Neuropathic Pain Phenotype Does Not Involve the NLRP3 Inflammasome and Its End Product Interleukin-1β in the Mice Spared Nerve Injury Model

RESEARCH ARTICLE Neuropathic Pain Phenotype Does Not Involve the NLRP3 Inflammasome and Its End Product Interleukin-1β in the Mice Spared Nerve Injury Model Verdad Curto-Reyes1,2, Guylène Kirschmann1,2, Marie Pertin1,2, Stephan K. Drexler3, Isabelle Decosterd1,2, Marc R. Suter1* a11111 1 Pain Center, Department of Anesthesiology, Lausanne University Hospital (CHUV) and University of Lausanne, Lausanne, Switzerland, 2 Department of Fundamental Neurosciences, University of Lausanne, Lausanne, Switzerland, 3 Department of Biochemistry, University of Lausanne, Epalinges, Switzerland * OPEN ACCESS Citation: Curto-Reyes V, Kirschmann G, Pertin M, Drexler SK, Decosterd I, Suter MR (2015) Neuropathic Pain Phenotype Does Not Involve the NLRP3 Inflammasome and Its End Product Interleukin-1β in the Mice Spared Nerve Injury Model. PLoS ONE 10(7): e0133707. doi:10.1371/journal. pone.0133707 Editor: Mario D. Cordero, University of Sevilla, SPAIN Received: October 14, 2014 Accepted: June 30, 2015 Published: July 28, 2015 Copyright: © 2015 Curto-Reyes et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are available from Figshare: http://dx.doi.org/10.6084/m9. figshare.1478064. Funding: Swiss National Science Foundation grants 310030A_124996 (ID) and 33CM30-124117 (MRS and ID), the 2011 IASP Early Career Grant funded by the ScanDesign Foundation BY INGER & JENS BRUUN (MRS) and the Swiss Society of Anesthesiology (MRS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is one of the main sources of interleukin-1β (IL-1β) and is involved in several inflammatory-related pathologies. To date, its relationship with pain has not been studied in depth. The aim of our study was to elucidate the role of NLRP3 inflammasome and IL-1β production on neuropathic pain. Results showed that basal pain sensitivity is unaltered in NLRP3-/- mice as well as responses to formalin test. Spared nerve injury (SNI) surgery induced the development of mechanical allodynia and thermal hyperalgesia in a similar way in both genotypes and did not modify mRNA levels of the NLRP3 inflammasome components in the spinal cord. Intrathecal lipopolysaccharide (LPS) injection increases apoptosis-associated speck like protein (ASC), caspase-1 and IL-1β expression in both wildtype and NLRP3-/- mice. Those data suggest that NLRP3 is not involved in neuropathic pain and also that other sources of IL-1β are implicated in neuroinflammatory responses induced by LPS. Introduction The inflammasomes are cytosolic protein complexes which act as intracellular sensors of disruption of homeostasis. Their stimulation leads to the proteolytic cleavage of the proinflammatory cytokines pro-IL-1β (interleukin-1 beta) and pro-IL-18 (interleukin-18) through the activation of caspase-1. The active complex consists of a central scaffold protein for which it is named (e.g. NLRP1, NLRP3, NLRP6, NLRC4, AIM2), an adaptor apoptosis-associated speck-like protein (ASC) containing a caspase activation and recruitment domain (CARD), which is mandatory for most inflammasomes, and the precursor form of caspase-1 enzyme, pro-caspase-1. How the inflammasomes are activated is still debated, but in the case of NLRP3 (NACHT, LRR and PYD domains-containing protein 3), one of the most studied caspase-1 activators, several signals PLOS ONE | DOI:10.1371/journal.pone.0133707 July 28, 2015 1 / 13 No NLRP3 Inflammasome Involvment in a Mice Neuropathic Pain Model Competing Interests: The authors have declared that no competing interests exist. related to cell damage and stress (generation of extracellular ATP, production of reactive oxygen species (ROS), activators that form crystalline/particulate ligands) are likely involved [1]. When the inflammasome is activated, pro-caspase-1 undergoes an autolysis leading to the formation of caspase-1, which in turn cleaves pro-IL-1β, generating the mature cytokine, which is subsequently released along with caspase-1 [2], via non-classical secretory pathways [3, 4]. The NLRP3 inflammasome is the most studied inflammasome in the central nervous system [5]. It has been linked to acute disorders (from infections [6] to acute brain injury [7]) and chronic diseases exhibiting an inflammatory component (experimental autoimmune encephalitis [8], Parkinson’s disease [9], Alzheimer’s disease [10], prion disease [11], etc.). NLRP3 was recently also implicated in fibromyalgia [12]. NLRP3 inflammasome has been studied in microglia and macrophages but has also been suggested to have functions in neurons [6, 13]. In the spinal cord, the expression of NLRP3 is increased in an experimental autoimmune encephalitis model and NLRP3 knockout mice show a delayed course of a less severe disease [14]. Following peripheral nerve injury, extensive inflammatory changes have been described in the central nervous system, which participate in the painful behaviour [15, 16]; this encompasses microglial and astrocytic reactivity and the involvement of multiple cytokines. Prototypic pro-inflammatory cytokines such as IL-1β, interleukin-6 or tumour necrosis factor-α are often cited as major contributors in the neuro-glial crosstalk during inflammatory reactions of the central nervous system [16]. A major behavioural consequence of peripheral nerve injury is pain. Pain relationship with IL-1β has been studied for a long time. IL-1β causes mechanical and thermal hyperalgesia when injected into peripheral and central tissues [17–22], and increased IL-1β expression in the spinal cord, DRG, and injured nerve in some animal models of neuropathic pain has been described [23–28]. Here we hypothesize NLRP3 inflammasome has a role in acute and chronic pain following peripheral nerve injury, as well as a role in IL-1β expression. The behavioural response and expression of the NLRP3 complex components were assessed after a nerve injury or after intrathecal injection of lipopolysaccharide (LPS). We demonstrate that pain-related behaviour in naive and neuropathic animals is unchanged in NLRP3 deficient mice and that IL-1β does not play a major role in the spinal cord in the spared nerve injury model in mice. Moreover, IL-1β expression following intrathecal LPS is independent of NLRP3. Methods Animals and surgery All experiments were approved by the Committee on Animal Experimentation of the Canton de Vaud, Lausanne, Switzerland, in accordance with Swiss Federal law on animal care, the guidelines of the International Association for the Study of Pain (IASP) and the ARRIVE guideline (S1 Text) [29]. Mice were housed 5/cage at constant temperature (21 ± 2°C) and a 12/12 dark/li (...truncated)