渡邊 総一郎

Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), have been implicated in the pathogenesis of skin disorders. We previously reported that Il36rn-/- mice exhibit an enhanced contact hypersensitivity (CHS) response through increased neutrophil recruitment. In addition, Il36rn-/- mice show severe imiquimod-induced psoriatic skin lesions and enhanced neutrophil extracellular trap (NET) formation. We hypothesized that NETs may play an important role in the CHS response. To confirm this, we examined the CHS response and NET formation in Il36rn-/- mice. Il36rn-/- mice showed enhanced CHS responses, increased infiltration of inflammatory cells, including neutrophils, CD4+ T cells, and CD8+ T cells, NET formation, and enhanced mRNA expression of cytokines and chemokines, including IL-1β, C-X-C motif chemokine ligand (CXCL)1, CXCL2, and IL-36γ. Furthermore, NET formation blockade improved the CHS response, which consequently decreased inflammatory cell infiltration and NET formation. Consistently, we observed decreased expression of these cytokines and chemokines. These findings indicate that IL-36Ra deficiency aggravates the CHS response caused by excessive inflammatory cell recruitment, NET formation, and cytokine and chemokine production, and that NET formation blockade alleviates the CHS response. Thus, NET formation may play a prominent role in the CHS response.

BACKGROUND: Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), has been implicated in the pathogenesis of skin disorders. However, the pathogenic role of IL-36Ra in cutaneous ischemia-reperfusion (I/R) injury remains unclear. OBJECTIVES: We investigated the role of IL36Ra in cutaneous I/R injury. METHODS: We examined I/R injury in Il36rn-/- mice. The area of wounds, numbers of infiltrated cells, apoptotic cells and neutrophil extracellular trap (NET) formation were assessed. The expression levels of various genes were analysed using real-time RT-PCR. The expression of high mobility group box 1 (HMGB1), an endogenous toll-like receptor (TLR) 4 ligand, was confirmed using immunohistology, and serum HMGB1 levels were measured by ELISA. Cytokine production by stimulated cultured J774A.1 and HaCaT cells was examined. RESULTS: IL-36Ra deficiency resulted in significantly delayed wound healing and increased neutrophil and macrophage infiltration into the wound tissues. Il36rn-/- mice had increased mRNA expression levels of CXCL1, CXCL2, CCL4, TNF-α, TGF-β, IL-1β, IL-6 and IL-36γ relative to wild-type mice. Apoptosis was identified in keratinocytes by TUNEL assay. HMGB1 expression in the I/R site was decreased in both keratinocytes and adnexal cells, while serum HMGB1 levels were significantly elevated after reperfusion. The mRNA levels of various cytokines, including IL-1β, were elevated in J774A.1 cells through TLR4 signalling by HMGB1 stimulation. In addition, HaCaT cells stimulated with IL-1β showed significantly increased CXCL1, TNF-α, IL-6, IL-36β and IL-36γ mRNA expression. Furthermore, NET formation was increased by IL-36Ra deficiency. Finally, either the blockade of TLR4 signalling by TAK-242 or inhibition of NET formation by Cl-amidine normalized exacerbated I/R injury in Il36rn-/- mice. CONCLUSIONS: This study indicated that IL-36Ra deficiency exacerbates cutaneous I/R injury due to excessive inflammatory cell recruitment, NET formation, and excessive cytokine and chemokine production via the TLR4 pathway by HMGB1 released from epidermal apoptotic cells.

