neurons during nerve injury culminates in neuropathic pain.

External application of lysozyme induces pain in healthy rats

During bacterial infections, the enzymatic activity of lysozyme releases multiple degradation products of bacterial cells (including lipoproteins, peptidoglycans, and DNA), which lead to proinflammatory response in the host immune cells (33). Sterile inflammation expected during nerve injury is not accompanied by any bacterial infection. Yet, to confirm that lysozyme itself, and not an exposure to any bacterial product, is responsible for heightened pain response in our nerve injury murine models, we investigated the effects of lysozyme injection in healthy animals. Intrathecal injections of lysozyme (34) (to achieve the CSF concentrations of 3, 5, 10, 50, 100, 1000, and 2000 μg/ml) sensitized the animals for the von Frey threshold (mechanical allodynia) and against the thermal stimulus at all the doses tested (n = 16 to 18 animals, P < 0.001 at 30 min; Fig. 2, A and B). The injections also reduced the motility of the animals (movies S4 and S5). Thus, lysozyme by itself is capable of inducing pain response in healthy animals.

Fig. 2 External lysozyme injections sensitize rats for pain and induce neuronal hyperexcitability in spinal cord slices from healthy animals.

(A and B) (A) Mechanical allodynia and (B) thermal hyperalgesia in male rats after injections of varying concentrations of lysozyme (3 to 2000 μg/ml; n = 16 to 18 animals, ***P < 0.001). AUC, area under the curve; a.u., arbitrary units. (C and D) (C) Mechanical allodynia and (D) thermal hyperalgesia in male rats treated with chemically inhibited or inactivated lysozyme or its fragment, HLH (n = 15 animals, ***P < 0.001). (E) Bar graph analyses of sEPSCs and EPSC amplitudes in spinal cord slice preparation upon lysozyme infusion (n = 29, **P < 0.005). (F to H) Representative traces of evoked excitatory synaptic input responses at indicated time points, (F) EPSCs, (G) EPSPs, and (H) their overlay, upon lysozyme treatment (n = 30). (I to K) Bar graph and overlay of normalized (I) EPSCs, (J) EPSPs, and (K) EPSCs in dorsal horn neurons of control and lysozyme-chitobiose complex–treated slices (n = 29, P = 0.925). CB, chitobiose; dCB, chitobiose injected 5 min after lysozyme treatment. In (E) and (I), error bars represent SEM. All behavioral data were analyzed using two-way ANOVA, followed by Bonferroni’s test.

Chitobiose binds to lysozyme’s active site, inhibiting its catalytic activity (30). We observed no reduction in mechanical pain threshold upon treatment with lysozyme-chitobiose complex (1:2 molar ratio) and diminished mechanical response upon administration of chitobiose 5 min after lysozyme injection (delayed chitobiose; 1:4 molar ratio) in healthy animal (n = 15 animals, P > 0.99 at 30 min; Fig. 2, C and D). To further probe the role of the bacteriolytic function of lysozyme, we injected intact lysozyme, its heat-denatured form, and an N-terminal peptide region of lysozyme [helix-loop-helix (HLH)], which accounts for the bacteriolytic activity without enzymatic activity and chitobiose binding (35). Intact lysozyme induced pain sensitivity in the healthy animals (P < 0.001 at 30 min), whereas both denatured lysozyme and its HLH fragment had no effect (n = 15 animals; Fig. 2, C and D). This led us to deduce that, although its catalytic site is involved in its pain modulation activity, the bacteriolytic activity of lysozyme is not instrumental for evoking pain. Other proteins of comparable isoelectric point (pI) and fold, cytochrome C and α-lactalbumin, respectively, did not induce pain, indicating the specificity of lysozyme in pain sensitization (fig. S1C).

A recent study described the involvement of different cell types in mediating neuropathic pain in males and females (36). We also examined the sex specificity of lysozyme-induced pain. Intrathecal lysozyme injections (to achieve the CSF concentration of 100 μg/ml) in female rats increased their mechanical and thermal sensitivity (n = 15 animals, P < 0.001 at 30 min; fig. S2, A and B) and exhibited reduced motility similar to their male counterparts (movie S6). To delineate whether lysozyme-induced pain is mediated by different cell types in males and females, we intrathecally injected minocycline (300 μg per animal) into both male and female PSL rats to inhibit glial cells and infiltrating macrophages. Other groups of male and female rats receiving minocycline were also injected with chitobiose. Consistent with previous findings (36), unlike males (P < 0.001 at 30 min), minocycline had no effects on female rats (P > 0.99 at 30 min). Both males and females treated with chitobiose, after minocycline injections, showed tolerance toward mechanical stimulus as compared with rats treated with minocycline alone (n = 16, P < 0.001; fig. S2, C and D). Above results indicate that lysozyme equally affects both male and female rats. Moreover, akin to male rats, females also did not show any pain sensitivity upon treatment with the inactive forms of lysozyme (fig. S2, E and F).

