Galvanic current activates the NLRP3 inflammasome to promote Type I collagen production in tendon
- Instituto Murciano de Investigación Biosanitaria IMIB-Arrixaca, Hospital Clínico Universitario Virgen de la Arrixaca, Spain
- Department of Physical Therapy, University of Murcia, Spain
- Laboratorio de Óptica, Instituto Universitario de Investigación en Óptica y Nanofísica, Universidad de Murcia, Spain
- CEU San Pablo University, Spain
- MVClinic Institute, Spain
- Invasive Physiotherapy Department, Getafe C.F, Spain
- Department of Biochemistry and Molecular Biology B and Immunology, University of Murcia, Spain
Figures
Figure 1 with 1 supplement
Galvanic current increases the M1 phenotype of macrophages.
(A) Quantitative PCR for M1 genes Cox2, Il6, Il1b and Tnfa expression from mouse bone-marrow-derived macrophages (BMDMs) treated for 2 h with LPS (1 μg/ml) or 4 h with IL-4 (20 ng/µl) as indicated and then 2 impacts of 12 mA of galvanic current for 6 s. Cells were then further cultured for 6 h before analysis. Center values represent the mean and error bars represent s.e.m.; n = 5–10 samples of five independent experiments; for Cox2 unpaired t-test except in the two first columns where Mann-Whitney tests were performed, for Il6 Mann-Whitney test except in LPS vs LPS+ galvanic current comparison where unpaired t-tests were performed, for Il1b Mann-Whitney test, for Tnfa unpaired t-test except in LPS vs LPS+ galvanic current comparison where Mann-Whitney were performed, ***p < 0.0005, **p < 0.005, *p < 0.05, and ns p > 0.05. (B) Quantitative PCR for M2 genes Arg1, Fizz1, Mrc1 and Ym1 expression from BMDMs treated as in (A). Center values represent the mean and error bars represent s.e.m.; n = 3–10 samples of five independent experiments; unpaired t-test except LPS vs LPS+ galvanic current comparison where Mann-Whitney tests were performed, ***p < 0.0005, *p < 0.05, and ns p > 0.05. (C) IL-6, TNF-α and IL-1β release from BMDMs treated as in (A) but with different intensities of galvanic current (3, 6, 12 mA); ND: non detected. Center values represent the mean and error bars represent s.e.m.; n = 2 for untreated cells and n = 4–6 for treatment groups from four independent experiments; one-way ANOVA were performed comparing treated groups with the control group, ****p < 0.0001, *p < 0.05, and ns p > 0.05.
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Figure 1—source data 1
Raw data of panels of Figure 1.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Device designed to apply galvanic current in 6-well plates.
The two poles are separated by plastic spacers and generate a homogeneous and constant galvanic current throughout the well.
Figure 2 with 1 supplement
IL-1β release induced by galvanic current is dependent on the NLRP3 inflammasome.
(A) IL-1β release from wild type, Casp1/11-/-, Pycard-/- and Nlrp3-/- mouse bone-marrow-derived macrophages (BMDMs) treated for 2 h with LPS (1 μg/ml) and then 2 impacts of different intensities of galvanic current (3, 6, 12 mA) for 6 s. Cells were then further cultured for 6 h before cytokine were measured in supernatant. Center values represent the mean and error bars represent s.e.m.; n = 6–16 samples of 10 independent experiments; LPS vs LPS+ galvanic current in wild type and wild type vs Nlrp3-/- unpaired t-test, wild type vs Casp1/11-/- and wild type vs Pycard-/- Mann-Whitney test, **p < 0.005. (B) IL-1β release from wild type BMDMs treated as in A but applying the NLRP3-specific inhibitor MCC950 (10 μM) 30 min before the galvanic current application and during the last 6 h of culture. As a control, cells were treated with nigericin (1.5 μM) rather than galvanic current. Center values represent the mean and error bars represent s.e.m.; n = 5–10 samples of 5 independent experiments; nigericin vs nigericin+ MCC950 Mann-Whitney test, galvanic current vs galvanic current+ MCC950 unpaired t-test, ****p < 0.0001 and ***p < 0.0005. (C) IL-18 release from BMDMs treated as in A. Center values represent the mean and error bars represent s.e.m.; n = 2–7 samples of at least two independent experiments. (D) IL-1β release from wild type BMDMs treated as in A but applying a buffer with 40 mM of KCl (high K+ buffer) during the last 6 h of culture. As controls, cells were treated with nigericin (1.5 μM) or Clostridium difficile toxin B (1 μg/ml) rather than galvanic current. Center values represent the mean and error bars represent s.e.m.; n = 3–12 samples of four independent experiments; unpaired t-test, ****p < 0.0001, **p < 0.005 and ns p > 0.05. (E) Intracellular K+ concentration from wild type BMDMs primed with LPS as in A, but then treated for 6 h with nigericin (1.5 μM) or two or 8 impacts of 12 mA for 6 or 12 s as indicated. Center values represent the mean and error bars represent s.e.m.; n = 3–8 samples of three independent experiments; Mann-Whitney test, **p < 0.005 and *p > 0.05. (F) Immunoblot of cell extract and supernatants for caspase-1, IL-1β, GSDMD and β-actin from wild type BMDMs treated as in B, but with eight impacts. Representative of n = 2 independent experiments. (G) Fluorescence microscopy of HEK293T cells stably expressing NLRP3–YFP after 6 h of application of 2 impacts of 12 mA of galvanic current for 6 s, the specific inhibitor MCC950 (10 μM) was added 30 min before the galvanic current; scale bar 10 μm; n = 3 independent experiments.