<title>Abstract</title>Loss-of-function mutations in the interleukin (IL)-36 gene <italic>IL36RN</italic> are associated with psoriasis. The importance of neutrophil extracellular traps (NETs), web-like structures composed of neutrophil DNA, in the pathogenesis of psoriasis has been unclear. Here, we aimed to clarify the role of NET signaling in the deficiency of IL36 receptor antagonist (DITRA). We evaluated the severity of psoriasis-like lesions induced by imiquimod cream treatment in <italic>Il36rn</italic>^−/− mice. The mRNA levels of psoriasis-related cytokines were measured via real-time reverse transcription polymerase chain reaction, and the effects of Cl-amidine, a peptidyl arginine deiminase 4 (PAD4) inhibitor, on psoriasis-like lesions were evaluated. PAD4 is a histone-modifying enzyme that is involved in NET formation. Psoriasis area and severity index scores, epidermal thickness, and infiltrated neutrophil counts were significantly increased in <italic>Il36rn</italic>^−/− mice; NET formation was confirmed pathologically. Several cytokines and chemokines were upregulated in the skin lesions of <italic>Il36rn</italic>^−/− mice and Cl-amidine treatment improved these psoriasis-like lesions. These results suggest that NET formation plays an important role in the pathology of psoriasis-like lesions in these mice and might represent a promising therapeutic target for DITRA.

Loss-of-function homozygous or compound heterozygous mutations in IL36RN, which encodes interleukin-36 receptor antagonist (IL-36Ra), have been implicated in the pathogenesis of various skin disorders. Previous findings showed that IL-36γ promoted wound healing in mice; however, the pathogenic role of IL-36Ra in wound healing remains unclear. We elucidated the role of IL-36Ra, a regulator of IL-36 in tissue repair by investigating the recruitment of inflammatory cells and cytokine production in the absence of IL-36Ra. Full-thickness excisional wounds were made on the back of Il36rn-/- mice and healing was assessed by monitoring macroscopic wound sizes, numbers of infiltrated cells, and gene expression of inflammatory cytokines. Macroscopic wound healing, re-epithelialization, and granulation tissue formation were delayed by 3 days post-injury in Il36rn-/- mice. This delay was associated with increased infiltrations of neutrophils and macrophages, and increased expression of cytokines, such as IL-36γ, C-X-C motif chemokine ligand 1 (CXCL1), and transforming growth factor (TGF)-β. Importantly, administration of TAK-242, a toll-like receptor 4 (TLR4) inhibitor, caused normalization of wound healing in Il36rn-/- mice, abrogating the initial delay in tissue repair. These results showed that targeting TLR4- mediated infiltrations of immune cells and cytokine production could be beneficial in regulating wound healing in IL-36Ra-deficient skin disorders.

Loss-of-function mutations in IL36RN cause generalized pustular psoriasis (GPP), which is characterized by neutrophil-infiltrated lesions. Neutrophils are important during contact hypersensitivity in mice. However, it has never been determined whether interleukin-36 receptor antagonist (IL-36Ra) deficiency is an exacerbating factor in contact dermatitis. We examined whether a loss-of-function IL36RN mutation exacerbates contact dermatitis and evaluated the changes in contact dermatitis-related cytokines. Wild-type and Il36rn-/- mice were treated with 1-fluoro-2,4-dinitorobenzene (DNFB) and evaluated for ear thickness, histopathological features, numbers of infiltrated neutrophils, and numbers of CD4 + and CD8 + T cells. Furthermore, mRNA levels of contact dermatitis-related cytokines were measured by real-time polymerase chain reaction, and effects of TAK-242, a toll-like receptor 4 (TLR4) inhibitor, on the contact hypersensitivity (CHS) response were evaluated. We found that the ear thickness, cytokine expression, and neutrophil infiltration significantly increased in Il36rn-/- mice compared with that in wild-type mice. TAK-242 alleviated CHS and prevented neutrophil infiltration, cytokine expression, and ear thickening in Il36rn-/- mice. These data indicate that Il36rn-/- mutations are an exacerbating factor for CHS and that TAK-242 can reduce the inflammatory responses that are associated with the CHS response.