Bath application of lysozyme to spinal cord slices evokes neuronal excitability

Neuropathic pain is associated with spontaneous, aberrant neuronal firing (37). Nerve fibers involved in pain relay, pass through the lamina I/II region of the spinal cord. To assess the effects of lysozyme treatment on neuronal excitability in this region, we performed whole-cell patch-clamp experiments with adult lamina I/II neurons in the dorsal horn of the spinal cord. Bath application of lysozyme did not alter the passive and active resting membrane properties (fig. S3A). However, lysozyme increased the spontaneous excitatory postsynaptic currents (sEPSCs) and evoked EPSCs in the dorsal horn neurons (Fig. 2E) thereby, increasing the amplitude of spontaneous synaptic activity in this region. Lysozyme also increased the amplitude of afferent-mediated evoked excitatory synaptic inputs to the dorsal horn neurons by twofold (n = 29, P < 0.005; Fig. 2E). These effects on both EPSCs and excitatory postsynaptic potentials (EPSPs) were slow in onset, and the enhanced synaptic activity was notable around 35 to 45 min after lysozyme treatment, ruling out any possibility of its direct effects on ion channel conductance (Fig. 2, F to H). Lysozyme mixed with chitobiose had no significant effect on the evoked afferent-mediated excitatory synaptic inputs and spontaneous postsynaptic potentials (n = 30, P = 0.925; Fig. 2, I to K), affirming that the effects of lysozyme on neuronal excitability and modulation of pain response in rats are through the same mechanism. Chitobiose alone had no effect on neuronal excitability in these preparations (fig. S3B). Inhibition of lysozyme-mediated effects by chitobiose emphasizes the significance of the catalytic site of lysozyme in both neuronal excitation and ensuing pain sensitization.

Lysozyme exerts its effects on neurons through interaction with annexin A2 at the cell surface

Slow onset of neuronal hyperexcitability by lysozyme prompted us to investigate the molecular mediators involved in lysozyme-induced neuronal hyperexcitability. Co-immunoprecipitation (Co-IP) of rat DRG lysates with antibody against lysozyme, followed by electrospray ionization mass spectrometry analysis of tryptic peptides from the identified band position, revealed annexin A2 as its interacting partner (fig. S4A). Interaction between lysozyme and annexin A2 was further confirmed by Co-IP experiments with rat DRG lysates and SH-SY5Y human neuronal cell lysates (fig. S4B). However, preincubation of lysozyme with chitobiose abolished its interaction with annexin A2 (fig. S4B), indicating the involvement of the catalytic site of lysozyme in its interaction with annexin A2.

Annexin A2 exists in two forms: monomeric, which is confined mostly in the cytoplasm, and heterotetrameric (AIIt), which is abundant at the cell surface (38). In surface plasmon resonance (SPR) experiments, AIIt showed ~45-fold higher binding affinity (KD ≈ 3.86 nM) for lysozyme than the monomeric form (KD ≈ 178.7 nM; Fig. 3, A and B, and Table 1), indicating that lysozyme interacts preferably with AIIt. In addition, annexin A2 did not interact with α-lactalbumin (similar fold) and cytochrome c (similar pI) (fig. S4C). Because AIIt exists at the cellular surface, we investigated the spatial interaction between lysozyme and AIIt. Confocal immunofluorescence experiment on human SH-SY5Y neuronal cells treated with fluorescently labeled lysozyme showed that lysozyme was able to recruit annexin A2 at the cell surface, and its pretreatment with chitobiose abolished this interaction (Fig. 3C), thus confirming the lysozyme-AIIt interaction at neuronal cell surface.

Fig. 3 Lysozyme exerts its effects on neurons by interaction with annexin A2.

