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Advances in dermatology and venereology Acta Dermato-Venereologica
Atopic Dermatitis Linked Cytokine Interleukin-31 Induced Itch Mediated via a Neuropeptide Natriuretic Polypeptide B
Saumitra PITAKE #, Patrick C. RALPH #, Jennifer DEBRECHT and Santosh K. MISHRA * Department of Molecular Biomedical Sciences, NC State Veterinary Medicine, 1060 William Moore Drive, Office 242, Raleigh, NC 27607, USA. * E-mail: skmishra @ ncsu. edu. # These authors contributed equally. Accepted May 22, 2018; Epub ahead of print May 24, 2018
Atopic dermatitis( AD) is one of the most common itchinducing allergic skin diseases in humans and animals( 1, 2). Interleukin( IL)-31, a cytokine involved in immune response in inflammatory diseases such as AD, has been demonstrated to cause itch by acting on receptors in a subset of transient receptor potential vanilloid( TRPV1) neurons( 3 – 5). We recently identified a small subset of neurons that function as the primary detectors of chemical pruritic stimuli. We showed that the neuropeptide natriuretic polypeptide b( NPPB) is required for the transmission of itch signals and marks the full complement of itch responsive neurons at the periphery( 6). However, it remains unknown how itch signals in AD are transmitted by the primary afferents of the sensory neurons in the dorsal root ganglia( DRG) to the spinal cord. This study is aimed to determine if NPPB is involved as a neuropeptide in IL-31-mediated itch in AD via natriuretic polypeptide receptor A( NPRA) in the spinal cord.
METHODS( see Appendix S1 1)
RESULTS
Cytokine IL-31 transmits its signals through a receptor complex made of IL31R and oncostatin M receptor( OSMR). We used double ISH to show that the IL31R is co-expressed with NPPB expressing sensory neurons in the DRG. Our data demonstrated that IL31R is selectively expressed in all NPPB-positive somatosensory neurons( Fig. 1A). We further showed that IL31R is exclusively expressed in all OSMR neurons in the DRG( Fig. 1B). We showed the co-expression of NPPB with IL31R in almost 98 % of the neurons: 198 / 204 IL31R / OSMR-neurons.
1 https:// www. medicaljournals. se / acta / content / abstract / 10.2340 / 00015555-2977
To assess whether the IL-31-induced pruritogenic response is mediated via NPPB, we compared age-matched wild-type littermates and NPPB knockout( KO) mice. We injected IL-31 subcutaneously in both genotypes and measured scratching bouts. We observed a significant reduction in scratching response from NPPB KO mice( Fig. 1C). To further demonstrate the IL-31-mediated itch response by NPRA receptors expressed in the spinal cord, we used conjugated NPPB-saporin toxin to eliminate neurons expressing NPRA receptors in the spinal cord( 6). We consistently found a drastic reduction in scratching response from the NPPB-saporin treated mice as compared to the control. To rule out immunogenic involvement of IL-31 in itch production, we administered IL-31 to NOD control mice and the mice lacking functional B and T- cells( NOD / SCID). We found no significant difference between NOD control( black bar) and NOD / SCID immune-deficient mice( white bar; Fig. 1D) suggesting IL-31-mediated itch is an immuneindependent mechanism.
Contributions of TRPV1- or TRPA1-expressing DRG neurons in itch are important( 8). Recently, individual TRPV1- or TRPA1- KO mice confirmed an important role of TRP-channels in IL31- induced itch( 5); however, single KO have any compensatory effect on itch behavior was unclear. We sought out whether knocking out both TRPV1 and TRPA1-expression might show potential compensatory effect of TRP-channels on itch behavior. To determine the role of TRPV1 / TRPA1 ion channels, we generated double KO mice. We first performed IL-31 induced calcium response on wild-type DRG sensory neurons( Fig. 2A). Quantification of the number of cells responding to IL-31( 0.3 μM) revealed a reduction( approximately 50 %) in calcium response in double KO mice( Fig. 2B). Interestingly, our scratching behavior data demonstrated a significant decrease in IL-31( 0.3 nmol) induced itch response( 80 %) in TRPV1 / TRPA1 double KO mice( Fig. 2C) compared to wild type mice. Next, we determined whether NPPB release is induced by cytokine IL-31 and if the release is mediated through TRPV1 / TRPA1-ion channels. To explore this possibility, we cultured mouse DRGs from double KO and control mice and incubated neurons with IL-31( 0.3 μM) over 8 h to measure NPPB release. Indeed, our data showed a reduction( approximately 20 %) in NPPB release in double KO mice as compared to control mice. These results demonstrate that both TRPV1 and TRPA1 channels are associated with NPPB-release in DRG sensory neurons.
Fig. 1.( A) Double in situ hybridization( ISH) reveals that the neuropeptide natriuretic polypeptide b( NPPB)( green) co-expresses interleukin( IL)-31- receptor( red).( B) Double ISH of IL-31Ra co-expresses oncostatin M receptor( OSMR).( C) Itch responses to subcutaneously injected IL-31( 1.5 nmol) were significantly attenuated in NPPB knockout mice and mice treated with NPPB-saporin.( D) No differences in itch responses were observed in mice lacking B- and T-cell receptors( NOD / SCID) compare to control mice( NOD). Itch behaviors were observed in mice within 30 min of administering IL-31. Each column represents the mean ± SEM, n ≥ 6 mice for each group, ** p < 0.01.
This is an open access article under the CC BY-NC license. www. medicaljournals. se / acta Journal Compilation © 2018 Acta Dermato-Venereologica. doi: 10.2340 / 00015555-2977 Acta Derm Venereol 2018; 98: 795 – 796