Probes for CCL2

The crosstalk between the nervous and immune systems has gained increasing attention for its emerging role in neurological diseases. Radiation-induced brain injury (RIBI) remains the most common medical complication of cranial radiotherapy, and its pathological mechanisms have yet to be elucidated. Here, using single-cell RNA and T cell receptor sequencing, we found infiltration and clonal expansion of CD8+ T lymphocytes in the lesioned brain tissues of RIBI patients. Furthermore, by strategies of genetic or pharmacologic interruption, we identified a chemotactic action of microglia-derived CCL2/CCL8 chemokines in mediating the infiltration of CCR2+/CCR5+ CD8+ T cells and tissue damage in RIBI mice. Such a chemotactic axis also participated in the progression of cerebral infarction in the mouse model of ischemic injury. Our findings therefore highlight the critical role of microglia in mediating the dysregulation of adaptive immune responses and reveal a potential therapeutic strategy for non-infectious brain diseases.

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.

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.