(A and B) SPR sensorgram overlays for binding of different concentrations of (A) monomeric and (B) AIIt forms to surface-immobilized lysozyme. RU, response units. (C and D) Enzymatic activity of lysozyme upon binding with increasing concentrations of (C) monomeric annexin A2 and (D) AIIt. (E) Immunofluorescence micrograph of SH-SY5Y cells showing localizations of lysozyme and annexin A2 (green) upon treatments with lysozyme alone (left) and lysozyme-chitobiose complex (right). Lysozyme labeled with Alexa Fluor 594 (red) was used for treatments. DAPI, 4′,6-diamidino-2-phenylindole. (F) Mechanical allodynia in annexin A2–siRNA–treated healthy rats upon lysozyme injection (n = 16 animals, ***P < 0.001, two-way ANOVA followed by Bonferroni’s test).

Table 1 Rate constants and equilibrium constant for the binding of different forms of annexin A2 to lysozyme, measured by SPR (Biacore).

ka and kd are rate constants for association and dissociation, respectively. KD is equilibrium dissociation constant.

View this table:

The role of the catalytic site of lysozyme in its interaction with annexin A2 was also corroborated by enzyme activity assay with Micrococcus lysodeikticus peptidoglycan, where AIIt was more potent, as compared to its monomeric counterpart, in inhibiting lysozyme activity (Fig. 3, D and E). The addition of bovine serum albumin as a control did not inhibit lysozyme activity (fig. S4D). These results confirm a specific interaction between the catalytic site of lysozyme and annexin A2.

We then investigated the role of annexin A2 in lysozyme-mediated pain in animals. Healthy rats pretreated with annexin A2–siRNA (400 μg/day intrathecally for 3 days before lysozyme injection) showed marginal effect of lysozyme injection on pain threshold (P < 0.05), whereas those injected with mismatched (mm)–siRNA exhibited reduced pain threshold upon lysozyme injection (n = 16 animals, P < 0.001 versus healthy rats; Fig. 3F). Because annexin A2 has not been shown to be involved in signaling relevant to lipopolysaccharide (LPS) or peptidoglycan-generated proinflammatory response, these studies highlight the distinct nature of lysozyme–annexin A2 partnership in eliciting neuropathic pain.

Selective activation of TLR4 by lysozyme in rat spinal DRGs induces pain

Monomeric annexin A2 activates TLR2, whereas AIIt activates TLR4 signaling (39, 40). To probe whether recruitment of annexin A2 by lysozyme proceeds through the activation of TLR2 or TLR4 signaling, we intrathecally injected a selective TLR4 inhibitor TAK-242 (1 μM) and a selective TLR2 inhibitor CuCPT-22 (1 μM) (41, 42) into healthy rats before lysozyme injections. Pretreatment with TAK-242 reduced the effects of intrathecal lysozyme injections [CSF, 100 μg/ml; n = 16 rats, P < 0.005 (Fig. 4A) and P < 0.05 (Fig. 4B)], whereas CuCPT-22 pretreatment did not affect lysozyme-induced heightened pain response [n = 12 animals, P = 0.147 (Fig. 4C) and P = 0.368 (Fig. 4D)]. To further substantiate these findings, we injected lysozyme (CSF, 100 μg/ml) into TLR2 knockout (B6.129-Tlr2tm1Kir/J) and TLR4 mutant (C3H/HeJ) mice (43, 44). Lysozyme injections did not affect TLR4 mutant mice but induced pain hypersensitivity in TLR2 knockout mice (n = 16, P < 0.001 versus aCSF at 30 min; Fig. 4E). These results demonstrate that lysozyme selectively activates TLR4 to induce pain in rats.

Fig. 4 Selective activation of TLR4 by lysozyme in rat spinal cord induces pain response.

(A and B) Effects of lysozyme injections after inhibition of TLR4 by TAK-242 in healthy rats on both (A) mechanical allodynia (n = 16 animals, ##P < 0.005 at 30 min) and (B) thermal hyperalgesia (n = 16 animals, #P < 0.05 at 30 min). (C) Mechanical allodynia (n = 12 animals, P = 0.147 versus lysozyme treatment) and (D) thermal hyperalgesia (n = 12 animals, P = 0.368 versus lysozyme treatment) upon lysozyme injections in healthy rats treated with CuCPT-22 (TLR2 inhibitor). (E) Mechanical allodynia in TLR2 knockout (B6.129-Tlr2tm1Kir/J) and TLR4 mutant (C3H/HeJ) mice subsequent to intrathecal lysozyme injections (n = 16 mice per group, ***P < 0.001). (F) Western blot analysis of lysozyme-mediated p65 expression in TAK-242–treated rat DRG lysates (n = 4, **P < 0.005). (G) Representative Western blots showing expression of p65 (**P < 0.005) and lysozyme (**P < 0.005) from mm-siRNA–, sham- and lysozyme-siRNA–treated PSL rat DRG lysates (n = 3 tissue samples). (H) qPCR analyses of lysozyme (***P < 0.001) and p65 (***P < 0.001) expression in mm-siRNA– and lysozyme-siRNA–treated PSL rats (n = 3 tissue samples). (I) Western blot analysis of p65 expression upon lysozyme treatment in annexin A2 knockdown animals (n = 5 DRG lysate samples, *** P < 0.001). (J) Representative Western blot showing p65 expression in SH-SY5Y cells treated with lysozyme-chitobiose complex (1:4 molar ratio) and inactivated forms of lysozyme (n = 4, ***P < 0.001). All behavioral data were analyzed using two-way ANOVA, followed by Bonferroni’s test.*P represents lysozyme versus inhibitor alone treatment, whereas #P stands for the combination of inhibitor and Lyz versus inhibitor alone. All Western blot repeats were performed with different tissue samples and analyzed using unpaired (two-tailed) t test.

