Epidermal Neuromedin U Attenuates IgE-Mediated Allergic Skin Inflammation

<div><p>Although keratinocyte-derived neuropeptide neuromedin U (NMU) mediates the proinflammatory effects of innate-type mast cell activation, no information is available on the physiological roles. Here, to investigate the effects of NMU on IgE-mediated allergic skin inflammation, we determined whether IgE-mediated inflammation associated with severe scratching was induced in <i>Nmu</i>^<i>-/-</i> mice administered repeated hapten applications to the ear or footpad. Dry skin was induced by targeted deletion of <i>Nmu</i>. Mice administered repeated hapten application developed IgE-mediated allergic inflammation characterized by severe scratching and increased serum IgE levels only when the ear, and not the footpad, was subjected to scratching, indicating that depletion of NMU from the epidermis alone does not drive such allergic inflammation. Thus, the susceptibility of <i>Nmu</i>^<i>-/-</i> mice to allergic inflammation depends primarily on scratching dry skin. Further, allergic skin inflammation mediated by FcεRI cross-linking in <i>Nmu</i>^<i>-/-</i>mice was inhibited by prior injection of NMU. These results indicate that NMU plays an important physiological role as a negative regulator during the late stage of IgE-mediated allergic skin inflammation.</p></div

Effects of NMU on ITH mediated by two types of FcεRI cross-linking.

<p>(A) ITH responses were induced by TNCB application to the footpad of B6 or <i>Nmu</i>^-/- mice previously injected with TNP-specific IgE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160122#pone.0160122.s001" target="_blank">S1A Fig</a>). Different concentrations of TNP-specific IgE (0.01–1.0 μg in 20 μl of PBS per footpad) were injected 3 days before applying TNCB as follows: 0.05 μg to 0.2 μg of IgE achieved concentrations equivalent to that achieved in the serum of these mice after repeated hapten exposure on days 7–21 (2.5–10 μg/ml). (B) ITH was induced by injecting 0.2 μg of TNP-specific IgE into the footpads of B6 or <i>Nmu</i>^-/- mice followed by the injection of 25 ng of anti-mouse IgE. NMU (50 pmol) or vehicle (PBS) was injected one day before injection of mice with an anti-mouse IgE (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160122#pone.0160122.s001" target="_blank">S1B Fig</a>). In B6 mice, ITH was decreased by NMU released from keratinocytes upon injection compared with that of similarly treated <i>Nmu</i>^-/- mice. (C) Numbers of histologically identifiable dermal mast cells (MC), eosinophils (Eo), and neutrophils (Ne) in the footpads of B6 and <i>Nmu</i>^-/- mice (n = 4). Data are expressed as the mean ± SEM (n = 10), *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Cytokine mRNA expression in the ears of B6 and <i>Nmu</i>^-/- mice after repeated hapten application.

<p>Results are expressed as fold-increase in expression in the ears of each mouse before hapten application, as assessed by the value of the mean ± SEM (n = 4–6). <i>P</i> < 0.05, * <i>P</i> < 0.01.</p

Histological analyses of the ears of B6 and <i>Nmu</i>^-/- mice after repeated hapten exposure.

<p>(A) Hematoxylin-eosin (HE) staining of B6 and (B) <i>Nmu</i>^-/- mice on day 28 (n = 5). Original magnification, ×100. (C), Numbers of histologically identifiable dermal mast cells (MC), eosinophils (Eo), and neutrophils (Ne) in the ears of B6 and <i>Nmu</i>^-/- mice (n = 5). *<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Frequencies of IL-17-producing cells in draining lymph nodes 23 days after repeated hapten application.

<p>Mean frequencies of IL-17-producing CD4⁺, CD8⁺, NK (A) and γδ⁺ T cells (B) from B6 (n = 3) and <i>Nmu</i>^-/- mice (n = 5). Data are expressed as the mean ± SEM.*<i>P</i> < 0.05, **<i>P</i> < 0.01.</p

Differences in the responses of mice after repeated hapten application to the ear or footpad.

<p>(A) A hapten-specific ITH reaction is indicated by rapid ear swelling 0.5 h after each elicitation (n = 10). (B) Ear thickness (n = 10). (C) Number of mast cells in the ear (n = 5). (D) Serum IgE levels of mice after repeated hapten application to the ear (n = 10). (E) Serum IgE levels of mice after repeated hapten application to the footpad (n = 10). (F) Total number of scratching episodes from 7:00 PM–7:00 AM (n = 10). (A–F) Data are expressed as the mean ± SEM. *<i>P</i> < 0.05, ** <i>P</i> < 0.01.</p