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Figure 2—source data 1
Uncropped western blots with the relevant bands boxed from Figure 2D.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig2-data1-v2.pdf
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Figure 2—source data 2
Original files of the full raw unedited blots from Figure 2D.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig2-data2-v2.zip
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Figure 2—source data 3
Raw data of panels from Figure 2.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig2-data3-v2.xlsx
Figure 2—figure supplement 1
Low number of galvanic current impacts does not induce a detectable decrease in intracellular K+.
(A) IL-1β release from wild type, Casp1/11-/-, Pycard-/- and Nlrp3-/- mouse bone-marrow-derived macrophages (BMDMs) treated for 2 h with LPS (1 μg/ml) and then for 6 h with nigericin (1 μM). Center values represent the mean and error bars represent s.e.m.; n = 2–8 samples of three independent experiments; unpaired t-test, **p < 0.005. (B) Intracellular K+ concentration from wild type BMDMs primed with LPS as in A, but then treated for 30 min with nigericin (1 μM) or for the indicated time after 2 impacts of 12 mA for 6 s. Center values represent the mean and error bars represent s.e.m.; n = 5–9 samples of nine independent experiments; LPS vs LPS+ nigericin unpaired t-test and LPS vs LPS+ galvanic current at different times one-way ANOVA, ***p < 0.0005 and ns p > 0.05.
Figure 3
Galvanic current does not induce inflammasome-mediated pyroptosis.
(A) Extracellular amount of LDH determining cell membrane damage from mouse bone-marrow-derived macrophages (BMDMs) treated for 2 h with LPS (1 μg/ml) and then two or 8 impacts of different intensities of galvanic current (3, 6, 12 mA) for 6 or 12 s were applied as indicated. Cells were then further cultured for 6 h before LDH determination in supernatant. Center values represent the mean and error bars represent s.e.m.; n = 3–4 samples of six independent experiments; Kruskal-Wallis test to compare LPS with increasing intensities of galvanic current, LPS vs LPS+ galvanic current (12mA-12s)x2 unpaired t-test, LPS vs LPS+ galvanic current (12mA-6s)x8, ****p < 0.0001, ***p < 0.0005 and *p < 0.05. (B) Extracellular amount of LDH from wild type, Nlrp3-/-, Pycard-/-, and Casp1/11 /- mouse BMDMs treated as in A. Center values represent the mean and error bars represent s.e.m.; n = 6–17 samples of 12 independent experiments; unpaired t-test except for Casp1/11-/- comparison, ***p < 0.0005 and *p < 0.05. (C) IL-1β (left) and IL-18 (right) release from wild type BMDMs treated as in A, but applying the NLRP3-specific inhibitor MCC950 (10 μM) during the last 6 h of culture. Center values represent the mean and error bars represent s.e.m.; n = 6–10 samples of 5 independent experiments; unpaired t-test, ****p < 0.0001 and *p < 0.005. (D) Extracellular amount of LDH from wild type BMDMs treated as in C. Center values represent the mean and error bars represent s.e.m.; n = 5–10 samples of five independent experiments; unpaired t-test, ns p > 0.005. (E) Kinetic of Yo-Pro-1 uptake (upper panel) or slope of the uptake (lower panel) in wild type BMDMs treated for 2 h with LPS (1 μg/ml) and then with different intensities of galvanic current (as indicated) or with the detergent triton X-100 (1 %) for 3.5 h. Center values represent the mean and error bars represent s.e.m.; n = 3–6 of three independent experiments; Kruskal-Wallis test, ***p < 0.0005, **p < 0.005 and ns p > 0.05. (F) Yo-Pro-1 uptake indicating plasma membrane pore formation and cell viability. Kinetic of Yo-Pro-1 uptake (upper panel) or slope of the uptake (lower panel) in wild type or Pycard-/- BMDMs treated as in E but, when indicated, the NLRP3 specific inhibitor MCC950 (10 μM) was added before galvanic current application. Center values represent the mean and error bars represent s.e.m.; n = 3–6 samples of three independent experiments; unpaired t-test, **p < 0.005, *p < 0.05 and ns p > 0.05.