Initiation of TLR4 signaling leads to activation of NFκB pathway. Lysozyme treatment up-regulated NFκB p65 in both rat and human neuronal cell lines (fig. S5, A to E), as well as its nuclear translocation in SH-SY5Y cells (fig. S5, F and G). Concurrently, lysozyme treatment induced NFκB p65 overexpression in rat DRGs; however, pretreatment of rats with TAK-242, followed by lysozyme injections, did not result in any changes in NFκB p65 expression (Fig. 4F). These results verified the initiation of NFκB pathway upon TLR4 activation by external application of lysozyme both ex vivo and in vivo. Next, we monitored the effect of siRNA-mediated down-regulation of endogenous lysozyme on NFκB expression in PSL rats. Western blots from the L4 and L5 DRGs show a concurrent reduction (~6-fold) in lysozyme and p65 subunit of NFκB upon lysozyme-siRNA treatment (n = 3; P < 0.005) but no change in p50 subunit (Fig. 4G). Real-time quantitative polymerase chain reaction (qPCR) data exhibit reduced expression of both lysozyme and NFκB p65 (~2.8- and 8-fold, respectively) in lysozyme-siRNA–treated rat DRGs (n = 3; P < 0.001), as compared to the scrambled siRNA–treated rat DRGs (Fig. 4H). DRG lysates from sham-operated rats were used as control. These results substantiate that endogenously overexpressed lysozyme specifically activates NFκB p65 subunit during nerve injury. Moreover, DRG extracts of rats treated with annexin A2–siRNA, as well as neuronal cells treated with annexin A2–siRNA or annexin A2–neutralizing antibody, did not show any lysozyme-mediated up-regulation of NFκB p65 (Fig. 4I and fig. S6, A and B). In addition, chitobiose or bromophenol blue–bound lysozyme or denatured lysozyme or its HLH fragment did not induce NFκB p65 expression in cell culture (Fig. 4J and fig. S6C). Thus, both externally applied and endogenously up-regulated lysozymes during nerve injury activate NFκB p65 in an annexin A2–TLR4–dependent manner in neurons.

Lysozyme activates neuronal TLR4

Our previous experiments show that minocycline had no effect on lysozyme-mediated pain induction. In addition, lysozyme treatments of SH-SY5Y human neuronal cells lead to the induction of p65 expression, indicating that lysozyme activates neuronal TLR4 and that, unlike LPS, its effects are not mediated by glial activation. LPS requires MD2 and CD14 for its binding and activation of TLR4. To further comprehend the neuronal TLR4 activation in the context of lysozyme-induced neuropathic pain, we intrathecally injected OxPAPC (a competitive inhibitor of CD14, MD2; 30 μg per animal) (45), followed by an injection of lysozyme (to achieve the CSF concentration of 100 μg/ml), into healthy rats, and LPS injections were used as control. Lysozyme injections induced pain response in the rats preinjected with OxPAPC; however, LPS injections had negligible effect on pain sensitivity in rats pretreated with OxPAPC (n = 6, P < 0.001 versus the combination of OxPAPC and LPS; Fig. 5A). Together, these findings demonstrate that neuronally overexpressed lysozyme activates neuronal TLR4 independent of MD2, during nerve injury, leading to heightened pain response. These results also underscore that the specificity of lysozyme-mediated neuronal TLR4 activation differs from the LPS-mediated TLR4 activation on the glia.

Fig. 5 Lysozyme activates TLR4 and induces hyperexcitability in both Aδ- and C-fiber nociceptors.

