Research ArticleCELLULAR NEUROSCIENCE

Inflammation up-regulates cochlear expression of TRPV1 to potentiate drug-induced hearing loss

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Science Advances  17 Jul 2019:
Vol. 5, no. 7, eaaw1836
DOI: 10.1126/sciadv.aaw1836
  • Fig. 1 TRPV1 immunoexpression in cell lines.

    (A) Immunoblot of lysates from kidney cell lines using TRPV1 antisera (cat# ACC-030; Alomone Labs). At ~95 kDa, the expected molecular mass of TRPV1, KPT2, and KPT11 cells exhibited weak band densities, while KDT3 and MDCT cells had strong band densities. At ~42 kDa, immunolabeling for actin served as a loading control. (B) TRPV1 immunofluorescence was detected in KPT11 and MDCT cells, with negligible fluorescence in KPT2 and KDT3 cells. (C) Robust TRPV1 immunofluorescence is present in KPT2-Trpv1, but not in control KPT2-pBabe, cells. Scale bars, 20 μm.

  • Fig. 2 Activation of heterologously expressed TRPV1 enhanced cellular uptake of gentamicin.

    (A) Following 1-min incubation with GTTR, KPT2-Trpv1 cells display more GTTR fluorescence compared to KPT2-pBabe cells. KPT2-Trpv1 cells treated with RTX (5 nM) exhibit increased GTTR fluorescence compared to RTX-treated KPT2-pBabe cells or KPT2-Trpv1 cells treated with GTTR only. Weak GTTR fluorescence in I-RTX–treated KPT2-Trpv1 cells was similar in intensity to KPT2-pBabe cells treated with GTTR only or with a TRPV1 antagonist (I-RTX, 100 nM) and was visibly less intense than in KPT2-Trpv1 cells treated with GTTR only. Scale bar, 20 μm. (B) Significantly increased pixel intensities for GTTR fluorescence in (i) KPT2-Trpv1 cells compared to KPT2-pBabe cells; (ii) RTX-treated KPT2-Trpv1 cells compared to RTX-treated KPT2-pBabe cells, non-RTX–treated KPT2-Trpv1 cells, or non-RTX–treated KPT2-pBabe cells; and (iii) KPT2-Trpv1 cells compared to I-RTX–treated KPT2-Trpv1 cells and I-RTX–treated KPT2-pBabe cells (*P < 0.05; n = 4 per group). ELISAs revealed significantly increased levels of GTTR (C) and native gentamicin (D) in (i) KPT2-Trpv1 cells compared to KPT2-pBabe cells; (ii) RTX-treated KPT2-Trpv1 cells compared to RTX-treated KPT2-pBabe cells, and untreated KPT2-Trpv1 or KPT2-pBabe cells; and (iii) KPT2-Trpv1 cells compared to I-RTX–treated KPT2-Trpv1 or KPT2-pBabe cells (*P < 0.05; n = 4 per group). Error bars, SD. a.u., arbitrary units.

  • Fig. 3 Heterologous TRPV1-mediated uptake of gentamicin is attenuated by extracellular [Ca2+].

    (A) KPT2-Trpv1 cells treated with GTTR (5 μg/ml) show reduced fluorescence when coincubated with 2 mM extracellular [Ca2+]. The TRPV1 agonist capsaicin (3 μM) visibly increased GTTR fluorescence over untreated KPT2-Trpv1 cells. Capsaicin-enhanced GTTR uptake is visibly attenuated by 2 mM extracellular [Ca2+]. Scale bar, 20 μm. (B) GTTR fluorescence in KPT2-pBabe cells was significantly less than that in KPT2-Trpv1 cells. Extracellular [Ca2+] (2 mM) diminished GTTR fluorescence in KPT2-Trpv1 cells. Capsaicin significantly increased GTTR fluorescence in KPT2-Trpv1 cells but not in KPT2-pBabe cells. Capsaicin-enhanced GTTR uptake in KPT2-Trpv1 cells is significantly attenuated by 2 mM extracellular [Ca2+], without modulating GTTR levels in KPT2-pBabe cells (P > 0.05; n = 4 per group). ELISA data for cellular levels of GTTR (C) and native gentamicin (D) support imaging data described in (A) and (B) (*P < 0.05; n = 4 per group). Error bars, SD.

