CGRP sensory neurons promote tissue healing via neutrophils and macrophages.

The immune system has a critical role in orchestrating tissue healing. As a result, regenerative strategies that control immune components have proved effective1,2. This is particularly relevant when immune dysregulation that results from conditions such as diabetes or advanced age impairs tissue healing following injury2,3. Nociceptive sensory neurons have a crucial role as immunoregulators and exert both protective and harmful effects depending on the context4-12. However, how neuro-immune interactions affect tissue repair and regeneration following acute injury is unclear. Here we show that ablation of the NaV1.8 nociceptor impairs skin wound repair and muscle regeneration after acute tissue injury. Nociceptor endings grow into injured skin and muscle tissues and signal to immune cells through the neuropeptide calcitonin gene-related peptide (CGRP) during the healing process. CGRP acts via receptor activity-modifying protein 1 (RAMP1) on neutrophils, monocytes and macrophages to inhibit recruitment, accelerate death, enhance efferocytosis and polarize macrophages towards a pro-repair phenotype. The effects of CGRP on neutrophils and macrophages are mediated via thrombospondin-1 release and its subsequent autocrine and/or paracrine effects. In mice without nociceptors and diabetic mice with peripheral neuropathies, delivery of an engineered version of CGRP accelerated wound healing and promoted muscle regeneration. Harnessing neuro-immune interactions has potential to treat non-healing tissues in which dysregulated neuro-immune interactions impair tissue healing.

Early Over-Expressing of microRNA-145 Effectively Precludes the Development of Neuropathic Mechanical Hyperalgesia via Suppressing Nav1.8 in Diabetic Rats.

BACKGROUND: Painful diabetic neuropathy (PDN) is a common complication secondary to diabetes mellitus. Nav1.8 is an isoform of voltage-gated sodium channels and its expression regulation is closely related with PDN. MicroRNA-145 (miR-145) is involved in the occurrence and development of neuropathic pain. TargetScan software has revealed that Nav1.8 (SCN10A) is the major target of miR-145. However, its function between miR-145 and Nav1.8 in PDN is unknown. OBJECTIVES: We aim to explore the regulatory effect of miR-145 on the expression and function of Nav1.8, which plays a pivotal role in precluding the advancement of neuropathic mechanical hyperalgesia in diabetic pain. STUDY DESIGN: An experimental, animal study. SETTING: An animal research facility at Nanjing Maternal and Child Health Institute, China. METHODS: The paw mechanical withdrawal threshold (PMWT) of rats was assessed with the von Frey test. The adverse regulation of Nav1.8 by miR-145 was confirmed by a dual luciferase detection system in HEK293T cells. The mRNA level and expression of Nav1.8 in dorsal root ganglion (DRG) neurons were assessed with real-time polymerase chain reaction (real-time PCR), western blotting and immunofluorescence assays following intrathecal injection of agomiR-145 in vitro and in vivo. Whole-cell patch-clamping was applied to assess alterations in the tetrodotoxin-resistant (TTX-R) sodium current (Nav1.8) in DRGs. RESULTS: The PMWT was significantly decreased in rats following streptozotocin (STZ) injection on Day 7 and was maintained at a lower level on Day 28; this change was accompanied by changes in the expression of Nav1.8 in DRG neurons, which was increased 3 days after STZ injection and reached a maximal level on Day 14. The early knockdown of Nav1.8 with siRNA or agomiR-145 treatment on Day 8 effectively precluded the deterioration of pain behaviors in STZ-treated rats. The luciferase intensity was significantly decreased in HEK293T cells expressing wild-type SCN10A infected with miR-145 mimic. In addition, Nav1.8 overexpression was significantly repressed via overexpression of miR-145 in cultured DRG neurons, and neuronal hyperexcitability was concomitantly decreased. Furthermore, the intrathecal administration of agomiR-145 elicited a significant decrease in Nav1.8 expression in DRG neurons from STZ-treated rats on Day 14. LIMITATIONS: The causes of PDN are likely to be multifactorial and inflammatory markers, such as IL-6, IL-2, and TNF-?, are elevated in hyperglycemia and might be the precipitating factors that contribute to miR-145 dysregulation. The curative effect of miR-145 upregulation in reversal of pain behaviors at the stage of well-established PDN wasn't investigated in this study. CONCLUSION: Early infection with a lentiviral vector overexpressing miR-145 adversely regulated the expression and function of TTX-resistant Nav1.8 and abrogated the development of PDN. Therefore, miR-145 might be a potential therapeutic target for preventing PDN in the near future.