(A) Comparison of mechanical allodynia in OxPAPC treated rats, followed by lysozyme or LPS injections (n = 6 animals per group, ***P < 0.001 versus rats treated with the combination of OxPAPC and LPS, two-way ANOVA followed by Bonferroni’s test). (B and C) Bar graph representation of the frequency of (B) large synaptic events (EPSCs) and (C) normalized sEPSC frequency in dorsal horn neurons upon lysozyme infusion (n = 30, *P < 0.05). (D and E) Paired-pulse depression of C-fiber–mediated synaptic inputs in control- and lysozyme-treated spinal cord slices: (D) representative current traces and (E) bar graph representation of the mean paired-pulse ratio (PPR; n = 30, *P < 0.05). (F and G) Representative traces and bar graph analyses of Aδ-fiber–mediated (F) EPSPs at different stimulation frequencies [0.1 Hz (*P < 0.05) and 10 Hz (**P < 0.005), n = 29] and (G) EPSCs in some dorsal horn neurons upon lysozyme infusion (n = 29, *P < 0.05). In (C), (E), (F), and (G), error bars represent SEM.

Lysozyme induces neuronal excitability in both Aδ- and C-fiber nociceptors

We further investigated the effects of lysozyme application on nociceptor sensitization through electrophysiological recordings on Aδ- and C-fibers. These fibers were identified on the basis of their firing characteristics and capsaicin sensitivity (fig. S7). The data showed a consistent increase in frequency of spontaneous synaptic events, as well as increase in the frequency of large-amplitude events in dorsal horn neurons (n = 33, P < 0.05; Fig. 5, B and C). Lysozyme also reduced paired-pulse depression of C-fiber–mediated synaptic inputs, suggesting a presynaptic enhancement of C-fiber–mediated EPSCs (n = 30, P < 0.05; Fig. 5, D and E). It also enhanced Aδ-fiber–mediated EPSPs and EPSCs in dorsal horn neurons, indicating a reduction in the threshold for activation of some Aδ-fibers (n = 30, P < 0.05; Fig. 5, F and G). The time required for the electrophysiological effects of lysozyme on the spinal cord slices was consistent with our animal experiments, where maximum activity was observed in 30 to 35 min, excluding a direct effect of lysozyme on ion channels.

Lysozyme expression is increased during spinal cord injury in humans and interacts with TLR4 to induce neuronal excitability

Lysozyme concentrations are high in the CSF of patients with poor recovery from spinal cord injury (46). To extend our understanding about the role of lysozyme in human patients, we tested lysozyme expression after spinal cord injury in human, by Western blotting from the spinal cord autopsy tissue samples (table S1). Lysozyme expression was increased in human patients after spinal cord injury (n = 4 Western blot technical repeats, P < 0.005; Fig. 6A). Next, we performed Co-IP experiments with TLR4 antibody, which further verified lysozyme-TLR4 interaction in human spinal cord injury samples (Fig. 6B). These results are consistent with the up-regulated lysozyme expression and the existence of its interaction with TLR4 during spinal cord injury in humans.

Fig. 6 Lysozyme interacts with TLR4 during spinal cord injury in humans and stimulates neuronal excitation in human primary neurons.

(A) Western blot analysis of lysozyme expression in patients with spinal cord injury (four spinal cord samples from unrelated spinal injury cases) and healthy participant samples (patients who died because of ailments other than spinal cord injury) [n = 4 Western blot repeats, **P < 0.005, unpaired (two-tailed) t test]. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (B) Co-IP of lysozyme with TLR4 antibody (IP: TLR4) in spinal cord tissue lysates from patients with spinal cord injury (n = 3 repeats). IgG, immunoglobulin G; IB, immunoblotting. (C) Immunofluorescence micrograph analysis of C-FOS expression in human primary neurons upon treatments with lysozyme (***P < 0.001, lysozyme versus PBS), Cu CPT-22 (TLR2 inhibitor, *P = 0.05 lysozyme versus Cu CPT-22), and TAK-242 (TLR4 inhibitor, ***P < 0.001 lysozyme versus TAK-242) [n = 8 fields from independent experiments for each treatment, unpaired (two-tailed) t test]. Differentiated primary neuronal cells were marked with β-III-tubulin (green).

