Probes for CCL2

Migraine, especially chronic migraine, is highly debilitating and still lacks effective treatment. The persistent headache arises from activation and sensitization of primary afferent neurons in the trigeminovascular pathway, but the underlying mechanisms remain incompletely understood. Animal studies indicate that signaling through chemokine C-C motif ligand 2 (CCL2) and C-C motif chemokine receptor 2 (CCR2) mediates the development of chronic pain after tissue or nerve injury. Some migraine patients had elevated CCL2 levels in CSF or cranial periosteum. However, whether the CCL2-CCR2 signaling pathway contributes to chronic migraine is not clear. Here, we modeled chronic headache with repeated administration of nitroglycerin (NTG, a reliable migraine trigger in migraineurs) and found that both Ccl2 and Ccr2 mRNA were upregulated in dura and trigeminal ganglion (TG) tissues that are implicated in migraine pathophysiology. In Ccl2 and Ccr2 global knockout mice, repeated NTG administration did not evoke acute or persistent facial skin hypersensitivity as in wild-type mice. Intraperitoneal injection of CCL2 neutralizing antibodies inhibited chronic headache-related behaviors induced by repeated NTG administration and repetitive restraint stress, suggesting that the peripheral CCL2-CCR2 signaling mediates headache chronification. We found that CCL2 was mainly expressed in TG neurons and cells associated with dura blood vessels, whereas CCR2 was expressed in subsets of macrophages and T cells in TG and dura but not in TG neurons under both control and disease states. Deletion of Ccr2 gene in primary afferent neurons did not alter NTG-induced sensitization, but eliminating CCR2 expression in either T cells or myeloid cells abolished NTG-induced behaviors, indicating that both CCL2-CCR2 signaling in T cells and macrophages are required to establish chronic headache-related sensitization. At cellular level, repeated NTG administration increased the number of TG neurons that responded to calcitonin-gene-related peptide (CGRP) and pituitary adenylate cyclase-activating polypeptide (PACAP) as well as the production of CGRP in wild-type but not Ccr2 global knockout mice. Lastly, co-administration of CCL2 and CGRP neutralizing antibodies was more effective in reversing NTG-induced behaviors than individual antibodies. Taken together, these results suggest that migraine triggers activate CCL2-CCR2 signaling in macrophages and T cells. This consequently enhances both CGRP and PACAP signaling in TG neurons, ultimately leading to persistent neuronal sensitization underlying chronic headache. Our work not only identifies the peripheral CCL2 and CCR2 as potential targets for chronic migraine therapy, but also provides proof-of-concept that inhibition of both peripheral CGRP and CCL2-CCR2 signaling is more effective than targeting either pathway alone.

Purpose: Surgical destabilization of the medial meniscus (DMM) is a widely used mouse model of knee osteoarthritis (OA). The cell bodies of primary sensory neurons innervating the knee joints are located in the lumbar dorsal root ganglia (L3-L5 DRG). Analysis of the gene expression profile of L3-L5 DRG after DMM or sham surgery revealed that innate neuro-immune pathways were strongly regulated, especially in the later stages of the model, 8-16 weeks after DMM, when persistent pain is associated with severe joint damage. In depth analysis of the microarray data further showed that a number of genes encoding molecules in the Notch signaling pathway were regulated, mostly in late-stage disease, along with the upregulation of the gene encoding monocyte chemoattractant protein (MCP)-1/C-C motif chemokine ligand 2 (CCL2). CCL2 is a proalgesic mediator that is released upon tolllike receptor (TLR) 2/4 activation, and plays a key role in initiating and maintaining pain in this model. The aim of this study was to investigate Notch signaling in the knee-innervating DRG of mice with experimental knee OA, and determine the effect of Notch signaling activation on TLR2/4-mediated CCL2 synthesis in cultured DRG cells. Methods: DMM or sham surgery was performed in the right knee of 10- week old male C57BL/6 mice. Ipsilateral L4 DRG from mice 26 weeks after DMM or sham surgery were collected and cryosectioned. Expression of the Notch downstream target gene, Hes1, was detected using RNA in situ hybridization (ISH) (RNAscope, Advanced Cell Diagnostics). Quantification of mRNA expression was performed as calculating H-score of each sample according to the 0-4 five-bin scoring system recommended by the manufacturer, based on the number of cells with the same range of number of dots per cell. Active Notch protein was detected via immunofluorescence (IF) staining using an antibody against Notch intracellular domain (NICD), which is only present after g-secretase cleavage of Notch at S3. For in vitro cultures of DRG cells, bilateral L3-L5 DRG were collected from 10-week old male naïve C57BL/6 mice. Following enzymatic digestion, DRG cells were plated on poly-L-lysine and laminin coated glass coverslips, and cultured in F12 medium supplemented with 1x N2 and 0.5% fetal bovine serum. Inhibition of Notch signaling was achieved by (1) g-secretase inhibitor, DAPT; (2) ADAM-17 inhibitor, TAPI-1; or (3) soluble form of the Jag1 peptide (sJag1). On day 4, cells were pre-treated with DAPT (25 mM), TAPI-1 (20 mM), or sJag1 (40 mM) for 1 hour, followed by addition of the TLR2 agonist, Pam3CSK4 (1 mg/ml), or the TLR4 agonist, LPS (1 mg/ ml). Then, RNA was collected 3 hours later for qRT-PCR to quantify Ccl2 mRNA expression, or culture supernatants were collected 24 hours later to measure the CCL2 protein level using Quantikine Mouse CCL2/JE/ MCP-1 Immunoassay kit from R&D Systems, Inc.