Authorship：Corresponding author   International / domestic magazine：International journal  

DOI： 10.1111/ijd.16640

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. The pathology of neovascular age-related macular degeneration (nAMD), also known as wet AMD, is associated with an abnormal blood vessel growth in the eye and involves an imbalance of proangiogenic and antiangiogenic factors. Thrombospondin (TSP)-1 and TSP-2 are endogenous matricellular proteins that inhibit angiogenesis. TSP-1 is significantly diminished in eyes with AMD, although the mechanisms involved in its reduction are unknown. Granzyme B (GzmB) is a serine protease with an increased extracellular activity in the outer retina and choroid of the human eyes with nAMD-related choroidal neovascularization (CNV). This study investigated whether TSP-1 and TSP-2 are GzmB substrates using in silico and cell-free cleavage assays and explored the relationship between GzmB and TSP-1 in the human eyes with nAMD-related CNV and the effect of GzmB on TSP-1 in retinal pigment epithelial culture and an explant choroid sprouting assay (CSA). In this study, TSP-1 and TSP-2 were identified as GzmB substrates. Cell-free cleavage assays substantiated the GzmB proteolysis of TSP-1 and TSP-2 by showing dose-dependent and time-dependent cleavage products. The proteolysis of TSP-1 and TSP-2 were hindered by the inhibition of GzmB. In the retinal pigment epithelium and choroid of human eyes with CNV, we observed a significant inverse correlation between TSP-1 and GzmB, as indicated by lower TSP-1 levels and higher GzmB immunoreactivity. In the CSA, the vascular sprouting area increased significantly with GzmB treatment and reduced significantly with TSP-1 treatment. Western blot showed significantly reduced expression of TSP-1 in GzmB-treated retinal pigment epithelial cell culture and CSA supernatant compared with that in controls. Together, our findings suggest that the proteolysis of antiangiogenic factors such as TSP-1 by extracellular GzmB might represent a mechanism through which GzmB may contribute to nAMD-related CNV. Future studies are needed to investigate whether pharmacologic inhibition of extracellular GzmB can mitigate nAMD-related CNV by preserving intact TSP-1.

DOI： 10.1016/j.labinv.2023.100123

PubMed

Authorship：Last author,　Corresponding author   International / domestic magazine：International journal  

DOI： 10.1111/1346-8138.16713

PubMed

Authorship：Lead author,　Corresponding author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Pruritus is a hallmark feature in pemphigoid diseases, where it can be severe and greatly impact the quality of life of affected patients. Despite being a key symptom, the exact pathophysiological mechanisms involved in pruritus in pemphigoid are yet to be fully elucidated and effective therapies addressing them are limited. This review summarizes the present understanding of pruritus specific to pemphigoid diseases, especially the pruritogens that induce it, and the therapeutic options that have been explored so far. The majority of the available evidence is on bullous pemphigoid and epidermolysis bullosa acquisita. Histamine derived from basophils correlates with pruritus severity, with omalizumab demonstrating promising efficacy in pruritus for bullous pemphigoid. IL-4/-13 contribute to itch in bullous pemphigoid with dupilumab being evaluated in clinical trials. Other pruritogens of interest include substance P, tryptase, and thymic stromal lymphopoetin, with therapies targeting them requiring further investigation. Scratching behaviors contribute directly to blister formation through various mechanisms, such as pathological autoantibody recruitment, T helper cell type 1 polarization, and exposure of intracellular autoantigens. Treatments addressing these pathways may contribute to decreasing disease severity. Additional studies are needed to fully characterize how pruritus is regulated in pemphigoid diseases, to help pave the way to develop novel and effective therapeutics that will not only address pruritic symptoms but also decrease disease severity.

DOI： 10.1111/1346-8138.16652

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Genetically modified mice are widely used to recapitulate human diseases. Atherosclerosis can be induced in mice with low-density lipoprotein receptor (Ldlr)-deficiency and a high-fat diet (HFD). Disintegrin and metalloproteinase-17 (ADAM17) in the smooth muscle cell (SMC) contribute to vascular pathologies, and hence its role in atherosclerosis was investigated. Adam17 deletion in SMCs by Sm22α-Cre driver (Ldlr-/-/Adam17Sm22Cre) and HFD resulted in severe skin lesions in >70% of mice, associated with skin inflammation, which was not observed in Ldlr-/--HFD, nor in mice with SMC deficiency of Adam17 by a different Cre driver (Ldlr-/-/Adam17Myh11Cre). We found that Sm22α is highly expressed in keratinocytes (compared with SMCs), which could underlie the observed skin lesion in Ldlr-/-/Adam17Sm22Cre-HFD. Although expression of Sm22α in non-SMCs has been reported, this is the first study demonstrating a severe side effect resulting from the off-target expression of Sm22α-Cre, resulting in ADAM17 loss in keratinocytes that led to a moribund state.NEW & NOTEWORTHY Although Sm22α-Cre is commonly used to target gene deletion in smooth muscle cells, Sm22α-derived Adam17 deletion resulted in unexpected severe skin lesions following high-fat diet feeding in a model of atherosclerosis. Adam17 deletion by a different SMC driver, Myh11-Cre, did not result in skin lesions in the same atherosclerosis model. Sm22α is highly expressed in keratinocytes, causing ectopic loss of ADAM17 in keratinocytes that caused significant epidermal lesions when combined with a high-fat diet.

