Zoltán Winter

Granulocytic myeloid-derived suppressor cells (G-MDSCs) are immunoregulatory cells of the neutrophil lineage. Here, we show that immunoregulation by G-MDSCs is imprinted by the joint tissue microenvironment in patients with axial spondyloarthritis and in animal models of inflammatory arthritis. We demonstrate that the suppressive function of G-MDSCs is significantly enhanced in the joint tissue, but not in the circulation. Analysis of public single-cell RNA sequencing datasets was consistent with Cd200r1 ^high Hmgb2 ⁺ precursors differentiating into G-MDSCs following interaction with Cd200 ⁺ fibroblasts. In hTNFtg/ Il23mc mice, treatment with a CD200R1 agonist promoted an immunoregulatory signature associated with PD-L1 expression, whereas CD200 blockade reversed the phenotype. Following adoptive transfer of G-MDSC precursor cells into arthritic mice, administration of a CD200-blocking antibody impaired the differentiation of PD-L1 ⁺ G-MDSCs and exacerbated disease severity. These findings reveal a tissue-intrinsic program that facilitates resolution of inflammation through fibroblast-myeloid crosstalk that enhances G-MDSC function.

Psoriatic disease initially affects the skin and later extends to the joints. Here, we show a two-step process that orchestrates the spread of inflammation from the skin to the joints. Induction of psoriatic skin disease in photoconvertible mice, followed by sequencing and computational characterization of skin-derived cells in the joints, was used to identify a population of CD2 ⁺ MHC-II ⁺ CCR2 ⁺ myeloid precursors that builds a skin-derived myeloid cell compartment in the joints. Single-cell cross-species reference mapping and mitochondrial variant tracing showed an orthologous human cell population. Interactome analysis of the joints showed that in a second step, resident regulatory CD200 ⁺ fibroblasts regulate the priming of CD2 ⁺ MHC-II ⁺ CCR2 ⁺ myeloid precursors, which subsequently control IL-17 expression in T cells. Hence, the spread of inflammation requires a distinct migratory myeloid precursor population and a permissive local tissue environment, similar to tumor metastasis.

Psoriatic disease initially affects the skin, but later extends to the joints. Herein, we describe a two-step process that orchestrates spreading of inflammation from the skin to the joints. Induction of psoriatic skin disease in photoconvertible mice, followed by sequencing and computational characterization of skin-derived cells in the joints, identified a unique population of CD2 ⁺ MHC-II ⁺ CCR2 ⁺ myeloid precursors that built up a skin-derived myeloid cell compartment in the joints. Single-cell cross-species reference mapping and mitochondrial variant tracing showed an orthologue human cell population. Interactome analyses in the joints showed that in a second step, resident regulatory CD200 ⁺ fibroblasts critically regulate the priming of the CD2 ⁺ MHC-II ⁺ CCR2 ⁺ myeloid precursors, which subsequently regulate the IL-17 expression in T cells. Hence, spreading of inflammation requires a distinct migratory myeloid precursor population and a permissive local tissue environment, similar to tumour metastasis. Graphical

Low temperatures and cooling agents like menthol induce cold sensation by activating the peripheral cold receptors TRPM8 and TRPA1, cation channels belonging to the TRP channel family, while the reduction of potassium currents provides an additional and/or synergistic mechanism of cold sensation. Despite extensive studies over the past decades to identify the molecular receptors that mediate thermosensation, cold sensation is still not fully understood and many cold-sensitive peripheral neurons do not express the well-established cold sensor TRPM8. We found that the voltage-gated potassium channel KCNQ1 (Kv7.1), which is defective in cardiac LQT1 syndrome, is, in addition to its known function in the heart, a highly relevant and sex-specific sensor of moderately cold temperatures. We found that KCNQ1 is expressed in skin and dorsal root ganglion neurons, is sensitive to menthol and cooling agents, and is highly sensitive to moderately cold temperatures, in a temperature range at which TRPM8 is not thermosensitive. C-fiber recordings from KCNQ1 ^−/− mice displayed altered action potential firing properties. Strikingly, only male KCNQ1 ^−/− mice showed substantial deficits in cold avoidance at moderately cold temperatures, with a strength of the phenotype similar to that observed in TRPM8 ^−/− animals. While sex-dependent differences in thermal sensitivity have been well documented in humans and mice, KCNQ1 is the first gene reported to play a role in sex-specific temperature sensation. Moreover, we propose that KCNQ1, together with TRPM8, is a key instrumentalist that orchestrates the range and intensity of cold sensation.

The odontoblast, with processes beyond the dentin-enamel boundary, is the initial site of TRPC5 cold transduction in teeth.

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8^EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Previous research identified TRPM8 and TRPA1 cold transducers with separate functions, one being functional in the non-noxious range and the second one being a nociceptive transducer. TRPM8-deficient mice present overt deficits in the detection of environmental cool, but not a lack of cold avoidance and TRPA1-deficient mice show clear deficits in some cold nocifensive assays. The extent of TRPA1's contribution to cold sensing in vivo is still unclear, because mice lacking both TRPM8 and TRPA1 (DKO) were described with unchanged cold avoidance from TRPM8-/- based on a two-temperature-choice assay and by c-fos measurement. The present study was designed to differentiate how much TRPM8 alone and combined TRPA1 and TRPM8 contribute to cold sensing. We analyzed behaviour in the thermal ring track assay adjusted between 30°C and 5°C and found a large reduction in cold avoidance of the double knockout mice as compared to the TRPM8-deficient mice. We also revisited skin-nerve recordings from saphenous-nerve skin preparations with regard to nociceptors and thermoreceptors. We compared the frequency and characteristics of the cold responses of TRPM8-expressing and TRPM8-negative C-fiber nociceptors in C57BL/6J mice with nociceptors of TRPM8-deficient and DKO mice and found that TRPM8 enables nociceptors to encode cold temperatures with higher firing rates and larger responses with sustained, static component. In TRPM8-/-, C-fiber cold nociceptors were markedly reduced and appeared further reduced in DKO. Nevertheless, the remaining cold responses in both knockout strains were similar in their characteristics and they were indifferent from the TRPM8-negative cold responses found in C57BL/6J mice. TRPM8 had a comparably essential role for encoding cold in thermoreceptors and lack of TRPM8 reduced response magnitude, peak and mean firing rates and the incidence of thermoreceptors. The encoding deficits were similar in the DKO strain. Our data illustrate that lack of TRPA1 in TRPM8-deficient mice results in a disproportionately large reduction in cold avoidance behaviour and also affects the incidence of cold encoding fiber types. Presumably TRPA1 compensates for lack of TRPM8 to a certain extent and both channels cooperate to cover the temperature range, making cold-temperature encoding by TRPA1 – although less powerful – synergistic to TRPM8.

There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron‐blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1–calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell‐regulatory calmodulin–protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain‐modulating behavior of these important pharmacons.

Loss-of-function mutations of Na_V1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of Na_V1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of Na_V1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of Na_V1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with Na_V1.7 inhibitors and significantly reduced in Na_V1.7^−/− mice. To validate the use of the model for profiling Na_V1.7 inhibitors, we determined the Na_V selectivity and tested the efficacy of the reported Na_V1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited Na_V1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited Na_V1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited Na_V channels and was only effective in the OD1 model when delivered systemically. Our novel model of Na_V1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of Na_V1.7 inhibitors.