Gut microbiota promotes pain chronicity in Myosin1A deficient male mice.

Chronic pain is a heavily debilitating condition and a huge socio-economic burden, with no efficient treatment. Over the past decade, the gut microbiota has emerged as an important regulator of nervous system's health and disease states. Yet, its contribution to the pathogenesis of chronic somatic pain remains poorly documented. Here, we report that male but not female mice lacking Myosin1a (KO) raised under single genotype housing conditions (KO-SGH) are predisposed to develop chronic pain in response to a peripheral tissue injury. We further underscore the potential of MYO1A loss-of-function to alter the composition of the gut microbiota and uncover a functional connection between the vulnerability to chronic pain and the dysbiotic gut microbiota of KO-SGH males. As such, parental antibiotic treatment modifies gut microbiota composition and completely rescues the injury-induced pain chronicity in male KO-SGH offspring. Furthermore, in KO-SGH males, this dysbiosis is accompanied by a transcriptomic activation signature in the dorsal root ganglia (DRG) macrophage compartment, in response to tissue injury. We identify CD206+CD163- and CD206+CD163+ as the main subsets of DRG resident macrophages and show that both are long-lived and self-maintained and exhibit the capacity to monitor the vasculature. Consistently, in vivo depletion of DRG macrophages rescues KO-SGH males from injury-induced chronic pain underscoring a deleterious role for DRG macrophages in a Myo1a-loss-of function context. Together, our findings reveal gene-sex-microbiota interactions in determining the predisposition to injury-induced chronic pain and point-out DRG macrophages as potential effector cells.

Formalin-evoked pain triggers sex-specific behavior and spinal immune response.

Mounting evidence shows sex-related differences in the experience of pain with women suffering more from chronic pain than men. Yet, our understanding of the biological basis underlying those differences remains incomplete. Using an adapted model of formalin-induced chemical/inflammatory pain, we report here that in contrast to male mice, females distinctly display two types of nocifensive responses to formalin, distinguishable by the duration of the interphase. Females in proestrus and in metestrus exhibited respectively a short-lasting and a long-lasting interphase, underscoring the influence of the estrus cycle on the duration of the interphase, rather than the transcriptional content of the dorsal horn of the spinal cord (DHSC). Additionally, deep RNA-sequencing of DHSC showed that formalin-evoked pain was accompanied by a male-preponderant enrichment in genes associated with the immune modulation of pain, revealing an unanticipated contribution of neutrophils. Taking advantage of the male-enriched transcript encoding the neutrophil associated protein Lipocalin 2 (Lcn2) and using flow cytometry, we confirmed that formalin triggered the recruitment of LCN2-expressing neutrophils in the pia mater of spinal meninges, preferentially in males. Our data consolidate the contribution of female estrus cycle to pain perception and provide evidence supporting a sex-specific immune regulation of formalin-evoked pain.

Tyrosine kinase type A-specific signalling pathways are critical for mechanical allodynia development and bone alterations in a mouse model of rheumatoid arthritis.

ABSTRACT: Rheumatoid arthritis is frequently associated with chronic pain that still remains difficult to treat. Targeting nerve growth factor (NGF) seems very effective to reduce pain in at least osteoarthritis and chronic low back pain but leads to some potential adverse events. Our aim was to better understand the involvement of the intracellular signalling pathways activated by NGF through its specific tyrosine kinase type A (TrkA) receptor in the pathophysiology of rheumatoid arthritis using the complete Freund adjuvant model in our knock-in TrkA/C mice. Our multimodal study demonstrated that knock-in TrkA/C mice exhibited a specific decrease of mechanical allodynia, weight-bearing deficit, peptidergic (CGRP+) and sympathetic (TH+) peripheral nerve sprouting in the joints, a reduction in osteoclast activity and bone resorption markers, and a decrease of CD68-positive cells in the joint with no apparent changes in joint inflammation compared with wild-type mice after arthritis. Finally, transcriptomic analysis shows several differences in dorsal root ganglion mRNA expression of putative mechanotransducers, such as acid-sensing ionic channel 3 and TWIK-related arachidonic acid activated K+ channel, as well as intracellular pathways, such as c-Jun, in the joint or dorsal root ganglia. These results suggest that TrkA-specific intracellular signalling pathways are specifically involved in mechanical hypersensitivity and bone alterations after arthritis using TrkA/C mice.

Sensory neuron-derived TAFA4 promotes macrophage tissue repair functions.

