Background
Neuropathic pain is associated with sensory abnormalities and altered stimulus-response function, such as allodynia, hyperalgesia, and loss of sensation in some areas [
1,
2]. The International Association for the Study of Pain defined neuropathic pain as “pain caused by a lesion or disease of the somatosensory nervous system” (
www.iasp-pain.org/Taxonomy#Neuropathicpain). Neuropathic pain poses a heavy burden on the quality of life of patients while currently available treatments are often ineffective. The underlying molecular and cellular mechanisms of neuropathic pain remain poorly elucidated. There are increasing evidences indicating a role of neuroimmune processes in the development of neuropathic pain [
3‐
6].
The myeloid differentiation factor-88 adaptor protein (MyD88) is involved in Toll-like receptors (TLRs, except for TLR3) signaling and interleukin-1 receptor (IL-1R) signaling [
7‐
10]. MyD88 mediates activation of TLRs or IL-1R and leads to NF-κB activation, inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) in immune cells [
8‐
10]. TLRs are danger-associated and pathogen-associated molecular pattern receptors that regulate innate immunity via activated NF-κB-dependent and mitogen-activated protein kinase (MAPK)-dependent cytokine production [
11,
12]. TLRs and IL-1R are not only expressed on immune cells but also found on sensory neurons in dorsal root ganglions (DRGs) and glial cells (microglia and astrocytes) in the spinal cord [
5,
13‐
17]. A number of previous studies have found that TLRs in the spinal cord played an important role in the model of neuropathic pain and nerve injury, in which microglia and astrocytes produced proinflammatory cytokines by activating TLRs [
18‐
20]. Nociceptors of DRG that express TLRs or IL-1R are also activated by LPS or inflammatory cytokine interleukin-1β, inducing pain hypersensitivity [
5,
18‐
22]. Recent publications show that MyD88, adaptor protein of TLRs and IL-1R, is also found in the expression in DRG and spinal cord [
18‐
20].We hypothesized that suppressed MyD88 adaptor protein in the DRG and spinal cord could alleviate peripheral nerve injury-induced neuropathic pain. Our findings reveal MyD88 adaptor protein involved in the neuropathic pain and may provide potential therapeutic strategies for treatment of neuropathic pain.
Discussion
In the present study, we explored the role of MyD88-dependent TIR signaling pathways in the DRG and SDH in a rat model of CCI-induced neuropathic pain. We found that CCI induced a rapid and long-term upregulation of MyD88 in the DRG and SDH of rat. Meanwhile, the proinflammatory cytokine IL-1β and HMGB1 were upregulated in the DRG and SDH. We also found that phospho-NF-κB p65 and phospho-p44/42 MAPK of Toll/interleukin-1 receptor downstream signal protein were rapidly and long-lastingly upregulated in the DRG and SDH after CCI. In contrast, CCI did not cause any significant change in TRIF. Intrathecal injection of MyD88 inhibitor MIP attenuated CCI-induced pain and decreased MyD88 expression in DRG and SDH. Inhibition of MyD88 suppressed the phospho-NF-κB p65, phospho-p44/42 MAPK, the production of TNF-α, and the activation of astrocytes and microglial cells in SDH. These results suggested that MyD88-dependent TIR/NF-κB p65 and p44/42 MAPK pathway, activated by IL-1β and HMGB1, was involved in CCI-induced neuroinflammation and neuropathic pain. In this study, we administrated MIP on preoperative days −3 to −1 (before CCI) to achieve a maximum and sustaining effect of the drug. Previous studies used a similar strategy of MIP application to inhibit MyD88 in vitro or in vivo [
31‐
33].
HMGB1 is a proinflammation mediator and endogenous ligand of TLRs such as TLR2 and TLR4 [
34]. HMGB1 can be induced in the DRG and/or SDH in multiple animal models of pain [
34‐
36]. IL-1β is a potent proinflammatory cytokine and endogenous ligand of IL-1R [
21]. HMGB1 and IL-1β can activate calcium mobilization in DRG neurons and stimulate astrocytes or microglia cells to produce proinflammatory mediators or cytokines such as TNF-α via activation of NF-kappa-B, MAPK, or other pathways [
35,
37]. Previous studies showed that intrathecal injection of HMGB1 or IL-1β induced pain hypersensitivity including heat hyperalgesia and mechanical allodynia [
34,
37]. Release of HMGB1 and IL-1β in the nociceptive pathway may play a crucial role for the development of pain via influencing adjacent neurons and glia [
38]. In this study, we also found that HMGB1 and IL-1β was robustly produced and sustained through POD 21 in the DRG and SDH of CCI rats.