  • Fig. 4 Activation of heterologous TRPV1 exacerbated gentamicin-induced cytotoxicity.

    (A) KPT2-Trpv1 cells are more susceptible to 10 mM gentamicin (GT) treatment for 3 hours in the presence of TRPV1 agonist capsaicin (CAP; *P < 0.05; **P < 0.01) but not when coincubated with a TRPV1 antagonist, capsazepine (CPZ). The viability of parental KPT2 or KPT2-pBabe cells (without heterologous TRPV1 expression) was not significantly altered by GT alone or by GT in the presence of capsaicin or capsazepine. (B) Strong TRPV1 immunofluorescence in MDCT cells (transfected with control siRNA) was greatly diminished in MDCT cells transfected with siRNA for TRPV1. Scale bar, 20 μm. (C) Expression of Trpv1 RNA in MDCT cells is significantly diminished by siRNA for Trpv1 (*P < 0.05; n ≥ 4). (D) MDCT cells transfected with siRNA for TRPV1 and then treated with GT were significantly more viable than MDCT cells transfected with control siRNA (**P < 0.01; n = 8). Error bars, SD.

  • Fig. 5 TRPV1 expression in kidney and inner ear.

    (A) In wild-type mice, TRPV1 immunofluorescence (green in color images) is present (arrows) near the apical brush border (red in color images) of proximal tubule cells, as well as in the soma of proximal, and as puncta in distal, tubule cells. Negligible immunofluorescence when the TRPV1 antibody was adsorbed with the antigenic peptide. Ab, antibody. (B) In whole mounts of the cochlear lateral wall, specific TRPV1 immunofluorescence near the lumenal surface of marginal cells (white or green) at the level of the actiniferous tight junctions (red) between adjacent marginal cells. Weak TRPV1 immunolabeling in intermediate cells, with stronger, more punctate, labeling in basal cells and fibrocytes. Negligible fluorescence detected in strial cells or fibrocytes when the TRPV1 antibody is adsorbed with the antigenic peptide. (C) In z-sections of the stria vascularis, TRPV1 immunofluorescence (green in the lower panel) is localized near the lumenal surface of the marginal cells (m), between the actiniferous tight junctions (red in the lower panel, arrows), and in basal cells (b) and fibrocytes (f) below the weakly labeled soma of intermediate (i) cells. (D) In the organ of Corti, punctate TRPV1 immunofluorescence (green, vertical arrows) is present in cytoplasmic channels in the cuticular plate, as well as a stripe near the lateral side (diagonal arrows), of OHCs. TRPV1 immunofluorescence is also localized in the inner pillar cell phalanges (ip) between IHCs and OHCs, outer pillar cell phalanges (op) between OHCs of the first row, and Deiters’ cells phalanges (d) between OHCs in row 2 and row 3 of OHCs. TRPV1 immunolabeling also lines the periphery of IHCs, especially on the modiolar side of IHC (yellow arrows). Scale bars, 20 μm.

  • Fig. 6 LPS enhanced TRPV1 expression and GTTR uptake in cochlear coil explants.

    (A) In organ of Corti explants from neonatal Pcdh15+/− and Pcdh15 KO mice, TRPV1 immunofluorescence was visibly more intense in the phalangeal apices of inner and outer pillar cells (ip and op, respectively), as well as Deiters’ cells (d), compared to IHC, and especially OHC, apices and inner sulcal cells (is). Twenty-four hours after a 1-hour exposure to LPS (1 μg/ml), enhanced TRPV1 immunofluorescence is observed throughout organ of Corti explants from Pcdh15+/− and Pcdh15 KO mice compared to untreated explants. (B) LPS significantly increased the pixel intensity of TRPV1 immunofluorescence in inner pillar cells, IHCs, outer pillar cells, OHCs, and Deiters’ cells, but not in inner sulcus cells, in organ of Corti explants from Pcdh15+/− (Het) and Pcdh15 KO (KO) mice over that in non–LPS-treated cochlear coils (*P < 0.05; n ≥ 3). (C) In untreated explants incubated with GTTR for 5 min, GTTR fluorescence was visibly more intense in Pcdh15+/− OHCs and IHCs than in control hair cells from Pcdh15 KO mice. GTTR fluorescence is also visible in pillar cells between the OHCs and IHCs, especially in Pcdh15 KO explants. Twenty-four hours after exposure to LPS, enhanced GTTR fluorescence is observed in Pcdh15+/− OHCs, IHCs, and pillar cell apices compared to control Pcdh15+/− explants. Enhanced GTTR fluorescence is also observed in OHCs, IHCs, and pillar cell apices of Pcdh15 KO explants exposed to LPS compared to control Pcdh15 KO explants. (D) LPS exposure significantly increased the intensity of GTTR fluorescence in OHCs of both Pcdh15+/− and Pcdh15 KO cochlear explants (****P < 0.0001; n ≥ 3). Scale bars, 20 μm. Error bars, SD.

