Galvanic current activates the NLRP3 inflammasome to promote Type I collagen production in tendon

The NLRP3 inflammasome coordinates inflammation in response to different pathogen- and damage-associated molecular patterns, being implicated in different infectious, chronic inflammatory, metabolic and degenerative diseases. In chronic tendinopathic lesions, different non-resolving mechanisms produce a degenerative condition that impairs tissue healing and which therefore complicates their clinical management. Percutaneous needle electrolysis consists of the application of a galvanic current and is an emerging treatment for tendinopathies. In the present study, we found that galvanic current activates the NLRP3 inflammasome and induces an inflammatory response that promotes a collagen-mediated regeneration of the tendon in mice. This study establishes the molecular mechanism of percutaneous electrolysis that can be used to treat chronic lesions and describes the beneficial effects of an induced inflammasome-related response.


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Tissue damage and infection triggers an inflammatory response that is coordinated by 41 the activation of the immune system. Inflammation promotes a response to recover 42 homeostasis by removing invading pathogens and repairing tissues (Medzhitov, 2008).

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Caspase-1 also process gasdermin D protein (GSDMD), and its amino-terminal fragment 63 (GSDMD NT ) oligomerize in the plasma membrane forming pores allowing the release of 64 IL-1b and IL-18 cytokines, as well as other intracellular content, including inflammasome 65 oligomers ( Baroja-Mazo et al., 2014;Broz et al., 2020). 66 diseases such as gout, type 2 diabetes or Alzheimer (Daniels et al., 2016;Heneka et al., 68 2013; Masters et al., 2010;Mayor et al., 2006), therefore selective small molecules that 69 block NLRP3 are emerging as novel anti-inflammatory therapies (Cocco et al., 2017;Coll 70 et al., 2015;Tapia-Abellán et al., 2019). However, in some pathological circumstances, 71 a boost, rather than an inhibition of NLRP3 would be beneficial to reduce clinical 72 complications, such as in immunosuppressed septic patients that accumulate high 73 mortality rates due to secondary infections associated to a profound deactivation of the 74 NLRP3 inflammasome (Martínez-García et al., 2019). In chronic non-resolving lesions, 75 as tendinopathies developed after prolonged extreme exercise, there are several 76 mechanisms establishing a degenerative condition of the tissue that impairs healing and 77 complicate clinical management (Cook and Purdam, 2009;Regan et al., 1992; 78 Soslowsky et al., n.d.). Anti-inflammatory therapies have shown inefficient in randomized 79 trials for the treating of this type of lesions (Bisset et al., 2006;Coombes et al., 2013)  not been yet described. In this study, we found that galvanic current applicated during 90 percutaneous needle electrolysis was able to activate the NLRP3 inflammasome and 91 induce the release of IL-1b from macrophages. Mice deficient on NLRP3 failed to 92 increase IL-1b in tendons after percutaneous needle electrolysis and resulted in a 93 reduction of TGF-b and type I collagen deposition, indicating that the NLRP3 inflammasome plays an important role in the regenerative response of the tendon 95 associated to percutaneous needle electrolysis.

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Galvanic current enhances macrophage pro-inflammatory M1 phenotype 98 We initially designed and produced a device to apply galvanic current to adherent 99 cultured cells in 6 well cell culture plates (Fig. S1), this device allowed us to explore the 100 effect of galvanic currents in bone marrow derived mouse macrophages. Application of 101 2 impacts of 12 mA of galvanic current for 6 seconds each over LPS stimulated 102 macrophages, induced an increase of the expression of Cox2 and Il6 genes (Fig. 1A).

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However, it did not affect LPS-induced Il1b or Tnfa pro-inflammatory gene expression 104 (Fig. 1A). Interestingly meanwhile Tnfa expression was upregulated with galvanic 105 current alone (Fig. 1A), galvanic currents were not inducing the expression of Cox2, Il6 106 or Il1b genes on non-LPS treated macrophages, or over IL-4 treated macrophages ( Fig.   107 1A). When macrophages were polarized to M2 by IL-4, galvanic currents decreased the 108 expression of the M2 markers Arg1, Fizz1 and Mrc1 (Fig. 1B). These data suggest that 109 galvanic current could enhance the pro-inflammatory signature of M1 macrophages 110 whilst decrease M2 polarization. We next studied the concentration of released pro-111 inflammatory cytokines from macrophages, and found that galvanic current was not able 112 to increase the concentration of IL-6 or TNF-a release after LPS stimulation (Fig. 1C), 113 but significantly augmented the release of IL-1b in an intensity dependent manner ( Fig.   114   1C). This data indicates that the increase of Il6 and Tnfa gene expression detected at 115 mRNA level would not be transcribing to higher amounts of released IL-6 and TNF-a 116 over LPS treatment, but galvanic current could be potentially activating an 117 inflammasome to induce the release of IL-1b.

