Spinal macrophage migration inhibitory factor and high mobility group box 1 mediate persistent bladder pain

Abstract

Repeated intravesical PAR4 (protease activated receptor 4) activation elicits persistent bladder pain lasting 5 days after the last treatment. Persistent bladder pain was fully reversed by a systemic HMGB1 (high mobility box 1) inhibitor while a MIF (macrophage migration inhibitory factor) antagonist partly reversed it. Since there is growing evidence that spinal MIF and HMGB1 mediate inflammatory and neuropathic pain we examined whether there were spinal changes occurring during persistent bladder pain that may be responsible for maintaining bladder pain. In addition, we tested whether we could modulate persistent bladder pain with spinal MIF or HMGB1 antagonists. Persistent bladder pain was elicited in female C57 mice by repeated (3x) intravesical instillation of PAR4-activating peptide while control animals received scramble peptide treatment. On day 4, spinal cord (L6-S1) changes in c-fos (non-specific marker of spinal activation) was assessed with immunofluorescence while MIF and HMGB1 were assessed with immunofluorescence, western blotting and real-time PCR. On day 7, mice received an intrathecal injection of a neutralizing MIF monoclonal antibody (15 μg in 5 μl PBS) or a HMGB1 inhibitor glycyrrhizin (25 μg in 5 μl of 5 % alcohol in PBS) and abdominal mechanical threshold was tested. On day 9, mice were treated with vehicle or control and abdominal mechanical threshold was tested. Immunofluorescence showed that c-fos and MIF in the dorsal horn, dorsal grey commissure and intermediolateral areas significantly increased in PAR4-treated mice while HMGB1 was decreased. In addition, intrathecal treatment with MIF neutralizing mAb or glycyrrhizin significantly alleviated abdominal mechanical hypersensitivity at 1 and 2 hours and the analgesic *

Intravesical CD74 and CXCR4, macrophage migration inhibitory factor (MIF) receptors, mediate bladder pain

“…We tested abdominal mechanical hypersensitivity in mice as previously described [18][19][20]. Briefly, 50% mechanical threshold [21][22][23] was calculated by measuring the response to von Frey fibers (0.008, 0.02, 0.07, 0.16, 0.4, 1.0, 2.0 and 6.0 g) applied to the lower abdominal region.…”

Role of high‐mobility group box 1 and its modulation by thrombomodulin/thrombin axis in neuropathic and inflammatory pain

“…Intravesical administration of a receptor‐activating peptide for proteinase‐activated receptor 4 (PAR4‐AP), a secondary thrombin receptor in human, causes prompt increase in bladder luminal HMGB1 levels at 1 h and delayed decrease in HMGB1 immunoreactivity in the isolated bladder urothelium at 24 h, accompanied by abdominal hypersensitivity, that is, referred hyperalgesia/allodynia, without development of cystitis (Kouzoukas et al, 2016), suggesting that the urothelial cell‐derived HMGB1 accumulates in the bladder lumen. Interestingly, repeated intravesical administration of PAR4‐AP on Days 0, 2 and 4 caused bladder pain (referred hyperalgesia/allodynia) 5 days after the last treatment, accompanied by decreased HMGB1 immunostaining in the spinal cord, which might reflect the release of HMGB1 to the extracellular space (F. Ma et al, 2019). Collectively, peripheral and spinal HMGB1 may play a role in the development of acute and chronic bladder pain respectively.…”

“…Bladder pain in patients with IC/BPS is largely resistant to currently available drug therapy, and there is thus urgent need for identification of appropriate therapeutic targets to treat bladder pain in IC/BPS patients. HMGB1 and its target receptors could serve as such therapeutic targets for treatment of bladder pain in patients with IC/BPS, as inactivation of endogenous HMGB1 with the anti‐HMGB1‐neutralizing antibody, TMα or glycyrrhizin prevents or suppresses bladder pain in different IC/BPS models (Kouzoukas et al, 2016; F. Ma et al, 2019; Tanaka et al, 2014; Table 2). Systemic administration of cyclophosphamide (CPA) causes acute cystitis accompanied by marked bladder pain in rodents, which is frequently used as an animal model to study the mechanisms underlying bladder pain accompanying IC/BPS.…”

“…Therefore, HMGB1 and its receptors including RAGE, TLR4 and CXCR4 could be promising therapeutic targets for prevention or treatment of pathological pain. It is of interest to test if low MW compounds that directly bind and inactivate HMGB1, such as methotrexate (Kuroiwa et al, 2013), metformin (Horiuchi et al, 2017), epigallocatechin gallate (Meng et al, 2016) and salicylic acid (Choi et al, 2015), could reduce HMGB1‐dependent pain, as did glycyrrhizin, a well‐known HMGB1 inactivator (Feldman et al, 2012; Kouzoukas et al, 2016; F. Ma et al, 2019; Sun et al, 2018), FPS‐ZM1, a RAGE antagonist, TAK242, a TLR4 signalling inhibitor, and AMD3100, a CXCR4 antagonist (Irie et al, 2017; Y. Li et al, 2017;Nishida et al, 2016 ; Sekiguchi et al, 2018 ; Tsubota et al, 2019). TMα capable of inactivating HMGB1 in a thrombin‐dependent manner prevents and reverses HMGB1‐dependent pathological pain, such as CIPN (Nishida et al, 2016; Sekiguchi et al, 2018; Tsubota et al, 2019) and visceral pain accompanying pancreatitis (Irie et al, 2017) or cystitis (Tanaka et al, 2014), suggesting its clinical usefulness.…”

“…administration of HMGB1 or ds-HMGB1 causes TLR4-dependent mechanical hyperalgesia/allodynia (Agalave et al, 2014;Morioka et al, 2019). Likewise, visceral nociception appears to involve the HMGB1/TLR4 axis in the peripheral tissue, because intravesical infusion of ds-HMGB1 produces TLR4-dependent abdominal mechanical hypersensitivity, that is, referred hyperalgesia/allodynia, in mice (F. Ma et al, 2017). Interestingly, in isolated and dissociated dorsal root ganglion (DRG) neurons, HMGB1 elicits an intracellular calcium influx mainly in capsaicin-sensitive neurons and increases the electrical excitability of small-to-medium diameter DRG neurons (Feldman, Due, Ripsch, Khanna, & White, 2012).…”