TLR4-mediated autophagic impairment contributes to neuropathic pain in chronic constriction injury mice
- Yibo Piao†1,
- Do Hyeong Gwon†2,
- Dong-Wook Kang†2,
- Tae Woong Hwang2,
- Nara Shin1, 2,
- Hyeok Hee Kwon1, 2,
- Hyo Jung Shin2,
- Yuhua Yin1, 2,
- Jwa-Jin Kim2, 3,
- Jinpyo Hong2,
- Hyun-Woo Kim2,
- Yonghyun Kim4,
- Sang Ryong Kim5,
- Sang-Ha Oh1, 2Email author and
- Dong Woon Kim2Email authorView ORCID ID profile
© The Author(s). 2018
Received: 23 August 2017
Accepted: 19 February 2018
Published: 27 February 2018
Neuropathic pain is a complex, chronic pain state characterized by hyperalgesia, allodynia, and spontaneous pain. Accumulating evidence has indicated that the microglial Toll-like receptor 4 (TLR4) and autophagy are implicated in neurodegenerative diseases, but their relationship and role in neuropathic pain remain unclear. In this study, we examined TLR4 and its association with autophagic activity using a chronic constriction injury (CCI)-induced neuropathic pain model in wild-type (WT) and TLR4-knockout (KO) mice. The mice were assigned into four groups: WT-Contralateral (Contra), WT-Ipsilateral (Ipsi), TLR4 KO-Contra, and TLR4 KO-Ipsi. Behavioral and mechanical allodynia tests and biochemical analysis of spinal cord tissue were conducted following CCI to the sciatic nerve. Compared with the Contra group, mechanical allodynia in both the WT- and TLR4 KO-Ipsi groups was significantly increased, and a marked decrease of allodynia was observed in the TLR4 KO-Ipsi group. Although glial cells were upregulated in the WT-Ipsi group, no significant change was observed in the TLR4 KO groups. Moreover, protein expression and immunoreactive cell regulation of autophagy (Beclin 1, p62) were significantly increased in the neurons, but not microglia, of WT-Ipsi group compared with the WT-Contra group. The level of PINK1, a marker for mitophagy was increased in the neurons of WT, but not in TLR4 KO mice. Together, these results show that TLR4-mediated p62 autophagic impairment plays an important role in the occurrence and development of neuropathic pain. And what is more, microglial TLR4-mediated microglial activation might be indirectly coupled to neuronal autophage.
Neuropathic pain is a complex, chronic pain state characterized by hyperalgesia, allodynia, and spontaneous pain . It is caused by a lesion or dysfunction of the peripheral or central nervous system (PNS and CNS, respectively) . Although it is undisputed that neurons play a fundamental role in neuropathic pain, the management of the suppression of aberrant neuronal activity has limited effectiveness and/or undesirable side effects . Thus, despite progress in the development of pharmacological agents, various therapeutic agents capable of blocking abnormal pain sensation without impairing normal abilities need to be proposed.
Recently, investigations that focus on the role of the PNS immune responses after nerve injuries have highlighted the active participation of glial cells in the maintenance of chronic pain in different pathological conditions. In particular, it has been confirmed that peripheral nerve injury can induce microglia and astrocyte activation in several chronic neuropathic pain models [4, 5]. Activated microglia release various algesic substances that enhance pain transmission by neurons; particularly, proinflammatory cytokines were shown to be common mediators of allodynia and hyperalgesia . Among these glial activation signals, Toll-like receptors (TLRs), particularly Toll-like receptor 4 (TLR4), have been demonstrated as initiators and mediators of neuropathic pain, .
TLR4 is an important pattern recognition receptor that has recently been implicated in chronic neuropathic pain [8, 9]. It recognizes pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and regulates the innate or adaptive immune response. TLR4 has been shown to be highly expressed by microglia in the CNS of rodents . Genetically altered mice with TLR4 deficiency have demonstrated significantly reduced microglia activation and pain hypersensitivity following nerve injury .