DOI： 10.1152/ajpheart.00325.2022

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

DOI： 10.1016/j.jid.2021.11.016

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

DOI： 10.1111/1346-8138.16244

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Dysregulated skin immunity is a hallmark of many skin diseases such as atopic dermatitis, autoimmune blistering diseases, and interface dermatitis. Current treatment options for the inflammatory skin diseases are limited and sometimes ineffective, therefore further understanding of pathomechanisms in the inflammatory skin conditions is necessary to develop new therapeutic alternatives. Recent studies suggest that the serine protease, granzyme B, is a key mediator in multiple inflammatory skin diseases, implying that strategies targeting granzyme B may be an attractive treatment option for such diseases. Specifically, granzyme B exhibits not only an intracellular apoptotic function but also extracellular proteolytic roles in inflammatory skin diseases including infectious diseases, pemphigoid diseases, atopic dermatitis, alopecia areata, and interface dermatitis. In this review, we summarize the current understanding with respect to the functions of granzyme B in the pathomechanism of various inflammatory skin diseases and evaluate the possibility of therapeutics targeting granzyme B.

DOI： 10.1016/j.jdermsci.2021.10.006

PubMed

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Pressure injuries (PIs), also known as bedsores or pressure ulcers, are a major cause of death and morbidity in the elderly. The serine protease, Granzyme B (GzmB), contributes to skin aging and impaired wound healing. Aging is a major risk factor for PIs; thus, the role of GzmB in PI pathogenesis was investigated. GzmB levels in human PI tissue and wound fluids were markedly elevated. A causative role for GzmB was assessed in GzmB knockout (GzmB-/-) and wild-type (WT) mice using a murine model of PI. An apolipoprotein E knockout (ApoE-/-) model of aging and vascular dysfunction was also utilized to assess GzmB in a relevant age-related model better resembling tissue perfusion in the elderly. PI severity displayed no difference between young GzmB-/- and WT mice. However, in aged mice, PI severity was reduced in mice lacking GzmB. Mechanistically, GzmB increased vascular wall inflammation and impaired extracellular matrix remodeling. Together, GzmB is an important contributor to age-dependent impaired PI healing.

DOI： 10.1038/s41514-021-00059-6

PubMed

Authorship：Lead author   Publishing type：Research paper (scientific journal)  

<title>Abstract</title>Pemphigoid diseases refer to a group of severe autoimmune skin blistering diseases characterized by subepidermal blistering and loss of dermal-epidermal adhesion induced by autoantibody and immune cell infiltrate at the dermal-epidermal junction and upper dermis. Here, we explore the role of the immune cell-secreted serine protease, granzyme B, in pemphigoid disease pathogenesis using three independent murine models. In all models, granzyme B knockout or topical pharmacological inhibition significantly reduces total blistering area compared to controls. In vivo and in vitro studies show that granzyme B contributes to blistering by degrading key anchoring proteins in the dermal-epidermal junction that are necessary for dermal-epidermal adhesion. Further, granzyme B mediates IL-8/macrophage inflammatory protein-2 secretion, lesional neutrophil infiltration, and lesional neutrophil elastase activity. Clinically, granzyme B is elevated and abundant in human pemphigoid disease blister fluids and lesional skin. Collectively, granzyme B is a potential therapeutic target in pemphigoid diseases.