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Galvanic current activates the NLRP3 inflammasome 120 Since IL-1b release is increased by the activation of caspase-1 after the canonical or 121 non-canonical inflammasome formation (Broz and Dixit, 2016), we next studied the 122 release of IL-1b induced by galvanic current in macrophages deficient on caspase-1 and 7 -11 to avoid both the canonical and non-canonical inflammasome signaling. We found 124 that Casp1/11 -/macrophages fail to release IL-1b induced by galvanic current (Fig. 2A).

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We then found that galvanic current application on Pycard -/macrophages also failed to 126 induce the release of IL-1b, denoting that the inflammasome adaptor protein ASC would 127 be also required for the inflammasome activation ( Fig. 2A). Since current application 128 could be considered a sterile danger signal, we next assessed the implication of NLRP3, 129 an inflammasome sensor important to elicit an immune response in sterile dangerous 130 situations (Broz and Dixit, 2016;Liston and Masters, 2017). Nlrp3 -/and the use of the 131 specific NLRP3 inhibitor MCC950 (Coll et al., 2015;Tapia-Abellán et al., 2019) impaired 132 the release of IL-1b induced by galvanic current ( Fig. 2A,B), demonstrating that the 133 NLRP3 inflammasome is activated during galvanic current application. As controls, 134 similar results were obtained in parallel with the specific NLRP3 activator nigericin ( Fig.   135 2B and S2A). Mechanistically, the use of an extracellular buffer with 40 mM of KCl 136 decreased IL-1b release induced by nigericin and galvanic current application, but not 137 the release of IL-1b induced by Clostridium difficile toxin B, that activate the Pyrin 138 inflammasome which is a K + -efflux independent inflammasome (Fig. 2C). However, 139 meanwhile we found a robust intracellular K + decrease in macrophages treated with the 140 K + ionophore nigericin, we fail to detect a decrease of intracellular K + when galvanic 141 current was applicated (Fig. S2B). This data suggests that either a small and/or transient 142 decrease of intracellular K + could be induced by galvanic current or alternatively a dilution 143 of intracellular K + concentration should occur when galvanic current is applicated, and 144 this could also explain the smaller concentration of IL-1b release induced by galvanic 145 current compared to nigericin application (Fig. 2C). After galvanic current application we 146 were able to detect the generation of the active p20 caspase-1 fragment, and processed 147 IL-1b and GSDMD NT (Fig. 2D). MCC950 was able to abrogate caspase-1 activation and 148 the processed forms of IL-1b and GSDMD NT (Fig. 2D), suggesting a functional caspase-149 1 activation and downstream signaling due to canonical NLRP3 activation and discarding the non-canonical NLRP3 activation that would result in GSDMD processing in the 151 presence of MCC950.

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Since GSDMD was processed and the N-terminus detected upon galvanic current 155 application, we next assessed pyroptosis by means of Yo-Pro-1 uptake to cells and LDH 156 leakage from the cell. Two impacts of galvanic currents of different intensities (3, 6, 12 157 mA) for a period of 6 seconds (conditions that induce IL-1b release) were only inducing 158 a significant, but slightly increase of cell death (Fig. 3A). This increase in cell death was 159 not associated with the activation of the inflammasome, since it was also present in 160 macrophages deficient on NLRP3, ASC or caspase-1/11 (Fig. 3B), suggesting that was 161 independently of pyroptosis. Increasing the number or the time of 12 mA impacts 162 applicated, resulted in a time-dependent increase of cell death (Fig. 3A), correlating with 163 higher concentrations of IL-1b release (Fig. 3C). However, meanwhile IL-1b release was 164 blocked by MCC950 (Fig. 3C), LDH release was not dependent on NLRP3 activation 165 ( Fig. 3D). This further corroborate that the NLRP3 activation is dependent on the 166 intensity and time of galvanic current application. Similarly, two impacts of 12 mA for a 167 period of 6 seconds were unable to induce plasma membrane permeabilization 168 measured by Yo-Pro-1 uptake during a period of 3 h (Fig. 3E). Yo-Pro uptake increased 169 over 3 h in an intensity dependent manner (3, 6, 12 mA) when 8 impacts were applicated 170 during 6 seconds (Fig. 3E). This increase of plasma membrane permeabilization was 171 not reverted after NLRP3 blocking with MCC950 or when ASC-deficient macrophages 172 were used (Fig. 3F). All these results demonstrate that doses of galvanic current of 3 or