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Figure 3—source data 1
Raw data of panels from Figure 3.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
Galvanic current induces polymorphonuclear and macrophage infiltrate in the calcaneal tendon of mice.
(A) Representative hematoxylin and eosin images of wild type mice calcaneal tendon after 3 days’ application of 3 punctures with needle (needling, green) or 3 impacts of 3 mA for 3 s (blue). Scale bar: 50 μm. Magnification show the presence of polymorphonuclear cells (arrowheads). (B) Quantification of polymorphonuclear cells per field of view of calcaneal tendon sections treated and stained as described in A. Center values represent the mean and error bars represent s.e.m.; n = 7–8 independent animals; unpaired t-test, *p < 0.005. (C) Representative immunostaining images for the macrophage marker F4/80 from the calcaneal tendon of wild type mice treated as described in A. Scale bar: 50 μm. Magnification show the presence of F4/80-positive cells (arrowheads). (D) Quantification of F4/80-positive cells per field of view of calcaneal tendon sections treated and stained as described in C. Center values represent the mean and error bars represent s.e.m.; n = 8 independent animals; Mann-Whitney test, *p < 0.005.
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Figure 4—source data 1
Raw data of panels from Figure 4C and D.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig4-data1-v2.xlsx
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Figure 4—source data 2
Representation of raw data of panels from Figure 4C and D.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig4-data2-v2.pdf
Figure 4—figure supplement 1
Galvanic current does not affect tendon mastocytes, tenocytes, or vascularity.
(A) Quantification of mastocytes per field of view of calcaneal tendon sections stained with toluidine blue from wild type mice after 3 days application of 3 punctures with needle (needling, green) or 3 impacts of 3 mA for 3 s (blue). Center values represent the mean and error bars represent s.e.m.; n = 6–15 independent animals; Mann-Whitney test, *p < 0.05. Images on the right show representative sections where mastocytes were counted, scale bar 50 μm. (B) IL-1β and LDH release from mice bone-marrow isolated neutrophils primed for 2 h with LPS (1 μg/ml) and then treated for 30 min with ATP (3 mM) or 6 h after application of 2 impacts of 12 mA of galvanic current for 6 s. Center values represent the mean and error bars represent s.e.m.; n = 3 independent animals. (C) Representative hematoxylin and eosin images of wild type mice calcaneal tendon untreated or after 3 days of 3 punctures with needle (needling) or 3 impacts of 3 mA for 3 s (percutaneous electrolysis), scale bar 200 µm. (D,E,F) Number, area and circularity of nuclei of tenocytes of calcaneal tendon sections of wild type mice stained with hematoxylin and eosin after 3 days of 3 punctures with needle (needling, green) or 3 impacts of 3 mA for 3 s (blue). Center values represent the mean and error bars represent s.e.m.; n = 4–14 independent animals; unpaired t-test, ****p < 0.0001, *p < 0.05 and ns p > 0.05. (G) Relative neovascularization assessed in calcaneal tendon sections stained with hematoxylin and eosin after different time (as indicated) of the application of 3 punctures with needle (needling, green) or 3 impacts of 3 mA for 3 s (blue). Center values represent the mean and error bars represent s.e.m.; n = 8–15 independent animals.
Figure 5
Galvanic current induces proinflammatory cytokine expression in the calcaneal tendon of mice.
(A,B) Quantitative PCR for the indicated genes normalized to Actb in the calcaneal tendon of wild type mice after 3 days of applying three punctures with needle (needling, green) or 3 impacts of 3 mA for 3 s (blue), and compared to the expression of genes in non-treated tendons. Center values represent the mean and error bars represent s.e.m.; n = 4–12 independent animals; for Il6, Nlrp3, Pycard, and Gsdmd unpaired t-test, for Il1b untreated vs galvanic current Mann-Whitney and puncture vs galvanic current unpaired t-test, for Cxcl10, Il1rn and Casp1/11 Mann-Whitney test, for Il1a one sample Wilcoxon test (ND: non detected), ***p < 0.0005, **p < 0.005, *p < 0.05 and ns p > 0.05.