Last, we probed the effect of lysozyme treatment in enhanced excitability in human neurons. Stimulated sensory neurons rapidly induce expression of an immediate early gene, c-Fos, in the postsynaptic spinal cord nerve cells of the nociceptive afferents (47, 48). Hence, C-FOS expression in neurons may be indicative of sustained neuronal activation associated with pain. We treated human primary neurons (derived from human neural progenitor cells; fig. S8) with TAK-242 (1 μM) and CuCPT-22 (1 μM), followed by lysozyme treatment (culture media, 100 μg/ml), and performed immunofluorescence with these cells. Cells treated with lysozyme alone or those treated with TLR2 inhibitor, followed by lysozyme treatment, exhibited enhanced expression of C-FOS (Fig. 6C). However, human primary neurons treated with TLR4 inhibitor, followed by lysozyme treatment, did not show any change in C-FOS expression as compared to phosphate-buffered saline (PBS)–treated cells (n = 4 experiments; Fig. 6C). β-III-tubulin (TUJ1) was used as the marker for differentiated neurons. These findings demonstrate that, during nerve injury, lysozyme interacts with TLR4 and induces neuronal excitation associated with pain in humans.

DISCUSSION

The intricacies of a link between innate immunity and nervous system during neuroinflammation are increasingly being appreciated. TLRs sense PAMPs and DAMPs during infections and sterile injury, respectively. TLR4 is one of the most extensively studied TLRs, yet its role and, more importantly, activation during neuronal injury remain underexplored. Moreover, there could be a divergence between the neuronal and glial, as well as endogenous and pathogen-derived TLR4 signaling. The activation of neuronal TLR4 during sterile neuroinflammation due to nerve injury and how it affects nociceptors are still unknown. The present study demonstrates that endogenously up-regulated expression of lysozyme (an innate immune component) by the neurons regulates their own TLR4 signaling, resulting in enhanced neuronal excitability leading to heightened neuropathic pain response in rodents.

During peripheral nerve injury, lysozyme expression was enhanced in the DRG neurons and not in glial cells in rodent models. Furthermore, chemical inhibition of lysozyme by chitobiose in PSL rats and lysozyme knockdown in multiple rodent models, for 6 days, relieved pain. Thus, a causal relationship between overexpression of endogenous lysozyme in neurons during nerve injury and development of neuropathic pain could be established unambiguously.

Nerve injury results in increased excitability of neurons in the spinal cord, which is the basis of enhanced pain sensitivity. To elicit pain, lysozyme by itself should be able to alter the excitability of the neurons. Bath application of lysozyme enhanced evoked EPSCs in a subpopulation of lamina I/II dorsal horn spinal cord neurons. Lysozyme also substantially increased the amplitudes of the dorsal root afferent-mediated excitatory synaptic inputs, as well as spontaneous EPSPs and sEPSCs in the dorsal horn neurons. Slow onset of synaptic activity, 30 to 35 min after lysozyme infusion, rules out the possibility of any direct ion channel activation by lysozyme.

We identified AIIt as the interacting partner of lysozyme at the neuronal cell surface. Failure of lysozyme to evoke pain in animals, where annexin A2 was knocked down, asserts to the indispensability of this interaction in lysozyme-induced neuropathic pain. Because annexin A2 is not known to be involved in LPS or peptidoglycan-mediated inflammatory signaling, abolition of pain upon its knockdown further rules out any role of the bacteriolytic products of lysozyme in the induction of neuropathic pain. Involvement of the catalytic site of lysozyme in its interaction with AIIt also opens the possibility of perturbing this interaction by inhibitors of lysozyme such as chitobiose and bromophenol blue. Because this interaction plays an important role in pathophysiology of neuropathic pain, these findings suggest that both chitobiose (and other lysozyme inhibitor sugar derivatives) and dyes, might be effective, not only against neuropathic pain but perhaps also against other neurogenerative conditions, where an enhanced expression of lysozyme is observed.

Blocking of TLR4, but not TLR2, signaling abolished lysozyme-mediated pain. The specificity of TLR4 activation by lysozyme was further corroborated by the inability of lysozyme to induce pain in C3H/HeJ mice, which harbors a TLR4 mutation. On the other hand, induction of pain response in TLR2 knockout mice excludes any role of TLR2 in lysozyme-mediated pain. Induction of NFκB p65 expression by lysozyme treatments and its inability to do the same in the presence of TAK-242 on both human neuronal cells and rat spinal cord show the efficacy and specificity of neuronal TLR4 activation by lysozyme. Furthermore, inhibition of p65 overexpression by knockdown of lysozyme gene in the neuropathic pain models and its associated pain relief in these animals confirm the link between endogenously up-regulated neuronal lysozyme, TLR4 activation, and NFκB p65 overexpression resulting in neuropathic pain.