Inflammation is a defence response to tissue damage that requires tight regulation in order to prevent impaired healing. Tissue-resident macrophages have a key role in tissue repair1, but the precise molecular mechanisms that regulate the balance between inflammatory and pro-repair macrophage responses during healing remain poorly understood. Here we demonstrate a major role for sensory neurons in promoting the tissue-repair function of macrophages. In a sunburn-like model of skin damage in mice, the conditional ablation of sensory neurons expressing the Gαi-interacting protein (GINIP) results in defective tissue regeneration and in dermal fibrosis. Elucidation of the underlying molecular mechanisms revealed a crucial role for the neuropeptide TAFA4, which is produced in the skin by C-low threshold mechanoreceptors-a subset of GINIP+ neurons. TAFA4 modulates the inflammatory profile of macrophages directly in vitro. In vivo studies in Tafa4-deficient mice revealed that TAFA4 promotes the production of IL-10 by dermal macrophages after UV-induced skin damage. This TAFA4-IL-10 axis also ensures the survival and maintenance of IL-10+TIM4+ dermal macrophages, reducing skin inflammation and promoting tissue regeneration. These results reveal a neuroimmune regulatory pathway driven by the neuropeptide TAFA4 that promotes the anti-inflammatory functions of macrophages and prevents fibrosis after tissue damage, and could lead to new therapeutic perspectives for inflammatory diseases.

Neurochemical and Ultrastructural Characterization of Unmyelinated Non-peptidergic C-Nociceptors and C-Low Threshold Mechanoreceptors Projecting to Lamina II of the Mouse Spinal Cord.

C-nociceptors (C-Ncs) and non-nociceptive C-low threshold mechanoreceptors (C-LTMRs) are two subpopulations of small unmyelinated non-peptidergic C-type neurons of the dorsal root ganglia (DRGs) with central projections displaying a specific pattern of termination in the spinal cord dorsal horn. Although these two subpopulations exist in several animals, remarkable neurochemical differences occur between mammals, particularly rat/humans from one side and mouse from the other. Mouse is widely investigated by transcriptomics. Therefore, we here studied the immunocytochemistry of murine C-type DRG neurons and their central terminals in spinal lamina II at light and electron microscopic levels. We used a panel of markers for peptidergic (CGRP), non-peptidergic (IB4), nociceptive (TRPV1), non-nociceptive (VGLUT3) C-type neurons and two strains of transgenic mice: the TAFA4Venus knock-in mouse to localize the TAFA4+ C-LTMRs, and a genetically engineered ginip mouse that allows an inducible and tissue-specific ablation of the DRG neurons expressing GINIP, a key modulator of GABABR-mediated analgesia. We confirmed that IB4 and TAFA4 did not coexist in small non-peptidergic C-type DRG neurons and separately tagged the C-Ncs and the C-LTMRs. We then showed that TRPV1 was expressed in only about 7% of the IB4+ non-peptidergic C-Ncs and their type Ia glomerular terminals within lamina II. Notably, the selective ablation of GINIP did not affect these neurons, whereas it reduced IB4 labeling in the medial part of lamina II and the density of C-LTMRs glomerular terminals to about one half throughout the entire lamina. We discuss the significance of these findings for interspecies differences and functional relevance.

c-Jun/p38MAPK/ASIC3 pathways specifically activated by nerve growth factor through TrkA are crucial for mechanical allodynia development.

Mechanical allodynia is a cardinal sign of several inflammatory pain disorders where nerve growth factor, a prototypic neurotrophin, plays a crucial role by binding to TrkA receptors. Here, we took the advantage of our generated knock-in mouse model expressing a chimeric TrkA/TrkC receptor that seems to not specifically develop mechanical allodynia after inflammation, to identify the TrkA downstream pathways involved in this phenomenon. We confirmed and extended that disrupting TrkA-specific pathways leads to a specific deficit in mechanical hypersensitivity development after somatic (systemic nerve growth factor administration and paw incision) and, to a lesser extent, visceral injuries. Despite a deficit in thin, mainly peptidergic, fibre innervation in TrkAC mice, thermal hyperalgesia development was not different from WT mice. Inflammatory reaction (oedema, IL-6 content), pain behaviours after intraplantar capsaicin, as well as TRPV1 calcium imaging response of dorsal root ganglion neurons were similar between TrkAC and WT mice. This deficiency in mechanical allodynia development in TrkAC mice is likely due to the alteration of the expression of different TrkA transduction pathways (ie, Akt, p38 MAPK, and c-Jun) especially p38 MAPK, in the dorsal root ganglion cell bodies, ultimately leading to an alteration of at least, ASIC3 channel overexpression, known to participate in nociceptor mechanosensory function.

A cell fitness selection model for neuronal survival during development.