Autophagy is a highly regulated process involved in the turnover of long-lived proteins and damaged organelles. It involves the sequestration of regions of the cytosol within double-membrane-bound compartments and delivery of the contents to the lysosome for degradation . Pain is a common feature of various neurodegenerative diseases, in which autophagy plays a critical role in the progression of the pathology and is being studied as a possible therapeutic target [12, 13]. A recent study demonstrated that autophagy is modulated differently in the spinal cord of mice in several neuropathic pain models . As the most thoroughly characterized type of pattern recognition receptor, TLR4 enhances the elimination of phagocytosed mycobacteria to activate autophagy and serves as an environmental sensor for autophagy. The stimulation of TLR4 with lipopolysaccharide (LPS) induces autophagosome formation in macrophages by the TIR-domain-containing adapter-inducing interferon β (TRIF)-p38 axis and its downstream signaling pathways . These results indicate that TLR4 and autophagy play a pivotal role in chronic neuropathic pain, but the mechanism remains poorly understood. Thus, in the present study, we investigated the spinal modulation of the main autophagic markers in chronic constriction injury (CCI)-induced neuropathic pain models established with wild-type and TLR4-knockout (KO) mice.
Spinal nerve injury following CCI surgery induces mechanical allodynia in mice
Different nociceptive effects between WT and TLR4 KO mice on CCI-induced CatWalk analysis
Activation of microglia in the spinal dorsal horn following CCI
Activation of astrocytes in the spinal dorsal horn following CCI
LC3 levels in the spinal dorsal horn following CCI
Beclin 1 levels in the spinal dorsal horn following CCI
p62 levels in the spinal dorsal horn following CCI
Expression of PINK1 in the spinal dorsal horn following CCI
Inhibition of autophagy reduced pain behavior
Damage or disease to the somatosensory nervous system that results in disorders of the PNS often leads to chronic neuropathic pain, a debilitating condition resulting from sensitization of the nociceptive pathway. A recent report suggested that the activation of glial cells, especially microglia located in the sensory laminae of the spinal dorsal horn, is the main cause of this process . Activated microglia following peripheral nerve injury changes the morphology and releases neuroactive factors and cytokines that contribute to neuropathic pain . Although the mechanism underlying microglial proliferation following nerve damage remains unclear, it has recently been reported that TLRs play a critical role in neuropathic pain after peripheral nerve injury [7, 32], particularly in microglia activation and driving pain hypersensitivity after nerve injury. TLR4 is an important PAMP and DAMP that regulates the innate or adaptive immune response. TLR4 has been shown to be highly expressed in the CNS of rodents by microglia , and genetically altered mice lacking TLR4 showed significantly reduced microglia activation and pain hypersensitivity following nerve injury . Our results showed that the TLR4 KO in mice reduced pain hypersensitivity and proliferation of microglia following CCI-induced nerve injury, verifying the relationship between TLR4 and microglia in neuropathic pain (Fig. 1). CatWalk analysis also showed the correlation between TLR4 and pain hypersensitivity in neuropathic pain. Nerve injury following CCI decreased the percentages of the print area and single stance on the ipsilateral side of mice, and the percentages were significantly increased in TLR4 KO compared with WT mice (Fig. 2).
Autophagy has recently been shown to be a mechanism by which host cells capture and eliminate intracellular pathogens. Pain is a common feature of various neurodegenerative diseases in which autophagy plays a critical role in the progression of the pathology and is being studied as a possible therapeutic target [12, 13]. A recent study demonstrated that autophagy was differently modulated in the spinal cord of mice in several neuropathic pain models . As the most thoroughly characterized type of pattern recognition receptor, TLR4 enhances the elimination of phagocytosed mycobacteria to activate autophagy and serves as an environmental sensor for autophagy. The stimulation of TLR4 with LPS induces autophagosome formation in macrophages by the TRIF-p38 axis and its downstream signaling pathways . Therefore, we hypothesized that the TLR4-mediated autophagy pathway may play a critical role in neuropathic pain. We investigated the spinal modulation of some autophagy markers (e.g., LC3, Beclin 1, and p62) in mice after CCI (Figs. 5–7). In WT mice, increased Beclin 1 levels were paralleled by strong p62 accumulation in the ipsilateral compared with the contralateral side but without a significant increase in LC3-I and LC3-II in both sides of the spinal dorsal horn, suggesting a block in the late phase of autophagic flux rather than an induction of the process. In TLR4 KO mice, however, no significant changes in the three markers were observed in either the ipsilateral or contralateral sides of the spinal dorsal horn, suggesting that the modulation of autophagy was almost blocked due to the lack of TLR4 signaling.