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Figure 5—source data 1
Raw data of panels from Figure 5.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig5-data1-v2.xlsx
Figure 6 with 1 supplement
Inflammatory response in the calcaneal tendon of Nlrp3-/- mice after galvanic current application.
(A–C) Quantitative PCR for the indicated genes in the calcaneal tendons of Nlrp3-/- mice (calculated as 2-ΔΔCt) normalized to the expression in wild type (calculated as 2-ΔΔCt) after 3 days of 3 impacts of 3 mA for 3 s. Center values represent the mean and error bars represent s.e.m.; n = 3–12 independent animals; for Il1b, Nlrp3, Pycard, Casp1/11, and Gsdmd unpaired t-test, for Il1rn and Cxcl10 Mann-whitney test, *p < 0.05 and ns p > 0.05. (D) Quantification of polymorphonuclear (top) and F4/80 positive cells (bottom) per field of view from wild type and Nlrp3-/- mice calcaneal tendon treated as in A. Center values represent the mean and error bars represent s.e.m.; n = 6–8 independent animals; unpaired t-test, *p < 0.05 and ns p > 0.05. Representative hematoxylin and eosin images (top) and F4/80 immunostaining (bottom) of calcaneal tendon quantified. Scale bar: 50 μm. Magnification shows the presence of polymorphonuclear (top) or F4/80 cells (bottom) denoted by arrowheads.
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Figure 6—source data 1
Raw data of panels from Figure 6.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Cytokine expression in the calcaneal tendon of Pycard-/- mice after galvanic current application.
(A,B) Quantitative PCR for Il1b (A) and Cxcl10 (B) in the calcaneal tendons of Pycard-/- mice (calculated as 2-ΔΔCt) normalized to the expression in wild type (calculated as 2-ΔΔCt) after 3 days of 3 impacts of 3 mA for 3 s. Center values represent the mean and error bars represent s.e.m.; n = 3–12 independent animals; unpaired t-test.
Figure 7 with 4 supplements
Galvanic current increase in type I collagen via NLRP3 inflammasome.
(A) Quantitative PCR for Tgfb1 in the calcaneal tendons of Nlrp3-/- mice (calculated as 2-ΔΔCt) normalized to the expression in wild type (calculated as 2-ΔΔCt) after 3 days of 3 impacts of 3 mA for 3 s. Center values represent the mean and error bars represent s.e.m.; n = 6–12 independent animals; Mann-Whitney test, ***P < 0.0005. (B,C) Quantification of the collagen type I and III in calcaneal tendon sections stained with picrosirius red from wild type (B,C) and Nlrp3-/- (C) mice after 3, 7 or 14 days (B) or 3 days (C) application of punctures with needle (needling, green) or 3 impacts of 3 mA for 3 s (blue), or in non-treated tendons (white). Center values represent the mean and error bars represent s.e.m.; n = 3–12 independent animals; for 3 days and panel C unpaired t-test, for 7 days non-treated vs needling Mann-Whitney test and non-treated vs percutaneous electrolysis unpaired t-test, for 14 days untreated vs needling unpaired t-test and untreated vs percutaneous electrolysis Mann-Whitney test, **p < 0.005, *p < 0.05 and ns p > 0.05. (D) Quantification of collagen structural dispersion in calcaneal tendon sections imaged with second harmonic generation microscopy and calculated with an algorithm based on the Hough transform from wild type and Nlrp3-/- mice after 7 days of 3 impacts of 3 mA for 3 s (blue), or in non-treated tendons (white). Center values represent the mean and error bars represent s.e.m.; n = 3 independent animals imaged at two or three different tendon areas, one-way ANOVA ***p < 0.0005 and *p < 0.05. (E) Biomechanical testing of calcaneal tendon from wild type and Nlrp3-/- mice after 14 days of 3 impacts of 3 mA for 3 s (blue) or from non-treated tendons (white). Center values represent the mean and error bars represent s.e.m.; n = 3–8 independent animals; unpaired t-test, *p < 0.05 and ns p > 0.05.
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Figure 7—source data 1
Raw data of panels from Figure 7.
- https://cdn.elifesciences.org/articles/73675/elife-73675-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Galvanic current do not change properties of the collagen.
