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
  • 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.

  • 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.

  • 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).

  • 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.

  • 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.

  • 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).

  • 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.

    Annexin A2
    forms
    ka (M−1 s−1)kd (s−1)KA (M−1)KD (M)
    (104)(10−4)(106)(10−9)
    Monomer0.37445.2746.928178.74
    Heterotetramer27.642012.886391.23.862

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)

  • The PDF file includes:

    • 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.
    • References (5362)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S3. Raw data (provided as separate Excel file).
    • Movie S1 (.mp4 format). Activity of a scrambled siRNA–treated Sprague Dawley rat model of neuropathic pain.
    • Movie S2 (.mp4 format). Activity of a lysozyme-siRNA–treated Sprague Dawley rat model of neuropathic pain.
    • Movie S3 (.mp4 format). Activity of a sham-operated Sprague Dawley rat.
    • Movie S4 (.mp4 format). Activity of aCSF-injected Sprague Dawley rat.
    • Movie S5 (.mp4 format). Activity of lysozyme-injected Sprague Dawley rat.
    • Movie S6 (.mp4 format). Activity of lysozyme-injected female Sprague Dawley rat.

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