TLRs play a pivotal role in innate immune responses. Increasing evidence suggests that TLRs are expressed in primary sensory neurons in DRG and TG [
5]. TLR4 mainly express in small-diameter sensory neurons of DRG and TG, which are mostly nociceptive sensor and regulate nociceptive sensation such as pain [
16,
20,
21]. Functional TLRs including TLR2 and TLR4 also express in microglia and astrocytes that modulate glial activation and spinal inflammatory of the spinal injury or chronic pain-induced central sensitization [
17,
39]. TLR4 mediates the hyperalgesia and neuroinflammation by damage/pathogen-associated molecular pattern components such as heat shock protein 90 (HSP90), HMGB1, and LPS [
16,
21,
34,
35,
40]. Peripheral neurons of DRG or glial cells of spinal cord may directly produce HMGB1 and/or HSP90 when peripheral and/or central nervous system are damaged or administrated with drugs [
18,
34,
35]. In this study, we measured total HMGB1 by Western blots, while only disulfide HMGB1 is known to be a TLR4 agonist [
41,
42]. This problem will be addressed in future studies. In addition, a recent study also found that HMGB1 activates pro-inflammatory signaling via TLR5, leading to allodynia [
43]. Further experiments using specific TLR2 or TLR4 antagonists or knockout mice could be performed to determine their roles in the MyD88-dependent signaling pathway.
IL-1R was an important receptor for regulating immune responses and inflammation [
44]. It expresses in nociceptive neurons of DRG and glial cells of SDH and mediates interleukin-1 including interleukin-1β-induced activation of cell [
27,
28,
45]. IL-1β can act directly on primary sensory neurons to evoke excitatory action on nociceptor neurons by IL-1R [
28]. IL-1β also activates IL-1R to contribute to hyperalgesia and the establishment of peripheral and central sensitization [
28,
46].
Two distinct signaling transduction pathways of TLRs were found [
13]. Activated TLRs and/or IL-1R signaling are involved in the recruitment of adaptor molecules such as MYD88 or TRIF, then phosphorylate NF-kappa-B or MAPK via IL-1 receptor-associated kinase1 (IRAK1) and TNF receptor-associated factors6 (TRAF6) [
7,
9,
10,
13]. MyD88 is the adaptor protein of Toll/interleukin-1 receptor (TIR) and plays an important role in the trafficking of TIR signal pathway [
7,
10]. MyD88 is expressed in DRG neurons and glial cells and could be significantly increased in the DRG from rat models of chronic pain induced by chemotherapy [
18]. SNL lesions produce chronic pain is approximately 50% reduced in MyD88-deficient mice [
19,
47]. Consistently, intrathecal administration of MyD88 inhibitor suppresses pain of paclitaxel-induced peripheral neuropathy when applied 14 days after paclitaxel administration [
18]. TRIF is an adaptor protein of the MyD88-independent signaling transduction pathways of TLRs and shares mostly by TLR3 and TLR4 signaling [
13]. The SNL-induced allodynia was significantly alleviated in the MyD88 knock-out mice with reduced glial activation in SDH and ATF3 expression in the DRG, but these effects were not observed in the TRIF deficient mice [
47]. TRIF expression was found not significantly changed in the SDH in a rat model of paclitaxel-induced peripheral neuropathic pain [
18]. Our results also showed a lack of change in TRIF protein in the DRG and SDH of CCI rats. Detailed mechanisms for the upregulation and activation of MyD88 without increases in TRIF with TLR signaling should be further investigated in the future.
Acknowledgements
We thank Dr. Wenyin Qiu, Xiaojin Qian, and Yongmei Chen in the Department of Anatomy, Histology and Embryology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences for their technical assistance in immunohistochemistry. We thank Dr. Xiaojing Liu, Department of Dermatology, Peking University People’s Hospital, China, for their generous help in providing the mouse anti-pNF-κB-p65 antibody.