Indeed, Beclin 1 upregulation in WT mice may indicate an increased autophagic flux but also defective autophagosome clearance. In the latter case, Beclin 1 upregulation will be associated with p62 accumulation because this autophagy substrate will not be efficiently degraded by the autophagosomes [23, 33]. Studies on the regulatory role of Beclin 1 in autophagy have suggested that the Beclin 1 complex is involved in autophagosome formation at an early stage , and this complex is essential for the recruitment of other autophagy-related proteins to the pre-autophagosomal structure .
No significant changes were observed in the expression of LC3-I and LC3-II in both WT and TLR4 KO mice (Fig. 5), and the lipidated form is known to be associated with autophagosomes . Monitoring LC3-II conversion is considered one of the most reliable methods for monitoring autophagy. However, a concomitant increase in both the rate of autophagosome formation and LC3 downstream degradation can show normal steady-state levels in LC3-II despite enhanced autophagy activity . Moreover, LC3 accumulation can result from autophagy induction, but also from impairment at one of the last steps such as fusion with the lysosomes or cargo degradation . Therefore, it is preferable to integrate LC3 studies with the analysis of other components of the autophagic machinery such as members of the initiation complex (e.g., Beclin 1) or autolysosome substrates (i.e., SQSTM1/p62) .
One of the best-known autophagic substrates is p62/SQSTM1, a key LC3-binding protein, which serves as a link between LC3 and ubiquitinated proteins . p62 and p62-bound polyubiquitinated proteins become incorporated into the completed autophagosome and are degraded in autolysosomes. Because of the correlation between autophagy modulation and p62 levels [27, 36, 37], this substrate is considered a useful readout of autophagic degradation [23, 38]. Indeed, p62 levels increase when autophagy is impaired . In WT mice, ipsilateral p62 accumulation was observed to significantly increase compared with the contralateral side, suggesting a block in the final degradative steps of autophagy. However, in the TLR4 KO mice, no significant change in the p62 level was observed between the two sides of the spinal dorsal horn (Fig. 7). Altogether, the analysis of LC3, Beclin 1, and p62 in this study indicated that autophagy impairment in CCI-induced neuropathic pain may be due to the occurrence of a block in the late phase of autophagic flux rather than in the induction of the process, and this autophagy seems to be mediated by TLR4 signaling. Moreover, mitophagy was assessed by monitoring the expression of the protein marker PINK1 (Fig. 8). Expression of PINK1 was increased in WT mice after CCI but showed no significant change in TLR4 KO mice, indirectly supporting our hypothesis regarding TLR4-mediated autophagy.
Although it was early reported that TLR4 is expressed primarily in microglia, but not astrocytes or neurons , it was also found that neurons do express TLR4 and that TLR signaling in neurons regulates neural precursor cell proliferation axonal growth, adult neurogenesis, and neuronal plasticity . In this study, we found that the expression of LC3, Beclin 1, and p62 associated with autophagy impairment in CCI-induced neuropathic pain was localized with neuronal cell, not astrocyte or microglia, in spinal dorsal horn. Previously, Tanga et al., reported that the genetically altered mice displayed significantly attenuated behavioral hypersensitivity and decreased expression of spinal microglial markers and proinflammatory cytokine . Therefore, it is reasonable that TLR4-mediated pro-inflammatory cytokine release in microglia and TLR4-mediated autophagic impairment in neurons contribute pain sensory hypersensitivity synergically.
