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: The synovial joint exhibits extraordinary biotribological properties allowing the articular cartilage layers to slide past each other at very low friction even under local pressures of up to 18 MPa (~180 atm). Articular cartilage is exquisitely mechanical sensitive. Compressive mechanical load contributes to articular cartilage homeostasis; however, overuse or destabilizing the joint increases surface shear stress, which promotes cartilage degradation. Our previous Results show that shear stress, induced by joint destabilization, regulates a number of inflammatory genes 6h post surgery, including Mmp3, Il1b, Arg1, Ccl2, and Il6. Immobilizing the joint by prolonged anesthesia or sciatic neurectomy abrogates the regulation of inflammatory genes and prevents development of OA. In this study, we use RNA Scope to identify which cells of the cartilage are activated by surface shear after joint destabilisation, and test whether this is modifiable by injection of a biocompatible phospholipid-based lubricant. Methods: Destabilization of the medial meniscus (DMM) or sham surgery was performed on the right knee of 10-week-old male C57BL/6 mice. 30 ml of lubricant (PMPC: poly(methacryloylphosphsphorylcholine)-functionalized lipid vesicles) or vehicle control (PBS) solution was injected in the joint two days before and at the time of surgery. Cartilage from naïve (no surgery) and DMM-operated knees of four mice per data point was collected by microdissection for bulk mRNA extraction. Expression levels of selected genes including shear-responsive genes Il1b and Mmp3 were tested by RT-PCR using TaqMan Low Density Arrays (TLDA) microfluidic cards. In addition, whole joints were collected and processed following the standard protocol for RNAscope (Advanced Cell Diagnostics). Coronal sections in the middle of the joints were sliced by a cryostat. Consecutive sections were used for Safranin O staining and RNAscope to identify anatomical tissues and detect the expression of genes of interest. Gene expression signals were collated from 11 stacks by confocal microscopy (Zeiss Confocal 880) focusing on the medial tibia cartilage, and were quantified by counting individual mRNA dots in the sham, DMM, vehicle and lubricant groups. Results: We observed the upregulation of injury-responsive genes Il1b, Mmp3, Ccl2, Adamts 4, Nos2, and Timp1 in the articular cartilage of DMM operated joints compared to Naïve (non-operated) animals. The injection of the lubricant in the joint significantly suppressed the expression of shear-responsive genes Il1b and Mmp3 after DMM, but did not influence the increase of other injury-induced inflammatory genes, such as Timp1, Adamts 4, Ccl2, Nos2. For RNAscope, focusing on Mmp3 expression, the number of Mmp3 positive cells increased two-fold in the DMM-vehicle group compared with the sham-vehicle group. Most of Mmp3 signal was expressed in the superficial region of the cartilage. DMM-PMPC groups showed a reduced number of Mmp3 positive cells compared with DMM-vehicle, with levels similar to sham-vehicle and sham-PMPC groups. Conclusions: Our data demonstrate that shear stress-induced inflammatory genes are regulated in the superficial layer of cartilage after joint destabilisation and can be suppressed by joint injection of a biocompatible engineered lubricant. As these lubricants have long retention times in the joint (data not presented), we believe that they may provide a potential novel therapeutic strategy for preventing the development of post-trauma OA. These studies are underway