Difelikefalin suppresses itch and reduces scratching independent of inflammation in a murine model of atopic dermatitis

BACKGROUND: Therapies specifically targeting nonhistaminergic pruritus are largely lacking. Difelikefalin (DFK) has been found to reduce itch in various chronic pruritic conditions, including atopic dermatitis (AD). OBJECTIVE: We sought to investigate the ability of DFK to impact scratching behavior, inflammatory mediators, and neuronal signaling in a murine model of AD. METHODS: The ears of C57BL/6 mice were topically treated with MC903 for 12 consecutive days to induce AD-like inflammation and itch. Before MC903 treatment, mice were treated with either DFK (0.5 mg/kg, intraperitoneal injection twice daily) or vehicle (saline). Skin ear thickness, histological analysis, flow cytometry, RNA-sequencing, and differential gene expression analyses of mouse ear skin were used to examine the effect of DFK on skin inflammation. Scratching behavior was quantified to measure itch behavior in mice that were topically treated with MC903 for 6 consecutive days; then, mice received a single injection of either DFK (1.0 mg/kg, intraperitoneal injection) or saline. Calcium imaging and single-cell RNA-sequencing were used in mouse dorsal root ganglia neurons to determine the size of the neurons activated with DFK treatment. Statistical significance was determined by Mann-Whitney test, unless otherwise noted. RESULTS: DFK rapidly suppressed itch without altering AD-like skin inflammation in MC903 (calcipotriol)-treated mice. In vitro Ca CONCLUSIONS: These studies support a potential neuromodulatory role of DFK for reducing itch associated with AD in mice

A TRPV4-dependent neuroimmune axis in the spinal cord promotes neuropathic pain

Microglia, resident macrophages of the CNS, are essential to brain development, homeostasis, and disease. Microglial activation and proliferation are hallmarks of many CNS diseases, including neuropathic pain. However, molecular mechanisms that govern the spinal neuroimmune axis in the setting of neuropathic pain remain incompletely understood. Here, we show that genetic ablation or pharmacological blockade of transient receptor potential vanilloid type 4 (TRPV4) markedly attenuated neuropathic pain-like behaviors in a mouse model of spared nerve injury. Mechanistically, microglia-expressed TRPV4 mediated microglial activation and proliferation and promoted functional and structural plasticity of excitatory spinal neurons through release of lipocalin-2. Our results suggest that microglial TRPV4 channels reside at the center of the neuroimmune axis in the spinal cord, which transforms peripheral nerve injury into central sensitization and neuropathic pain, thereby identifying TRPV4 as a potential new target for the treatment of chronic pain

Crystal structure of a complex of NOD1 CARD and ubiquitin.

The Caspase Recruitment Domain (CARD) from the innate immune receptor NOD1 was crystallized with Ubiquitin (Ub). NOD1 CARD was present as a helix-swapped homodimer similar to other structures of NOD1 CARD, and Ub monomers formed a homodimer similar in conformation to Lys48-linked di-Ub. The interaction between NOD1 CARD and Ub in the crystal was mediated by novel binding sites on each molecule. Comparisons of these sites to previously identified interaction surfaces on both molecules were made along with discussion of their potential functional significance

Cartoon view of the NOD1 CARD-Ub complex.

<p><b>A</b>) NOD1 CARD and Ub crystallized as a dimer of dimers. NOD1 CARD A (cyan) and Ub C (mauve taupe) are in the asymmetric unit. Symmetry-related subunits NOD1 CARD B (blue) and Ub D (yellow) complete the full complex. The NOD1 CARD homodimer demonstrates swapping of the sixth helices, similar to other reported NOD1 CARD dimers (2NSN and 2NZ7). The ubiquitin dimer chelates a phosphate anion (shown as spheres), similar to chelation of sulfate seen in another structure of Lys48-linked tetra-Ub (2O6V). Phe4 of Ub (green spheres) and the modified Cys59 of NOD1 CARD (orange spheres) are key residues mediating the NOD1 CARD-Ub interaction. The Lys48 residues of both Ub molecules are shown as magenta spheres. The N- and C- termini are highlighted as blue and red spheres, respectively. <b>B</b>) Close-up view of the dimethyl-arsenic adduct on NOD1 CARD. The Cys59 sidechain of NOD1 CARD (cyan) is shown in isolation. The adduct is within ∼3.5 Å of the aromatic Phe4 sidechain on Ub (mauve taupe), also shown in isolation. Electron density contoured at 2.0 sigma and carved at 2.3 Å shows the Cys59 sidechain is modified (left), while the nearby solvent-exposed Cys61 is not.</p

X-ray data collection and refinement statistics.

<p>Values for the highest resolution shell are given in parentheses.</p

Cartoon view of a model for how monomeric NOD1 CARD could bind linear di-Ub generated by the ZDOCK server (see Materials and Methods).