Our immunohistochemical studies were supported with autophagic inhibitor, Chlorquine treatment. Chloroquine (Sigma-Aldrich, St. Louis, MO) is one of many compounds which have shown to reverse autophagy by accumulating in lysosomes, disturbing the vacuolar H+ ATPase, which is responsible for lysosomal acidification and blocking autophagy . When injected intrathecally in WT, Chloroquine induced a significant reduction in threshold of mechanical sensitivity (Fig. 9b). This data is in perfect agreement with previous paper . In that paper, the authors showed chloroquine was able to modulate the spinal autophagic machinery by the increase in p62, indicative of autophagosome accumulation. However, they did not show data on comparison with TLR4 KO mice. Our data showed that CCI-induced mechanical allodynia in TLR4 KO with Chloroquine treatment attenuated pain threshold compared to TLR4 KO. This data strongly supported TLR4-mediated autophagic impairment in neurons contribute pain sensory hypersensitivity with microglia activation, whereas, microglial TLR4-mediated microglial activation might be indirectly coupled to autophage.
Eight-week-old male C57BL/6j mice (Narabiotech, Seoul, Korea) were used in this study. C57BL/10ScNJ TLR4-KO (TLR4−/−) mice were purchased from the Jackson Laboratories (Bar Harbor, ME, USA). All the mice were individually housed in cages on a standard 12 h/12 h light/dark cycle, and water and food were available ad libitum.
Mechanical allodynia assay
To assess the sensitization to innocuous mechanical stimulation (mechanical allodynia), we measured the paw withdrawal response frequency (PWF) using a von Frey filament (North Coast Medical, Morgan Hill, CA, USA) as described in a previous study . Based on that study, a von Frey filament with a force of 2.0 g was selected for testing. Mice were placed on a metal mesh flooring, and the von Frey filament was applied from underneath the metal mesh flooring to each plantar of the hind paw. The filament was applied 10 times to each paw at intervals of 10 s, and the number of paw withdrawal responses following each filament stimulus was counted. The result of each experimental animal was expressed as a percentage of the paw withdrawal response frequency (% PWF). Paw withdrawal responses were measured day 0 (baseline), 1, 3, 5, and 7 after CCI surgery in each set.
CCI-induced neuropathic pain
CCI of the common sciatic nerve was performed based on the method described by Bennett and Xie . Mice were anesthetized with an intraperitoneal injection (i.p.) of Avertin (2,2,2-tribromoethanol, 50% w/v in tertiary amyl alcohol, diluted 1:40 in H2O; 20 ml/kg, i.p.; Sigma-Aldrich, St. Louis, MO, USA). The right common sciatic nerve was exposed at the mid-thigh level and was dissected from the connective tissue. Three loose ligatures of the 4–0 chromic gut were tied around the nerve with an interval of 1.0 to 1.5 mm between each ligature. After surgery, mice recovered on the heating pad at 27 °C.
CatWalk-automated gait analysis
The CatWalk XT system (Noldus Information Technology, Wageningen, The Netherlands) was used for the quantitative assessment of the gait parameter and footfalls in rodents. CatWalk is a verified system in the research of several pain models such as spinal cord injury, traumatic brain injury, and neuropathic pain. During the test, the mice traversed a dark tunnel with a glass plate from one side to the other. Their footprints were illuminated by fluorescent light from the glass plate and were captured by a high-speed camera positioned underneath the plate. The captured images were immediately processed by CatWalk XT software, and numerous parameters were analyzed, such as the print area, swing speed, and single stance. In this study, we measured the print area and single stance to assess differences in nociceptive responses between WT and TLR4-KO CCI mice. The print area is the contacting area between the hind paw and glass, and a single stance is the duration of the contralateral or ipsilateral hind paw touching the glass plate in the step cycle. CatWalk gait analysis was measured before and 1, 3, 5, and 7 days after CCI surgery in each set.