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In order to study the effect of galvanic current in vivo, we found that application of 3 180 impacts of 3 mA of galvanic current during 3 seconds in the calcaneal tendon of mice 181 resulted in an increase of the number of polymorphonuclear cells after 3 days when 182 compared with tendons treated with needling alone (a puncture without current 183 application, Fig. 4A,B). This increase returned to basal after 7 days and stayed low up 184 to 21 days after galvanic current application (Fig. 4B). Similarly, the number of F4/80 + 185 macrophages increased after 3 days of galvanic current application when compared to 186 needling alone and returned to basal levels after 7 days (Fig. 4C,D). Other immune cell 187 types detected in the tendon, as mastocytes, were not significantly increased by galvanic    In order to evaluate if the NLRP3 inflammasome mediates the inflammatory response in 207 tendons after percutaneous electrolysis, we applied galvanic currents in the calcaneal 208 tendon of Nlrp3 -/mice. Application of 3 impacts of 3 mA of galvanic current for 3 seconds 209 in the calcaneal tendon of Nlrp3 -/mice resulted in a significant reduction of Il1b, Il1rn 210 and Cxcl10 expression after 3 days when compared to wild-type mice (Fig. 6A). Specific 211 inflammasome associated genes, as Pycard, Casp1 or Gsdmd (except for Nlrp3) where 212 not affecting their expression in the calcaneal tendon of Nlrp3 -/mice after 3 days of 213 galvanic current application when compared to wild type mice (Fig. 6B). Surprisingly, 214 galvanic current produced a tendency to increase the expression of Il6 in the tendons of 215 Nlrp3 -/after 3 days (Fig. 6C) and in parallel, the number of polymorphonuclear cells was 216 also increased ( Fig. 6D). However, the number of macrophages was not affected in the 217 Nlrp3 -/calcaneal tendon when galvanic current was applicated (Fig. 6D). We also 218 confirmed a decrease of Il1b and Cxcl10 expression in the tendons of Pycard -/mice after 219 3 days of galvanic current application (Fig. S4), suggesting that the NLRP3 220 inflammasome is important to modulate part of the inflammatory response after galvanic 221 current application. tissue regeneration, the production of new extracellular matrix is a key process (Shook 232 et al., 2018;Wynn, 2008). In order to investigate if the inflammatory response mediated 233 by the NLRP3 inflammasome after galvanic current application is important for tissue regeneration, we measured Tgfb1 expression as a key factor inducing collagen 235 production. We found that in vivo the expression of Tgfb1 after 3 days of galvanic current 236 application in the calcaneal tendon of mice was dependent on NLRP3 (Fig. 7B). In line, 237 after 7 days of percutaneous electrolysis the levels of type III collagen were decreased, 238 with a parallel increase of type I collagen when compared to needling alone (Fig. 7C, 239 S5E). However, percutaneous electrolysis did not affect collagen fiber properties 240 measured (width, length, strength or angle) when compared to needling alone ( Fig. S5A-241 D). The increase of type I collagen after 7 days of galvanic current application was 242 reduced in Nlrp3 -/mice ( Fig. 7D), suggesting that the NLRP3 inflammasome could 243 control the response of galvanic current inducing type I collagen. Overall, we found that 244 galvanic current application is able to activate the NLRP3 inflammasome and induce the 245 release of IL-1b, initiating an inflammatory response that could lead to the regeneration 246 of the tendon (Fig. S6).

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In this study we demonstrate how galvanic current application induces in macrophages 249 a pro-inflammatory signature, mainly characterized by the activation of the NLRP3

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The activation of the NLRP3 inflammasome induced by galvanic currents was found 286 dependent on K + efflux, as extracellular high concentrations of K + was able to block IL-287 1b release, but surprisingly galvanic current application was not resulting in a detectable 288 intracellular K + decrease. This oppose the effect of the well-studied K + ionophore 289 nigericin that was able to induce a dramatic decrease of intracellular K + in accordance to

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Therefore, this study reports how galvanic current is a feasible technique applicated in 308 vivo to activate the NLRP3 inflammasome and induce a local inflammatory response to 309 enhance a collagen-mediated regeneration process in the tendon, establishing the 310 molecular mechanism of percutaneous electrolysis for the treatment of chronic lesions.

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Total RNA extraction was performed using macrophages or mice tendons dissected as 394 described above. Macrophage total RNA extraction was performed using the RNeasy tendons was performed using Qiazol lysis reagent (Qiagen) and samples were 397 homogenized using an Omni THQ homogenizer. After homogenization, samples were 398 incubated 5 min at room temperature and centrifuged at 12000 xg for 15 min at 4ºC.

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After centrifugation, upper phase was collected and one volume of 70% ethanol was

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The adaptor ASC has extracellular and "prionoid" activities that propagate