Chemotherapy-induced peripheral neuropathy (CIPN) affects over 3 million people in the US without effective treatment. CIPN predominantly affects nociceptive (pain-sensing) nerve terminals, and often manifests as chronic debilitating pain in patients. Chemotherapy-induced inflammation strongly correlates with the onset of and persistent neuropathic pain. We propose to investigate peripheral interactions between macrophages, the most abundant immune cell type residing in skin, and nociceptive nerve terminals in CIPN. Molecular profiles of macrophages change continuously depending on their extracellular environment, allowing them to transition from resident to activated states. While pro- and anti-inflammatory activation of macrophages has been extensively studied, recent studies revealed many intermediate forms during the transition. How these are linked to pathological progression of nerves is still poorly understood, and we propose to fill this gap by using two complementary systems, Drosophila and mouse CIPN models. We will test the hypothesis that modulating macrophage activation could prevent peripheral sensory neuron hypersensitivity in CIPN. In our preliminary work, we established a Drosophila model to study neuron-macrophage interaction in vivo and found that macrophages activate and transition into different intermediate forms in a time-dependent manner that corresponds to nociceptive neuron phenotypes. Leveraging a single-cell quantitative approach using deep learning cell detection in situ, we found that expansion of activated macrophages peaks preceding morphological degeneration of neurons with a robust increase in selected pro- and anti-inflammatory genes. We further found a significant reduction in anti-inflammatory macrophages at a later stage, indicating an immune state switch between intermediate and late pathological stages. In search of genes that could modulate macrophage activation to promote neuronal health while not interfering with their roles in host defense, we identified mesencephalic astrocyte-derived neurotrophic factor, MANF, as a potential gene target for CIPN prevention. MANF is a potent regulator of immune activity, and its overexpression reduces the pro-inflammatory pathway and promotes activation of the anti-inflammatory pathway. In support, our preliminary results demonstrate that augmenting MANF prevents neuron phenotypes in Drosophila and mouse CIPN models, consistent with our hypothesis. We propose to characterize chemotherapy-induced macrophage profiles and investigate how MANF modulates macrophages to prevent neuronal toxicity in Drosophila in vivo and mouse neuron-macrophage coculture CIPN models. Our proposal has a high potential to contribute to effective anti-inflammatory treatment for promoting neuronal health in CIPN.