Unlike LPS, the ability of lysozyme to elicit pain response in the presence of OxPAPC, an inhibitor of TLR4-MD2 interaction (45), shows that lysozyme can interact with the neuronal TLR4 in an MD2-independent manner. Furthermore, unlike other endogenous TLR4 agonists (49), lysozyme-induced pain response is not dependent on glial cell activation, as evident from the inability of minocycline injections to affect chitobiose-mediated pain relief.

Nociceptor sensitization in the spinal cord dorsal horn is central to the pathophysiology of neuropathic pain. Bath application of lysozyme to the spinal cord slices enhanced the amplitude of Aδ-mediated synaptic inputs in a number of dorsal horn neurons, possibly by reducing the threshold for activation of Aδ-fibers. A consistent increase in the frequency of sEPSCs in dorsal horn neurons by lysozyme is suggestive of an effect principally on presynaptic terminals and neurotransmitters release. This also explains the delay between the time of lysozyme infusion to the spinal cord slices and the nerve fiber stimulation. Thus, lysozyme can affect the dorsal root afferent terminals to enhance excitatory neurotransmitter release. These, together with a shift in the paired-pulse ratio selectively in C-fiber–mediated synaptic inputs, demonstrate that lysozyme affects neurons in a fiber-specific manner.

Spinal cord injury is one of the leading causes of neuropathic pain in humans. An inverse correlation between lysozyme concentration in the spinal cord CSF and recovery from spinal cord injury has been indicated in a recent study (46). In agreement with our findings in animal models, we found an increased expression of lysozyme in spinal cord tissue samples from patients who died from spinal cord injury. Co-IP from these tissue lysates also confirmed the existence of lysozyme-TLR4 interaction in humans during spinal cord injury.

Induction of C-FOS is an index of enhanced neuronal activity in the nociceptive afferents. This increased neuronal activity is central to heightened pain sensitivity during neuropathy. Our observation that lysozyme application induced C-FOS expression in human primary neuronal cells, in a TLR4-dependent manner, points that lysozyme-TLR4 interaction can lead to neuronal hyperexcitability in humans.

Together, this study demonstrates that lysozyme regulates neuronal excitability during nerve injury in murine models and in humans. With a plethora of techniques and multiple combinations of knockdown and knockout approaches, we identified the existence of neuronal lysozyme–annexin A2–TLR4 axis during neuropathy. Induction of neuropathic pain by lysozyme through activation of neuronal TLR4 without the participation of MD2 makes it a distinct mechanism from that mediated by LPS. The role of TLR4 in neuroinflammation and pain has often been studied with externally infused LPS as the agonist, which mostly activates glial TLR4, leading to underrepresentation of the contribution of neuronal TLR4 in pain pathology. Other endogenous TLR4 agonists (including HMGB1 and HSP60) have been shown to modulate neuronal physiology only in the presence of glial TLR4 (50, 51). The present study identifies an unexplored function of lysozyme in pain physiology and proposes it to be an activator of neuronal TLR4 signaling culminating in heightened neuronal excitability.

The fact that lysozyme can excite neuronal cells can be of interest because it can have an important role in neuromuscular and neuroimmune interactions, especially during infections. Furthermore, lysozyme expression is extremely low in nervous system under healthy conditions (52) and gets up-regulated during nerve injury and neurodegenerative diseases, such as multiple sclerosis, Parkinson’s disease, Alzheimer’s disease, and spinal cord and peripheral nerve injuries, both in CNS and PNS, respectively (1, 2, 2629). Thus, blocking lysozyme in the nervous system might be safer than perturbing the core cellular signaling molecules such as TLR4 or NFκB because it might not have an adverse effect on normal neuronal physiology.

In conclusion, the study suggests that lysozyme inhibitors might be promising drugs for treating neuropathic pain. This study also advances our understanding of neuroinflammation, where neurons, by up-regulating the expression of an endogenous ligand lysozyme, alter their own excitability without engaging microglia or any other immune cell.

The study has some limitations that are worth discussing. We chose three widely used murine models of nerve injury during the study because none of these completely recreates the human conditions during nerve injury. The human data section of the study is limited by its small sample size. The study highlights a plausible role of lysozyme in the neuroinflammatory process. However, further investigations will be required to establish the role of lysozyme in the individual disease conditions involving different regions of the CNS and to explore lysozyme-mediated TLR4 activation and the downstream signaling events involved in neuroinflammation.

