Cystitis-Related Bladder Pain Involves ATP-Dependent HMGB1 Release from Macrophages and Its Downstream H2S/Cav3.2 Signaling in Mice

Abstract

Cystitis-related bladder pain involves RAGE activation by HMGB1, and increased Cav3.2 T-type Ca2+ channel activity by H2S, generated by upregulated cystathionine-γ-lyase (CSE) in mice treated with cyclophosphamide (CPA). We, thus, investigated possible crosstalk between the HMGB1/RAGE and CSE/H2S/Cav3.2 pathways in the bladder pain development. Bladder pain (nociceptive behavior/referred hyperalgesia) and immuno-reactive CSE expression in the bladder were determined in CPA-treated female mice. Cell signaling was analyzed in urothelial T24 and macrophage-like RAW264.7 cells. The CPA-induced bladder pain was abolished by pharmacological inhibition of T-type Ca2+ channels or CSE, and genetic deletion of Cav3.2. The CPA-induced CSE upregulation, as well as bladder pain was prevented by HMGB1 inactivation, inhibition of HMGB1 release from macrophages, antagonists of RAGE or P2X4/P2X7 receptors, and N-acetylcysteine, an antioxidant. Acrolein, a metabolite of CPA, triggered ATP release from T24 cells. Adenosine triphosphate (ATP) stimulated cell migration via P2X7/P2X4, and caused HMGB1 release via P2X7 in RAW264.7 cells, which was dependent on p38MAPK/NF-κB signaling and reactive oxygen species (ROS) accumulation. Together, our data suggest that CPA, once metabolized to acrolein, causes urothelial ATP-mediated, redox-dependent HMGB1 release from macrophages, which in turn causes RAGE-mediated CSE upregulation and subsequent H2S-targeted Cav3.2-dependent nociceptor excitation, resulting in bladder pain.

Role of HMGB1 in Chemotherapy-Induced Peripheral Neuropathy

“…HMGB1 is passively released from necrotic cells and can also be actively secreted by immune cells ( Figure 1 C) such as macrophages [ 44 ], microglia [ 45 ], neutrophils [ 46 ], and natural killer cells [ 47 ], and also by non-immune cells, such as fibroblasts [ 48 ], epithelial cells [ 49 , 50 ], neurons [ 33 ], platelets [ 51 ], hepatocytes [ 52 ], and cardiomyocytes [ 53 ]. The active secretion of HMGB1 is triggered by microbial pathogens such as lipopolysaccharide (LPS) and polyinosinic-polycytidylic acid (poly(I:C)) [ 44 , 52 , 54 ], or endogenous substances, such as ROS [ 55 ], reactive nitrogen species (RNS) [ 56 ], hyperglycemia [ 53 ], inflammatory cytokines (e.g., TNF-α [ 49 ], interferon (IFN)-α [ 54 ], INF-γ [ 57 ], ATP [ 19 , 58 ], nitric oxide [ 54 ], calcium phosphate-based mineralo-organic particles [ 46 ], and also by cell-to-cell interaction [ 47 ]. Cytoplasmic translocation of nuclear HMGB1 is triggered and/or modulated by post-translational molecular modifications of HMGB1, such as acetylation [ 39 , 59 ], phosphorylation [ 60 ], ADP-ribosylation [ 61 ], methylation [ 62 ], glycosylation [ 63 ], and ROS-induced oxidation [ 64 ] ( Figure 1 C).…”

“…RAGE, when stimulated, activates cell signals including mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) ( Figure 1 C) [ 68 , 69 ]. A number of studies demonstrate that RAGE is required for HMGB1-induced inflammation [ 70 ], pain [ 16 , 19 , 30 , 32 ], cell migration [ 71 ], neuritogenesis [ 72 ], autophagy [ 73 ], and proliferation [ 74 , 75 ]. We have demonstrated that RAGE mediates the allodynia/hyperalgesia following intraplantar administration of at-HMGB1, but not ds-HMGB1 [ 16 ], and that the HMGB1/RAGE pathway plays a crucial role in the neuropathic pain caused by spinal nerve injury [ 76 ] and visceral pain accompanying cystitis [ 19 , 28 ] and pancreatitis [ 32 ] ( Figure 1 C).…”

“…In 2001, it was reported for the first time that injection of HMGB1 around the sciatic nerve induced mechanical allodynia in rats [ 90 ]. Recently, increasing evidence has unveiled the pro-nociceptive role of HMGB1 in the peripheral tissue and spinal cord [ 16 , 18 , 29 , 80 , 89 ], and demonstrated that endogenous HMGB1 is involved in the pathogenesis of various types of intractable pain [ 11 , 15 ], including inflammatory pain [ 28 , 29 , 80 ], visceral pain [ 19 , 20 , 30 , 32 ], neuropathic pain [ 23 , 31 , 76 , 91 , 92 ], cancer pain [ 33 ], and post-stroke pain [ 34 ]. Endogenous HMGB1 also appears to play a key role in the development of CIPN in rats or mice treated with cancer chemotherapeutics, such as paclitaxel, oxaliplatin, and vincristine, considering the complete prevention of CIPN by inactivation of HMGB1 with HMGB1-nAb or TMα ( Figure 2 and Table 1) [ 8 , 9 , 10 , 11 ].…”

“…Many studies from different groups, including ours, have shown that HMGB1-nAb, developed by Nishibori et al (Table 1) [ 21 , 22 ], strongly suppresses inflammatory pain [ 28 ], neuropathic pain by surgical injury of the sciatic nerve [ 31 , 91 ] or spinal nerve [ 76 ], and visceral pain following cystitis or pancreatitis [ 19 , 20 , 30 , 32 ]. The same HMGB1-nAb completely prevents the development of CIPN in rodents treated with paclitaxel, oxaliplatin, or vincristine (Table 1) [ 8 , 9 , 10 ].…”

“…On the other hand, HMGB1 is actively secreted by activated immune cells and passively released from necrotic cells in pathological conditions including inflammation. Extracellular HMGB1 acts as a damage-associated molecular pattern (DAMP) protein via activation of several pattern recognition receptors (PRRs) including Toll-like receptors (TLRs), receptors for advance glycosylation end products (RAGE), C-X-C motif chemokine receptor 4 (CXCR4), etc., leading to acceleration of inflammation and pain [ 11 , 12 , 14 , 15 , 16 , 17 , 18 , 19 , 20 ]. There are plenty of papers showing that an anti-HMGB1-neutralizing antibody (HMGB1-nAb) [ 21 , 22 ] strongly suppresses somatic or visceral pathological pain with inflammatory and/or neuropathic components [ 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ].…”