  • Fig. 7 LPS enhances cochlear TRPV1 expression and GTTR uptake in vivo.

    (A) LPS enhanced TRPV1 expression in wild-type (WT) marginal cells compared to DPBS-treated wild-type mice. LPS did not enhance TRPV1 expression in marginal cells of Tlr4 KO mice. (B) LPS significantly enhanced TRPV1 immunofluorescence in marginal cells (MC) of wild-type, but not Tlr4 KO, mice (**P < 0.01; n ≥ 6). TRPV1 immunofluorescence in intra-strial (IS) and basal cells (BC), as well as spiral ligament fibrocytes (SL), was not significantly altered. (C) LPS significantly enhanced TRPV1 immunofluorescence in OHCs of wild-type, but not Tlr4 KO, mice (*P < 0.05; n ≥ 5). (D) LPS enhanced TRPV1 expression in the reticular lamina of wild-type mice compared to DPBS-treated wild-type mice. LPS did not enhance TRPV1 expression in the organ of Corti of Tlr4 KO mice. (E) Significantly increased Trpv1 mRNA expression in the cochleae of wild-type, but not in Tlr4 KO, cochleae following LPS exposure (*P < 0.05; n ≥ 5). (F) Significantly increased Trpv1 mRNA expression in the cochleae of wild-type, but not Trpv1 KO, cochleae following LPS exposure (**P < 0.01; n ≥ 3). (G) Intravenous LPS 24 hours before GTTR injection significantly increased OHC uptake of GTTR in wild-type mice (****P < 0.0001; n ≥ 3) but not in Trpv1 KO mice (H; n ≥ 3). Error bars, SD. Scale bars, 20 μm.

  • Fig. 8 LPS exacerbates kanamycin-induced hearing loss in wild-type, but not in Trpv1+/−or Trpv1 KO, mice.

    (A) ABR threshold shifts plotted by treatment group at 3 weeks after treatment. Wild-type (WT), Trpv1+/−, or Trpv1 KO mice treated with DPBS had similar PTS, except for Trpv1+/− mice with significantly improved thresholds at 12 kHz (P < 0.001) and larger PTS at 32 kHz (P < 0.001). LPS-treated Trpv1 KO mice had significant PTS compared to wild-type or Trpv1+/− mice at 24 and 48 kHz (P < 0.001). After kanamycin treatment, wild-type mice generally had greater threshold shifts than Trpv1 KO mice (except at 48 kHz) and significant threshold shifts compared to Trpv1+/− mice at 8, 12, 16, 24, and 32 kHz (P < 0.05). Following LPS + kanamycin, wild-type mice had significantly greater threshold shifts (P < 0.05) compared to Trpv1+/− or Trpv1 KO mice at all frequencies (except 48 kHz). See tables S4 to S7 for all P values; error bars, SD; n ≥ 5 mice (10 cochleae) per group. (B) OHC survival plotted by treatment group at 3 weeks after treatment. Wild-type, Trpv1+/−, or Trpv1 KO mice treated with DPBS had negligible OHC losses, except at the extreme basal region of the cochlea, and did not differ from each other. LPS-treated mice also had negligible OHC loss except at the extreme basal 10% region of the cochlea and did not substantially differ from other mouse strains. After kanamycin treatment, wild-type mice generally had greater OHC loss across the majority of the cochlea, except in the apical and extreme basal regions of the cochlea. Following LPS + kanamycin, wild-type mice had greater OHC loss compared to Trpv1+/− or Trpv1 KO mice in all, except the extreme basal regions of the cochlea. n ≥ 5 mice per group (one cochlea per mouse); error bars, 95% confidence interval derived from two-way ANOVA Tukey’s multiple comparisons tests.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/7/eaaw1836/DC1

    Fig. S1. Stable heterologous expression of TRPV1 in selected cell lines.