(A) Ultrasound scanning of the right elbow of a patient of with lateral epicondylalgia after 6 months, with pain and functional impairment. Baseline represents the image before galvanic current. Discharge represents the image after 4 sessions of percutaneous electrolysis with an intensity of 3 mA of galvanic current for 3 s, 3 times (3:3:3). 6 w, represents the image after 6 weeks of the last session of percutaneous electrolysis. (B,C) Quantification of the collagen fiber properties (width or length) in calcaneal tendon sections stained with picrosirius red from wild type mice after 3 and 7 days of punctures with needle (needling, green) or 3 impacts of 3 mA for 3 s (blue), or in untreated tendons (white). Center values represent the mean and error bars represent s.e.m.; n = 6–8 independent animals; (B) Mann-Whitney test, (C) non-treated vs needling Mann-Whitney test and non-treated vs percutaneous electrolysis unpaired t-test, ***p < 0.0005, **p < 0.005, *p < 0.05 and ns p > 0.05. (D) Representative images of calcaneal tendons sections stained with picrosirius red and viewed with polarized light from wild type mice used for quantifying the collagen type I and III in main Figure 7C. Images are after 7 days of punctures with needle (needling) or 3 impacts of 3 mA for 3 s of percutaneous electrolysis, or non-treated tendons, scale bar 100 μm. (E) Raw representative images of second harmonic generation microscopy of calcaneal tendons from wild type and Nlrp3-/- mice untreated or after 7 days of 3 impacts of 3 mA for 3 s, scale bar 50 μm.
Figure 7—figure supplement 2
Model summarizing the action of galvanic current in tendon regeneration.
Figure 7—figure supplement 3
Clamps designed to measure tendon tension.
(A) Image illustrating mice paws after dissection, maintaining intact the calcaneus and the gastrocnemius/soleus muscles. (B) Image illustrating mice paws after muscle fibers were removed to expose the intramuscular tendon fibers of the calcaneal tendon. (C) Image illustrating the orientation and the position of mice paws in the biochemical testing of the calcaneal tendon before starting the load.
Figure 7—figure supplement 4
Accumulation matrix of the algorithm based on the Hough transform from collagen fibers imaged with second harmonic generation microscopy of calcaneal tendons from wild type and Nlrp3-/- mice after 7 days of 3 impacts of 3 mA for 3 s.
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus musculus, male) | NLRP3-deficient mice (Nlrp3-/-) - C57 BL/6 Nlrp3tm1Vmd | Jackson laboratories | RRID:MGI:5468973 | In vivo mouse models and biological samples. |
| Genetic reagent (Mus musculus, male) | ASC-deficient mice (Pycard-/-) - C57 BL/6 Pycardtm1Vmd | Jackson laboratories | RRID:MGI:3047277 | In vivo mouse models and biological samples. |
| Genetic reagent (Mus musculus, male) | Casp1/11-deficient mice (Casp1/11-/-) - B6N.129S2-Casp1tm1Flv | Jackson laboratories | RRID:MGI:5467384 | Biological samples. |
| Antibody | Anti-GSDMD (rabbit monoclonal, clone EPR19828) | Abcam | Cat#: ab209845, RRID:AB_2783550 | WB (1:2000) |
| Antibody | Anti-Caspase 1 p20 (mose monoclonal, clone Casper-1) | Adipogen | casper-1, Cat# AG-20B-0042, RRID:AB_2490248 | WB (1:1000) |
| Antibody | Anti-IL-1β (rabbit polyclonal) | Santa Cruz | H-153Cat#: SC-7884, RRID:AB_2124476 | WB (1:1000) |
| Sequence-based reagent | KiCqStart SYBR Green Primers | Sigma-Aldrich | Tnfa(NM_013693)Il6(NM_031168)Il1b(NM_008361)Actb(NM_007393)Cox2 (Ptgs2)(NM_011198)Arg1(NM_007482)Fizz1 (Retnlb)(NM_023881)Mrc1(NM_ 008625)Ym1 (Chil1)(NM_007695001080219) | qRT-PCR |
| Commercial assay or kit | ELISA for IL-1β | R&D SystemsThermo Fisher Scientific | Cat# 88-7013-88, RRID:AB_257494688-7013-88xxx | ELISA for cell supernatant |
| Commercial assay or kit | ELISA for TNFα | Thermo Fisher Scientific | Cat# 88-7324-88, RRID:AB_257508088-7324-88xxx | ELISA for cell supernatant |
| Commercial assay or kit | ELISA for IL-6 | R&D Systems | Cat# M6000B, RRID:AB_2877063 M6000Bxxx | ELISA for cell supernatant |
| Commercial assay or kit | ELISA for IL-18 | Thermo Fisher Scientific | Cat# BMS618/3, RRID:AB_2575692BMS618-3xxx | ELISA for cell supernatant |
| Chemical compound, drug | MCC950 | Sigma-Aldrich | PZ0280 | Cell culture:10 µM |
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Galvanic current activates the NLRP3 inflammasome to promote Type I collagen production in tendon
eLife 11:e73675.
https://doi.org/10.7554/eLife.73675