<p>The sidechains of Phe4 (purple) and Ile44 (black) from Ub and Cys59 (red) and Tyr88 (green) are shown as spheres. The combination of the previously identified NMR Ub-binding site (Tyr88:Ile44) with the new Ub-binding site observed in our crystal structure (Cys59:Phe4) may explain the mechanism by which NOD1 CARD preferentially binds linear and Lys63-linked poly-Ub.</p

Ub interaction sites on NOD1 CARD.

<p><b>A</b>) The NOD1 CARD dimer from our crystal structure (CARD A, cyan and CARD B, blue) is depicted in cartoon view. Residues mediating interaction with Ub (red) are shown as sticks. The asterisk on Pro62 indicates that only the main chain of this residue is involved in the interaction. The Cys59 sulfhydryl is modified with a dimethyl-As adduct (C59-mod – see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104017#pone-0104017-g004" target="_blank">figure 4</a>). The green box around Tyr88 signifies that this residue is also involved in the previously identified NMR Ub-binding site. <b>B</b>) Surface views of the NOD1 CARD dimer showing the Ub binding site in the crystal. The majority of the binding site is on CARD A in the asymmetric unit, however Tyr88 from the CARD B subunit of the dimer is also involved in the interaction. The symmetrical binding site on the opposite side of the dimer is shown (pink) on the rotated view. <b>C</b>) Surface views of the NOD1 CARD dimer showing the Ub binding site previously identified by NMR (green and light green). This site is located primarily on helix α5, which is on a different surface of NOD1 CARD than the one in the crystal structure. In the dimer, the two sites occupy the same overall surface formed from both subunits, while in the monomer they are on different surfaces (see D). Due to helix swapping in the dimer, the Leu104 sidechain is derived from helix 6 on the opposite subunit. The red box around Tyr88 signifies that it is also involved in the Ub-binding site observed in the crystal. <b>D</b>) Surface views of the NOD1 CARD monomer (PDB ID: 2DBD) with both Ub-binding sites mapped onto it. This demonstrates that the sites occupy different surfaces on the monomer, with the implication that the full interaction observed in the crystal could not occur with the monomeric form of NOD1 CARD unless Tyr88 was not involved. This arrangement of two different binding sites on two different surfaces is similar to that seen in the structure of the HOIL-1L NZF domain bound to linear di-Ub <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104017#pone.0104017-Sato1" target="_blank">[34]</a>.</p

Mono-Ub in our structure formed dimers similar in conformation to other structures of K48-linked di-Ub.

<p><b>A</b>) Ribbon representation of an alignment of K48-linked diubiquitin structures. The proximal di-Ub (2O6V-1, green) and distal di-Ub (2O6V-2, magenta) from tetra-Ub and di-Ub from 1AAR (blue) were aligned to the Ub dimer structure reported in this study (Ub C, mauve taupe and Ub D, yellow). Main chain RMSDs for this alignment were 1.309, 0.892 and 0.737 Å, respectively. <b>B</b>) Loop representation of the Ub dimer from our structure shown chelating a phosphate anion. This chelation is mediated mainly by Arg42 and Arg72 from each subunit. <b>C</b>) Loop representation of the proximal di-Ub moiety from K48-linked tetra-Ub (2O6V-1). In this case, Arg42 and Arg72 mediate chelation of a sulfate anion. In both structures, the Arg residues, which are located at the periphery of the hydrophobic pocket and often participate in interactions with UBDs, are engaged instead through these chelation interactions. This may serve as a mechanism by which UBDs discriminate between K48-linked poly-Ub and other forms of poly-Ub. K48 of the distal Ub moiety and the N-terminus (NT) of the proximal Ub moiety of each dimer are shown, demonstrating the close proximity in the dimer relative to the covalently linked chain.</p

Identified UBD binding sites in Ub.

<p>Ub is colored in mauve taupe. <b>A</b>) Cartoon representation of Ub. Residues mediating interaction with NOD1 CARD are shown as sticks and are colored red. The canonical hydrophobic pocket residues are shown as sticks and colored black. <b>B</b>) Surface views of the NOD1 CARD interaction site on Ub. <b>C</b>) Surface views of the Phe4 hydrophobic patch (F4 patch) on Ub alone (green) and merged with the NOD1 CARD site (yellow). <b>D</b>) Surface views of one site on Ub recognized by the NZF domain of HOIL-1L alone (green) and merged with the NOD1 CARD site (yellow). This domain specifically recognizes linear chains of Ub by binding to the hydrophobic pocket of the distal Ub and to an area surrounding Phe4 of the proximal Ub of a linear di-Ub moiety. Residues on the proximal Ub interacting within 3.6 Å of the NZF domain in a crystal structure of its complex with linear di-Ub (PDB ID: 3B08) are shown. <b>E</b>) Surface views of a second site on Ub recognized by the Vps23 UEV domain (Vps23 site) alone (green) and merged with the NOD1 CARD site (yellow). The Vps23 UEV domain binds both the hydrophobic pocket and this second site on Ub. Residues on Ub interacting within 3.6 Å of the UEV domain in a crystal structure of its complex with mono-Ub (PDB ID: 1UZX) are shown.</p