    Fig. S2. RTX-enhanced TRPV1-mediated uptake of gentamicin is attenuated by extracellular [Ca2+].

    Fig. S3. Viability of cell lines incubated with a TRPV1 agonist or antagonist.

    Fig. S4. Agonists and antagonists of TRPV1 do not affect the bactericidal efficacy of aminoglycosides.

    Fig. S5. LPS enhanced TRPV1 expression in renal cells in vivo.

    Fig. S6. LPS did not enhance strial uptake of GTTR in Trpv1 KO mice in vivo.

    Fig. S7. LPS exacerbates kanamycin-induced hearing loss in wild-type, but not in Trpv1+/− or Trpv1 KO, mice.

    Fig. S8. LPS increases cochlear (but not serum) levels of gentamicin.

    Fig. S9. LPS exacerbates kanamycin-induced OHC loss in wild-type, but not in Trpv1+/− or Trpv1 KO, mice.

    Fig. S10. TRPV1 immunolabeling in cochlear tissues from wild-type and Trpv1 KO mice.

    Table S1. Significant differences in threshold at 3 weeks after treatment from baseline for wild-type (C57) mice.

    Table S2. Significant differences in threshold at 3 weeks after treatment from baseline for Trpv1+/− mice.

    Table S3. Significant differences in threshold at 3 weeks after treatment from baseline for Trpv1 KO mice.

    Table S4. Significant differences in thresholds between strains at 3 weeks after treatment with DPBS only.

    Table S5. Significant differences in thresholds between strains at 3 weeks after treatment with LPS only.

    Table S6. Significant differences in thresholds between strains at 3 weeks after treatment with kanamycin only.

    Table S7. Significant differences in thresholds between strains at 3 weeks after treatment with LPS + kanamycin.

    Table S8. Significant differences in thresholds between treatment groups at 3 weeks after treatment for wild-type (C57) mice.

    Table S9. Significant differences in thresholds between treatment groups at 3 weeks after treatment for Trpv1+/− mice.

    Table S10. Significant differences in thresholds between treatment groups at 3 weeks after treatment for Trpv1 KO mice.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Stable heterologous expression of TRPV1 in selected cell lines.
    • Fig. S2. RTX-enhanced TRPV1-mediated uptake of gentamicin is attenuated by extracellular Ca2+.
    • Fig. S3. Viability of cell lines incubated with a TRPV1 agonist or antagonist.
    • Fig. S4. Agonists and antagonists of TRPV1 do not affect the bactericidal efficacy of aminoglycosides.
    • Fig. S5. LPS enhanced TRPV1 expression in renal cells in vivo.
    • Fig. S6. LPS did not enhance strial uptake of GTTR in Trpv1 KO mice in vivo.
    • Fig. S7. LPS exacerbates kanamycin-induced hearing loss in wild-type, but not in Trpv1+/− or Trpv1 KO, mice.
    • Fig. S8. LPS increases cochlear (but not serum) levels of gentamicin.
    • Fig. S9. LPS exacerbates kanamycin-induced OHC loss in wild-type, but not in Trpv1+/− or Trpv1 KO, mice.
    • Fig. S10. TRPV1 immunolabeling in cochlear tissues from wild-type and Trpv1 KO mice.
    • Table S1. Significant differences in threshold at 3 weeks after treatment from baseline for wild-type (C57) mice.
    • Table S2. Significant differences in threshold at 3 weeks after treatment from baseline for Trpv1+/− mice.
    • Table S3. Significant differences in threshold at 3 weeks after treatment from baseline for Trpv1 KO mice.
    • Table S4. Significant differences in thresholds between strains at 3 weeks after treatment with DPBS only.
    • Table S5. Significant differences in thresholds between strains at 3 weeks after treatment with LPS only.
    • Table S6. Significant differences in thresholds between strains at 3 weeks after treatment with kanamycin only.
    • Table S7. Significant differences in thresholds between strains at 3 weeks after treatment with LPS + kanamycin.
    • Table S8. Significant differences in thresholds between treatment groups at 3 weeks after treatment for wild-type (C57) mice.
    • Table S9. Significant differences in thresholds between treatment groups at 3 weeks after treatment for Trpv1+/− mice.
    • Table S10. Significant differences in thresholds between treatment groups at 3 weeks after treatment for Trpv1 KO mice.

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