Immunohistochemistry was performed 7 days after surgery. The mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and perfused transcardially with heparinized phosphate-buffered saline (PBS, pH 7.4), followed by perfusion with 4% paraformaldehyde for 15 min. The lumbar enlargement (L4–L6) regions of the spinal cords were removed immediately, immersed in the same fixative overnight, and embedded in paraffin. The paraffin-embedded tissue arrays were performed in 4-μm sections and deparaffinized and rehydrated in a graded alcohol solution. The sections were soaked in 0.01 M citrate buffer (pH 6.0) and heated in a microwave vacuum histoprocessor (RHS-1, Milestone, Bergamo, Italy) at a controlled final temperature of 121 °C for 15 min for antigen retrieval. For immunohistochemical analyses as previously , endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. After primary antibody reaction (4 °C, overnight) as follows; Becline1(1:400; #AP1818a, ABGENT), p62 (1:400; #p0067, Sigma-Aldrich), PINK1 (1:400; #NBP2–36488, Novus Biologicals, Littleton, CO, USA), LC3 (1:200; #sc376404, Santa Cruz Biotechnology, Santa Cruz, CA, USA), NeuN (1:200; #24307S, Bioncompare), NeuN (1:200; #MAB377, Millpore), GFAP (1:2000, #Z0334, Dako), GFAP (1:2000; #MAB360, Millpore), Iba-1 (1:400; #019–19,741, Wako), Iba-1 (1:400; #016–26,721, Wako), the tissues were exposed to biotinylated anti-rabbit IgG and streptavidin peroxidase complex (Vector Laboratories, Inc., Burlingame, CA, USA). Immunostaining was visualized with diaminobenzidine (DAB), and the specimens were mounted using Polymount (Polysciences, Inc., Warrington, PA, USA).
Western blot analysis
The lumbar enlargement (L4-L6) regions of the spinal cord from WT and TLR4 KO mice were dissected and homogenized in lysis buffer. The lysates of the spinal cord (20 μg) were separated by 12 or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membranes. The blots were probed with the following primary antibodies: Becline 1 (1:1000; #sc-11,427, Santa Cruz Biotechnology, Santa Cruz, CA, USA), LC3 (1:1000; #L8918, Sigma-Aldrich), p62 (1:1000; #P0067, Sigma-Aldrich), PINK1 (1:1000; #NBP1–39667, Novus Biologicals, Littleton, CO, USA), and β-actin (1:500; #2965, Cell Signaling Technology, Danvers, MA, USA). The immune complexes were identified using an enhanced chemiluminescence (ECL) detection system (Habersham, Little Chalfont, United Kingdom).
All the data are presented as mean ± standard error of the mean. Quantitative analysis of immunostaining was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA) as previously . Statistical analyses were performed using the statistical software Prism 6.0 program (Graph Pad Software, San Diego, CA, USA), and repeated measurements from behavioral studies were analyzed by two-way analysis of variance. The results were considered significant at *P < 0.05, **P < 0.01, and ***P < 0.001.
We gratefully acknowledge Juhee Shin and Hyewon Park for expert technical assistance.
This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning (NRF-2016M3C7A1905074), and by the Korea government (MSIP) (2016R1A2B4009409, 2013R1A1A1057928, 2017R1D1A1B03028839).
Availability of data and materials
The datasets supporting the conclusion of this article are included within article.
YP, SO and DWK designed and performed experiments, analyzed the data and wrote the manuscript; GDH and DK performed Chloroquine experiments; TWH, NS, DHG, HHK and HJS performed histological and behavioral analysis; DK, YY and JK generated mouse model and performed genotype analysis; JH and HK analyzed the data and revised the manuscript; YK and SRK designed experiments and wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All the animal-related procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Chungnam National University (CNU-00781) and were consistent with the ethical guidelines of the National Institutes of Health and the International Association for the Study of Pain. All efforts were made to minimize animal suffering and to reduce the number of animals used.
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