Furthermore, we also importantly demonstrate that the decrease in macrophage recruitment to DRG by clodronate or LPS-RS, and subsequent decrease in pro-inflammatory cytokine expression, may contribute to the preservation of IENF following paclitaxel treatment. expression in DRG. Intrathecal injection of MCP-1 neutralizing antibodies reduced paclitaxel-induced macrophage recruitment into the DRG and also blocked the behavioral signs of CIPN. Intrathecal treatment with the TLR4 antagonist LPS-RS blocked mechanical hypersensitivity, reduced MCP-1 expression, and blocked the infiltration of macrophages into the DRG in paclitaxel treated rats. Finally, the inhibition of macrophage infiltration into DRG following paclitaxel treatment with clodronate or LPS-RS prevented the loss of intra-epidermal nerve fibers (IENFs) observed following paclitaxel treatment alone. Taken altogether, these results are the first to indicate a mechanistic link such that activation of TLR4 by paclitaxel leads to increased expression of MCP-1 by DRG neurons resulting in macrophage infiltration to the DRG that express inflammatory cytokines and the combination of these events results in IENF loss and the development of behavioral signs of CIPN. Keywords: Cancer, Neuropathic pain, PF-CBP1 TLR4, MCP-1, TNF 1. INTRODUCTION Chemotherapy induced peripheral neuropathy (CIPN) represents a dose-limiting adverse effect of cancer treatment which affects as many as half of cancer patients treated with single agents, and over 75% when combination therapies are utilized30,36. CIPN is observed following the administration of several types of drugs commonly used for the treatment of many of the most common solid and hematologic malignancies, including vinca alkaloids, taxanes, platinum derivatives, and bortezomib62,17,30. Furthermore, CIPN represents a clinical problem that is steadily on the rise as the number of long-term cancer survivors increases. CIPN EPHB2 most often presents as a sensory neuropathy with complaints of burning and shooting pains, tingling, and numbness, and observed as a length-dependent neuropathy with a stocking and glove distribution; and common analgesics aimed at reducing the painful symptoms are often ineffective8,9,16,20,26,25,40. The anti-cancer modes of action for the various chemotherapeutic drugs are largely understood, but the neurotoxic mechanisms contributing to the selectivity of the damage to sensory neurons alone and the clinical severity of CIPN remain unclear62,17,30. Currently, there are no pharmacologic or other means available to inhibit the occurrence of CIPN. Hence, dose reduction and ultimately withdrawal of the offending agent is the only option to slow the development of CIPN, potentially impacting optimal treatment62,17,30. Paclitaxel is one of the most effective chemotherapeutic drugs widely used for the treatment of solid tumors such as ovarian, breast, and non-small cell lung carcinoma; and also associated with the development of CIPN36. Although the specific mechanisms underlying the development of paclitaxel CIPN remain undefined, there are several lines of evidence indicating that engagement of innate immunity plays a key role48,37,43,42,41. For example, application of minocycline, an inhibitor of pro-inflammatory cytokine release, prevents mechanical allodynia induced by paclitaxel15,44, and we have demonstrated that intrathecal treatment with the TLR4 antagonist lipopolysaccharide-RS (LPS-RS) transiently reversed pre-established CIPN mechanical hypersensitivity and prevented the development of any behavioral signs of CIPN when given as a protective agent during chemotherapy43. Further, it was shown that paclitaxel treatment induces increased expression of monocyte chemoattractant protein-1 (MCP-1) in DRG and spinal cord and blockade of MCP-1/CCR2 signaling by anti-MCP-1 antibody or CCR2 antisense oligodeoxynucleotides significantly attenuated paclitaxel induced mechanical hypersensitivity, as well as the loss of distal intra-epidermal nerve fibers (IENF)64. MCP-1/CCL2 is a potent chemokine that regulates migration and infiltration of monocytes/macrophages22, PF-CBP1 and macrophages have been observed in DRG and the spinal dorsal horn in models of paclitaxel-induced CIPN48,44. Since a characteristic role of innate immunity involves monocyte/macrophage secretion of pro-inflammatory mediators, including TNF-, IL-1, IL-6, MIP-1, MIP-1 and MCP-1, that are widely recognized to contribute to an array of persistent pain states27,46,35, we hypothesized that paclitaxel treatment activates innate immunity resulting in macrophage recruitment to DRG and that these then drive the induction and maintenance of paclitaxel-induced peripheral hypersensitivity. 2. MATERIALS AND METHODS 2.1 Animals Adult male Sprague-Dawley rats (weighing 250C300g, Harlan, Houston, TX, USA) housed in a 12 h light/dark cycle with free access to food and water were used in all experiments. The studies were approved by the Institutional Animal Care and Use Committee at The University of Texas M. D. Anderson Cancer Center and were performed in accordance with the National Institutes of Health Guidelines for Use and Care of Laboratory Animals. 2.2 Paclitaxel CIPN model Animals were treated with paclitaxel as previously described49,23,14,7,43. Briefly, 6 mg/ml PF-CBP1 stock pharmaceutical grade paclitaxel (TEVA Pharmaceuticals, Inc. USA) was diluted with sterile 0.9% saline to 1 1 mg/ml and given at a dosage of 2 mg/kg intraperitoneally (i.p.) every other day for a total of four injections (days 1,.