58歳、男性。幼少よりアトピー性皮膚炎。ステロイド外用薬、抗ヒスタミン薬の内服で治療していたが、コントロール不良であった。2008年からシクロスポリン200mg/日を導入したが、2012年4月より紅皮症を呈し当科へ紹介。表在リンパ節腫脹および可溶性IL-2R高値(7,765U/mL)、異型リンパ球高値(20%)のため、Sezary syndromeを疑い、シクロスポリンを中止したところ異型リンパ球は速やかに減少し皮疹も消退傾向を示した。腹部の病理組織では異型リンパ球は認められず、表皮内の浸潤も明らかではなかったが、血液中のTCR遺伝子再構成を検索したところ、cβ1遺伝子の再構成バンドが確認された。経過および検査所見よりatopic dermatitiss like pre-Sezary syndromeと診断した。現在はプレドニゾロン5mg/日および紫外線療法で小康状態を保っている。(著者抄録)

<p>61歳，男性。2014年1月より陰部に皮膚腫瘤を自覚。徐々に増大し腫瘤の表面から出血を伴うようになったため，近医受診し同年8月当科紹介受診。初診時，陰部に25×20 mm大の一部血痂を伴う紅色隆起性腫瘤を認め，両鼠径に母指頭大〜小児手拳大までの複数のリンパ節腫大を伴っていた。陰部腫瘊の全摘切除病理所見では，真皮浅層から脂肪織深層まで好酸性顆粒状の胞体を有する大型な異型細胞が管状構造を呈しつつ深部に浸潤しており，腫瘊病変中に断頭分泌像が認められた。免疫組織化学染色では，GCDFP-15，CEA，ER，PgRが陽性であった。画像検索において内臓悪性腫瘊の皮膚転移が否定的であり，皮膚原発アポクリン腺癌と確定診断した。原発巣の拡大切除と両側鼠径リンパ節郭清術を施行し，現在術後17ヵ月経過したが，再発転移を認めず経過良好である。</p>

<p>62歳，男性。初診の1年前から急激に増大し，初診時，右大腿後面に18×11 cm大の腫瘊を認め，部分生検の組織所見より有棘細胞癌と診断した。MRI所見では，腫瘊深部は筋に近接しており，右鼠径および外腸骨のリンパ節が腫脹していた。CDDPと5-FUの術前化学療法による腫瘊縮小を確認後に筋膜を含めた腫瘊切除と右鼠径リンパ節廓清を施行した。全摘標本の水平・深部断端は陰性で，リンパ節転移はみられず，Stage II（T2N0M0）と確定した。術後7日目に一部椊皮部が離開し再縫合と床上安静を要した。術後20日目に抗生剤上応性の高熱を生じ，血清CRP，D-dimerが上昇したため，緊急造影CT検査を施行し両側肺塞栓症が判明した。直ちに抗凝固療法を開始することで，術後41日目に退院可能となった。肺塞栓症は致死的となりうる重要な周術期合併症であり，皮膚外科領域の手術においても常に留意すべき合併症である。</p>

46歳，男性。右母指外側の爪変形を認め，近医を受診しglomus tumor疑いで当科受診した。初診時，右母指外側の爪甲は縦裂しており下部に紅色腫瘍を認めた。病変内や周囲に色素斑は認められなかった。エコーでは，homogeneous，hypoechoic lesionで，豊富な血流も伴っていた。病理所見では，核小体が明瞭な異型腫瘍細胞が胞巣状に真皮に浸潤し，S−100，HMB−45，Melan−Aに陽性であった。悪性黒色腫と診断し，拡大切除と右腋窩センチネルリンパ節生検を行った。術後4ヵ月から右母指内側に爪が再生し切除標本で局所再発と判明した。さらに拡大切除を行い，1年10ヵ月経過し再発・転移はない。無色素性爪下黒色腫は，臨床・画像診断が困難であり，診断の遅れに注意が必要である。また，病変の範囲の把握が困難であり，爪全体に病変が広がっている可能性を念頭において慎重な切除範囲の設定と経過観察をしていくことが大切と考え報告した。