METHODS

Study design

The objective of this study was to explore the role of lysozyme overexpression during nerve injury. We compared the lysozyme expression in the DRGs from the extensively used rat nerve ligation models and patients with spinal cord injury upon nerve injury with that of healthy controls. We further identified the mechanistic details of the role of lysozyme during nerve injury–associated neuropathic pain by genetic and pharmacological manipulations, as well as biochemical, biophysical, and electrophysiological approaches. Last, we conclude that enhanced lysozyme leads to increased neuronal activity and pain during nerve injury. Control and neuropathic pain rat models were from the same litter and were randomly selected for experiments. Replicates and statistical tests are cited with each result. All behavioral testing was performed blind to the identity of the inhibitors administered in the animal groups. All behavioral studies were performed during the day in a quiet and temperature-controlled room.

Statistical analysis

All the experiments were carried out at least thrice independently. All behavioral data are presented as means ± SD. Animals of similar age, weight, and sex were grouped randomly, and the number of animals per group was 6 to 18. Statistical analyses were conducted using GraphPad Prism 6 (GraphPad Software Inc.). The behavioral data were analyzed using two-way ANOVA, followed by Bonferroni posttest for multiple comparisons. Western blots were analyzed by unpaired Student’s t test (two-tailed), unless specified. Electrophysiology data were expressed as means ± SEM. Statistical analysis was performed using Student’s t test, paired or unpaired as appropriate. All other data were analyzed with univariate ANOVA or ANOVA for repeated measurements, followed by Bonferroni posttest for multiple comparisons. P < 0.05 was considered significant for all tests.

SUPPLEMENTARY MATERIALS

stm.sciencemag.org/cgi/content/full/11/504/eaav4176/DC1

Materials and Methods

Fig. S1. Lysozyme contributes to pain sensitivity in PSL rats.

Fig. S2. Lysozyme also induces pain in female rats.

Fig. S3. Chitobiose treatment did not affect neuronal firing properties.

Fig. S4. Lysozyme interacts specifically with annexin A2.

Fig. S5. Lysozyme treatment increases NFκB p65 expression and its nuclear localization in neuronal cell lines.

Fig. S6. Lysozyme specifically regulates NFκB p65 through annexin A2 in SH-SY5Y human neuronal cells.

Fig. S7. Lysozyme activates both Aδ- and C-fiber nociceptors.

Fig. S8. Micrograph showing different stages of human primary neuronal cell culture.

Fig. S9. Schematic diagram representing the proposed mechanism of lysozyme action on DRG neurons.

Table S1. Clinical details of human patients.

Table S2. List of reagents.

Table S3. Raw data (provided as separate Excel file).

Movie S1. Activity of a scrambled siRNA–treated Sprague Dawley rat model of neuropathic pain.

Movie S2. Activity of a lysozyme-siRNA–treated Sprague Dawley rat model of neuropathic pain.

Movie S3. Activity of a sham-operated Sprague Dawley rat.

Movie S4. Activity of aCSF-injected Sprague Dawley rat.

Movie S5. Activity of lysozyme-injected Sprague Dawley rat.

Movie S6. Activity of lysozyme-injected female Sprague Dawley rat.

References (5362)

REFERENCES AND NOTES

Acknowledgments: We thank D. Spanswick and A. Whyment for help in conducting electrophysiology experiments in a double-blinded fashion at NeuroSolutions Ltd., Coventry, CV4 7ZS, UK; R. Chaturvedi of Indian Institute of Toxicology Research (IITR), Lucknow, India, for critical discussions on the findings; and S. Mayor of National Centre for Biological Sciences, Bangalore, and S.K. Sikdar of Molecular Biophysics Unit for critical comments on confocal microscopy images. We also thank A. Mahadevan of National Institute of Mental Health and Neurosciences (NIMHANS) Bangalore, India, for providing human spinal cord samples. A. Singh is acknowledged for help in data acquisition, compilation, and plotting. Funding: This work was supported by CSIR (grant number HRD/Bhatnagar Fellow/2011) and SERB (grant number SB/DF-003/2018) through extramural grants to A.S. A.S. is a Bhatnagar Fellow of Council of Scientific and Industrial Research (CSIR), India and Science and Engineering Research Board India (SERB) Distinguished Fellow. Author contributions: S.Y. designed the study, performed the experiments, and analyzed the data. A.S. provided the infrastructure and supervised the study. Both authors conceived the investigations and prepared, read, and contributed to the final manuscript. Competing interests: Authors declare that they have no competing interests. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials.
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