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.

The neuromuscular junction is a synapse critical for muscle strength and coordinated motor function. Unlike CNS injuries, motor neurons mount robust regenerative responses after peripheral nerve injuries. Conversely, motor neurons selectively degenerate in diseases such as amyotrophic lateral sclerosis (ALS). To assess how these insults affect motor neurons in vivo, we performed ribosomal profiling of mouse motor neurons. Motor neuron-specific transcripts were isolated from spinal cords following sciatic nerve crush, a model of acute injury and regeneration, and in the SOD1G93A ALS model. Of the 267 transcripts upregulated after nerve crush, 38% were also upregulated in SOD1G93A motor neurons. However, most upregulated genes in injured and ALS motor neurons were context specific. Some of the most significantly upregulated transcripts in both paradigms were chemokines such as Ccl2 and Ccl7, suggesting an important role for neuroimmune modulation. Collectively these data will aid in defining pro-regenerative and pro-degenerative mechanisms in motor neurons.

Triple-negative breast cancer (TNBC) has a high propensity for organ-specific metastasis. However, the underlying mechanisms are not well understood. Here, we show that the primary TNBC tumor-derived C-X-C motif chemokines 1/2/8 (CXCL1/2/8) stimulate lung resident fibroblasts to produce C-C motif chemokines 2/7 (CCL2/7), which in turn activate cholesterol synthesis in lung-colonizing TNBC cells and induce angiogenesis at lung metastatic sites. Inhibiting cholesterol synthesis in lung-colonizing breast tumor cells by the pulmonary administration of simvastatin-carrying HER3-targeting nanoparticles reduces the angiogenesis and growth of lung metastases in a syngeneic TNBC mouse model. Our findings reveal a novel, chemokine-regulated mechanism for the cholesterol synthesis pathway and a critical role of metastatic site-specific cholesterol synthesis in the pulmonary tropism of TNBC metastasis. The study has implications for the unresolved epidemiological observation that the use of cholesterol-lowering drugs has no effect on breast cancer incidence but can unexpectedly reduce breast cancer mortality, suggesting interventions of cholesterol synthesis in lung metastases as an effective treatment to improve survival in TNBC patients.

Immune surveillance of the brain plays an important role in health and disease. Peripheral leukocytes patrol blood-brain barrier interfaces, and after injury, monocytes cross the cerebrovasculature and follow a pattern of pro- and anti-inflammatory activity leading to tissue repair. We have shown that chronic social defeat (CSD) causes scattered vasculature disruptions. Here, we assessed CCR2+ monocyte trafficking to the vascular injury sites in Ccr2wt/rfp reporter mice both during CSD and one week following CSD cessation. We found that CSD for 14 days induced microhemorrhages where plasma fibrinogen leaked into perivascular spaces, but it did not affect the distribution or density of CCR2rfp+ monocytes in the brain. However, after recovery from CSD, many vascularly adhered CCR2+ cells were detected, and gene expression of the CCR2 chemokine receptor ligands CCL7 and CCL12, but not CCL2, was elevated in endothelial cells. Adhered CCR2+ cells were mostly the non-classical, anti-inflammatory Ly6Clo type, and they phagocytosed fibrinogen in perivascular spaces. In CCR2-deficient Ccr2rfp/rfp mice, fibrinogen levels remained elevated in recovery. Fibrinogen infused intracerebroventricularly induced CCR2+ cells to adhere to the vasculature and phagocytose perivascular fibrinogen in Ccr2wt/rfp but not Ccr2rfp/rfp mice. Depletion of monocytes with clodronate liposomes during CSD recovery prevented fibrinogen clearance and blocked behavioral recovery. We hypothesize that peripheral CCR2+ monocytes are not elevated in the brain on day 14 at the end of CSD and do not contribute to its behavioral effects at that time, but in recovery following cessation of stress, they enter the brain and exert restorative functions mediating vascular repair and normalization of behavior.