Developmental cell death plays an important role in the construction of functional neural circuits. In vertebrates, the canonical view proposes a selection of the surviving neurons through stochastic competition for target-derived neurotrophic signals, implying an equal potential for neurons to compete. Here we show an alternative cell fitness selection of neurons that is defined by a specific neuronal heterogeneity code. Proprioceptive sensory neurons that will undergo cell death and those that will survive exhibit different molecular signatures that are regulated by retinoic acid and transcription factors, and are independent of the target and neurotrophins. These molecular features are genetically encoded, representing two distinct subgroups of neurons with contrasted functional maturation states and survival outcome. Thus, in this model, a heterogeneous code of intrinsic cell fitness in neighboring neurons provides differential competitive advantage resulting in the selection of cells with higher capacity to survive and functionally integrate into neural networks.

Uncoupling of molecular maturation from peripheral target innervation in nociceptors expressing a chimeric TrkA/TrkC receptor.

Neurotrophins and their receptors control a number of cellular processes, such as survival, gene expression and axonal growth, by activating multiple signalling pathways in peripheral neurons. Whether each of these pathways controls a distinct developmental process remains unknown. Here we describe a novel knock-in mouse model expressing a chimeric TrkA/TrkC (TrkAC) receptor from TrkA locus. In these mice, prospective nociceptors survived, segregated into appropriate peptidergic and nonpeptidergic subsets, projected normally to distinct laminae of the dorsal spinal cord, but displayed aberrant peripheral target innervation. This study provides the first in vivo evidence that intracellular parts of different Trk receptors are interchangeable to promote survival and maturation of nociceptors and shows that these developmental processes can be uncoupled from peripheral target innervation. Moreover, adult homozygous TrkAC knock-in mice displayed severe deficits in acute and tissue injury-induced pain, representing the first viable adult Trk mouse mutant with a pain phenotype.

Hepatocyte growth factor-Met signaling is required for Runx1 extinction and peptidergic differentiation in primary nociceptive neurons.

Nociceptors in peripheral ganglia display a remarkable functional heterogeneity. They can be divided into the following two major classes: peptidergic and nonpeptidergic neurons. Although RUNX1 has been shown to play a pivotal role in the specification of nonpeptidergic neurons, the mechanisms driving peptidergic differentiation remain elusive. Here, we show that hepatocyte growth factor (HGF)-Met signaling acts synergistically with nerve growth factor-tyrosine kinase receptor A to promote peptidergic identity in a subset of prospective nociceptors. We provide in vivo evidence that a population of peptidergic neurons, derived from the RUNX1 lineage, require Met activity for the proper extinction of Runx1 and optimal activation of CGRP (calcitonin gene-related peptide). Moreover, we show that RUNX1 in turn represses Met expression in nonpeptidergic neurons, revealing a bidirectional cross talk between Met and RUNX1. Together, our novel findings support a model in which peptidergic versus nonpeptidergic specification depends on a balance between HGF-Met signaling and Runx1 extinction/maintenance.

TRPV3 in keratinocytes transmits temperature information to sensory neurons via ATP.

Transient receptor potential V3 (TRPV3) and TRPV4 are heat-activated cation channels expressed in keratinocytes. It has been proposed that heat-activation of TRPV3 and/or TRPV4 in the skin may release diffusible molecules which would then activate termini of neighboring dorsal root ganglion (DRG) neurons. Here we show that adenosine triphosphate (ATP) is such a candidate molecule released from keratinocytes upon heating in the co-culture systems. Using TRPV1-deficient DRG neurons, we found that increase in cytosolic Ca(2+)-concentration in DRG neurons upon heating was observed only when neurons were co-cultured with keratinocytes, and this increase was blocked by P2 purinoreceptor antagonists, PPADS and suramin. In a co-culture of keratinocytes with HEK293 cells (transfected with P2X(2) cDNA to serve as a bio-sensor), we observed that heat-activated keratinocytes secretes ATP, and that ATP release is compromised in keratinocytes from TRPV3-deficient mice. This study provides evidence that ATP is a messenger molecule for mainly TRPV3-mediated thermotransduction in skin.

Expressing TrkC from the TrkA locus causes a subset of dorsal root ganglia neurons to switch fate.

Tactile information is perceived by a heterogeneous population of specialized neurons. Neurotrophin receptors (the receptor tyrosine kinases, Trks) mark the major classes of these sensory neurons: TrkA is expressed in neurons that sense temperature and noxious stimuli, and TrkC is expressed in proprioceptive neurons that sense body position. Neurotrophin signaling through these receptors is required for cell survival. To test whether neurotrophins have an instructive role in sensory specification, we expressed rat TrkC from the TrkA (also known as Ntrk1) locus in mice. The surviving presumptive TrkA-expressing neurons adopted a proprioceptive phenotype, indicating that neurotrophin signaling can specify sensory neuron subtypes.