Absence of Functional Nav1.8 Channels in Non-diseased Atrial and Ventricular Cardiomyocytes.

PURPOSE: Several studies have indicated a potential role for SCN10A/NaV1.8 in modulating cardiac electrophysiology and arrhythmia susceptibility. However, by which mechanism SCN10A/NaV1.8 impacts on cardiac electrical function is still a matter of debate. To address this, we here investigated the functional relevance of NaV1.8 in atrial and ventricular cardiomyocytes (CMs), focusing on the contribution of NaV1.8 to the peak and late sodium current (INa) under normal conditions in different species. METHODS: The effects of the NaV1.8 blocker A-803467 were investigated through patch-clamp analysis in freshly isolated rabbit left ventricular CMs, human left atrial CMs and human-induced pluripotent stem cell-derived CMs (hiPSC-CMs). RESULTS: A-803467 treatment caused a slight shortening of the action potential duration (APD) in rabbit CMs and hiPSC-CMs, while it had no effect on APD in human atrial cells. Resting membrane potential, action potential (AP) amplitude, and AP upstroke velocity were unaffected by A-803467 application. Similarly, INa density was unchanged after exposure to A-803467 and NaV1.8-based late INa was undetectable in all cell types analysed. Finally, low to absent expression levels of SCN10A were observed in human atrial tissue, rabbit ventricular tissue and hiPSC-CMs. CONCLUSION: We here demonstrate the absence of functional NaV1.8 channels in non-diseased atrial and ventricular CMs. Hence, the association of SCN10A variants with cardiac electrophysiology observed in, e.g. genome wide association studies, is likely the result of indirect effects on SCN5A expression and/or NaV1.8 activity in cell types other than CMs.

Activity and connectivity changes of central projection areas revealed by functional magnetic resonance imaging in NaV1.8-deficient mice upon cold signaling.

The voltage-gated sodium channel subtype NaV1.8 is expressed in the peripheral nervous system in primary afferent nociceptive C-fibers and is essential for noxious cold signaling. We utilized functional magnetic resonance imaging on NaV1.8-deficient (NaV1.8-/-) compared with wildtype (WT) mice to identify brain structures decoding noxious cold and/or heat signals. In NaV1.8-/- mice functional activity patterns, activated volumes and BOLD signal amplitudes are significantly reduced upon noxious cold stimulation whereas differences of noxious heat processing are less pronounced. Graph-theoretical analysis of the functional connectivity also shows dramatic alterations in noxious cold sensation in NaV1.8-/- mice and clearly reduced interactions between certain brain structures. In contrast, upon heat stimulation qualitatively quite the same functional connectivity pattern and consequently less prominent connectivity differences were observed between NaV1.8-/- and WT mice. Thus, the fact that NaV1.8-/- mice do not perceive nociceptive aspects of strong cooling in contrast to their WT littermates seems not only to be a pure peripheral phenomenon with diminished peripheral transmission, but also consists of upstream effects leading to altered subsequent nociceptive processing in the central nervous system and consequently altered connectivity between pain-relevant brain structures.

Bacteria activate sensory neurons that modulate pain and inflammation.

Nociceptor sensory neurons are specialized to detect potentially damaging stimuli, protecting the organism by initiating the sensation of pain and eliciting defensive behaviours. Bacterial infections produce pain by unknown molecular mechanisms, although they are presumed to be secondary to immune activation. Here we demonstrate that bacteria directly activate nociceptors, and that the immune response mediated through TLR2, MyD88, T cells, B cells, and neutrophils and monocytes is not necessary for Staphylococcus aureus-induced pain in mice. Mechanical and thermal hyperalgesia in mice is correlated with live bacterial load rather than tissue swelling or immune activation. Bacteria induce calcium flux and action potentials in nociceptor neurons, in part via bacterial N-formylated peptides and the pore-forming toxin α-haemolysin, through distinct mechanisms. Specific ablation of Nav1.8-lineage neurons, which include nociceptors, abrogated pain during bacterial infection, but concurrently increased local immune infiltration and lymphadenopathy of the draining lymph node. Thus, bacterial pathogens produce pain by directly activating sensory neurons that modulate inflammation, an unsuspected role for the nervous system in host-pathogen interactions.