soichiro watanabe

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.

<p>A 61-year-old man noticed an erythematous tumor with blood crusting on his genital area in January 2014. However, he had not undergone any medical care for the lesion for 8 months after noticing it. On the first examination, the tumor measured 25×20 mm and he had several swollen lymph nodes on both sides of the groin areas. A histological examination revealed adenocarcinoma composed of atypical cells with abundant eosinophilic cytoplasm and a decapitation in some parts of the tumor. The tumor cells were positive for GCDFP-15, CEA, ER, and PgR. No evidence of metastasis to other organs was observed. Based on these findings, the tumor was diagnosed as an apocrine carcinoma. It was broadly excised, and total superficial inguinal lymphadenectomy was performed. At present, 17 months after surgery, no recurrence has been noted.[Skin Cancer (Japan) 2016 ; 31 : 144-149]</p>

<p>A 62-year-old man presented with an 18 × 11-cm tumor in the posterior femoral region. A diagnosis of squamous cell carcinoma was made on the basis of the pathological findings. Whole body examination, including computed tomography (CT), demonstrated swollen inguinal lymph nodes, although no evidence of distant metastasis was found. We administered a chemotherapy regimen with 5-fluorouracil and cisplatin as neoadjuvant agents. After chemotherapy, a decrease in the size of the tumor was observed and we performed resection of the primary lesion and the swollen inguinal lymph nodes. No tumor cells were observed in the surgical margins or the lymph nodes. Wound dehiscence occurred 7 days after surgery, which required prolonged bed rest. Twenty days after surgery, owing to elevated serum C-reactive protein and D-dimer levels, we performed contrast-enhanced CT, and a bilateral pulmonary embolism (PE) was diagnosed. Anticoagulant therapy was initiated promptly, and the patient was discharged from the hospital 41 days after the surgery. PE is an important and potentially fatal perioperative complication and should always be considered even after the dermatological surgery.[Skin Cancer (Japan) 2016 ; 31 : 133-138]</p>

A 49-year-old man noticed deformation of the nail of his right thumb 2 years ago, which was diagnosed as a glomus tumor in a local hospital. He presented to our hospital with a linear defect on the nail plate of the right thumb, with a red mass underneath. On ultrasonography, the mass was visualized as a homogeneous, hypoechoic, highly vascular lesion. Pathological examination showed dermal infiltration of atypical tumor cells. Immunostaining revealed tumor cells positive for S-100, HMB-45, and Melan-A. A diagnosis of subungual amelanotic melanoma was made, and resection and sentinel lymph node biopsy of the right axilla was performed. Four months after the operation, partial nail regeneration was observed, and a biopsy specimen showed tumor recurrence. After extensive resection, no recurrence or metastasis has been observed for 22 months after the initial visit. Dermatologists should diagnose subungual tumors carefully, because the diagnosis of amelanotic melanoma is difficult and misdiagnosis or delayed diagnosis often occurs.[Skin Cancer (Japan) 2015 ; 30 : 168-173]