Exosome Mediated Neuro-immune Communication in Chronic Pain

Renee Jean-Toussaint

doi:10.17918/63qg-t114

Chronic pain, both inflammatory and neuropathic, is extremely difficult to treat. There is a need for investigating novel mechanisms underlying pain to identify new therapeutic targets. Growing evidence supports a role for immune signaling in chronic pain mechanisms. Pronociceptive and proinflammatory mediators secreted by resident immune cells, peripheral immune cells and sensory neurons contribute to the sensitization of neurons during chronic pain states. Cell secrete 30-150 nm extracellular vesicles or exosomes to communicate with both adjacent and distant cells. Exosomes transport regulatory biomolecules including proteins and RNAs and the contents vary depending on the source and the physiological states of cells releasing them. Disease states can alter exosome composition affecting the message conveyed and thus, uptake of exosomes can impact gene expression and function in recipient cells. The overall objective of this study was to assess the utility of exosomes as a therapeutic for chronic inflammatory pain and to characterize exosomes released during neuropathic pain. Exosomes derived from antigen-presenting cells such as macrophages can induce therapeutically relevant adaptive immune responses, but the mechanistic basis of this protection is not well understood. Exosomes secreted by RAW 264.7 mouse macrophages after lipopolysaccharide (LPS) stimulation contain elevated levels of anti-inflammatory miRNAs. We observed efficient uptake of these exosomes by primary mouse cortical neurons, microglia and astrocytes and downregulation of miRNA target genes in recipient cells. Pre-treating recipient cells with these exosomes decreased pro-inflammatory cytokine expression upon LPS stimulation. A single intrathecal injection of 1[mu]g exosomes derived from LPS stimulated RAW 264.7 cells attenuated mechanical hyperalgesia in the complete Freund's adjuvant (CFA) mouse model of inflammatory pain. Importantly, exosomes did not alter the normal pain threshold in control mice. RNA sequencing of dorsal horn of the spinal cord showed exosome-induced modulation of ion channels and immune regulatory pathways. Interestingly, a single prophylactic intrathecal injection of 1[mu]g exosomes two weeks prior, attenuated pain hypersensitivity in CFA model without impacting the protective physiological inflammatory pain. Our findings provide strong basis for further investigating the use of exosomes from antigen presenting cells both as a pain therapeutic and as a prophylactic agent that can function as a vaccine against chronic pain. Recent studies indicate significant dysregulation of miRNAs and proteins in the dorsal root ganglion and spinal cord after nerve injury. However, there are no studies to date investigating alterations in exosome composition, function, and signaling mechanisms in a neuropathic pain state. We hypothesized exosome content would be altered following nerve injury and these alterations can provide insight into signaling mechanisms involved in neuropathic pain. To characterize exosome composition following nerve injury, exosomes were purified from mouse serum four weeks after spared nerve injury (SNI) or sham surgery. Our miRNA profiling showed a distinct exosomal miRNA signature in SNI model compared to sham control. Proteomics analysis using tandem mass spectrometry detected 274 gene products. Of these, 24 were unique to SNI model, 100 to sham surgery control and five to naïve control samples. In addition to commonly expressed exosome proteins, multiple members of serpin and complement family were detected in exosomes. Neuropathic pain can induce the activation of the complement cascade and our cytokine profiling using membrane-based antibody array showed significant upregulation of complement component 5a (C5a). Intercellular Adhesion Molecule 1 (ICAM-1), required for the leukocyte recruitment, adhesion and homing of exosomes was also upregulated in exosomes from SNI model compared to sham control. We observed a differential distribution of C5a and ICAM-1 within serum and exosomes between sham and SNI, indicating changes from local or paracrine to long distance signaling under neuropathic pain. Our studies suggest critical roles for cargo sorting of vesicular proteins in mediating neuropathic pain. Collectively, our studies show the therapeutic potential of exosomes derived from antigen presenting cells and shed light on novel mode of cellular communication mediated by exosomes after nerve injury.

Exosome Mediated Neuro-immune Communication in Chronic Pain

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Exosome Mediated Neuro-immune Communication in Chronic Pain

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