DOI： 10.1038/s41467-020-20604-3

Other URL： http://www.nature.com/articles/s41467-020-20604-3

Publishing type：Research paper (scientific journal)   International / domestic magazine：International journal  

Atopic dermatitis (AD) is the most common inflammatory skin condition. Skin barrier dysfunction is of major importance in AD because it facilitates allergen sensitization and systemic allergic responses. Long regarded as a pro-apoptotic protease, emerging studies indicate granzyme B (GzmB) to have extracellular roles involving the proteolytic cleavage of extracellular matrix, cell adhesion proteins, and basement membrane proteins. Minimally expressed in normal skin, GzmB is elevated in AD and is positively correlated with disease severity and pruritus. We hypothesized that GzmB contributes to AD through extracellular protein cleavage. A causative role for GzmB was assessed in an oxazolone-induced murine model of dermatitis, comparing GzmB-/- mice with wild-type mice, showing significant reductions in inflammation, epidermal thickness, and lesion formation in GzmB-/- mice. Topical administration of a small-molecule GzmB inhibitor reduced disease severity compared with vehicle-treated controls. Mechanistically, GzmB impaired epithelial barrier function through E-cadherin and FLG cleavage. GzmB proteolytic activity contributes to impaired epidermal barrier function and represents a valid therapeutic target for AD.

DOI： 10.1016/j.jid.2020.05.095

PubMed

Publishing type：Research paper (scientific journal)  

Granzymes are a family of serine proteases first shown to be intracellular initiators of immune-mediated cell death in target pathogenic cells. In addition to its intracellular role, Granzyme B (GzmB) has important extracellular functions in immune regulation and extracellular matrix (ECM) degradation. Verified substrates of extracellular GzmB activity include tight junctional and ECM proteins. Interestingly, little is known about the activity of GzmB in the outer human retina, a tissue in which the degradation of the tight junctional contacts of retinal pigment epithelial (RPE) cells and within the external limiting membrane, as well as remodeling of the ECM in Bruch's membrane, cause the breakdown of the blood-retinal barrier and slowing of metabolite transport between neuroretina and choroidal blood supply. Such pathological changes in outer retina signal early events in the development of age-related macular degeneration (AMD), a multifactorial, chronic inflammatory eye disease. This study is the first to focus on the distribution of GzmB in the outer retina of the healthy and diseased post-mortem human eye. Our results revealed that GzmB is present in RPE and choroidal mast cells. More immunoreactive cells are present in older (>65 years) compared to younger (<55 years) donor eyes, and choroidal immunoreactive cells are more numerous in eyes with choroidal neovascularization (CNV), while RPE immunoreactive cells are more numerous in eyes with soft drusen, an early AMD event. In vitro studies demonstrated that RPE-derived tight junctional and ECM proteins are cleaved by exogenous GzmB stimulation. These results suggest that the increased presence of GzmB immunoreactive cells in outer retina of older (healthy) eyes as well as in diseased eyes with CNV (from AMD) and eyes with soft drusen exacerbate ECM remodeling in the Bruch's membrane and degradation of the blood-retinal barrier. Currently there are no treatments that prevent remodeling of the Bruch's membrane and/or the loss of function of the outer blood-retinal barrier, known to promote early AMD changes, such as drusen deposition, RPE dysfunction and pro-inflammation. Specific inhibitors of GzmB, already in preclinical studies for non-ocular diseases, may provide new strategies to stop these early events associated with the development of AMD.

DOI： 10.3389/fimmu.2020.00574

PubMed

Publishing type：Research paper (scientific journal)  

DOI： 10.3389/fimmu.2019.01454

Publishing type：Research paper (scientific journal)  

DOI： 10.1080/14728222.2019.1661380

自己免疫性水疱症の一つである水疱性類天疱瘡(BP)の病変部には好酸球が浸潤しているが、その病理学的意義は不明な点が多い。好酸球リクルートメントにはIgE型自己抗体が関与している可能性が示唆されているが、IgE以外の因子も関与している可能性がある。BPの紅斑・水疱とmatrix metalloprotease-9との関係、痒みと好酸球との関係について概説した。現在、bertilimumabなどの好酸球を標的とした抗体製剤の臨床試験が進行中である。

＜文献概要＞類天疱瘡疾患は,真皮表皮接合部の蛋白に対する病原性自己抗体を持つ,自己免疫性表皮下水疱症である.コルチコステロイドの内服がその標準治療であるが,高い再発率や死亡率が臨床上の問題であり,より効果的で安全な治療が臨床現場において求められている.われわれは,類天疱瘡疾患において増加していることが知られていたがその病理学的意義が不明であった,セリンプロテアーゼの一種であるグランザイムBの病理学的役割を,モデルマウスを用いて検討した.その結果,グランザイムBは類天疱瘡疾患において,真皮表皮背接合部接着因子の切断と好中球リクルートメントに関与していることを明らかにし,さらにグランザイムBの阻害薬外用が類天疱瘡疾患の治療になりうることを示した.

J-GLOBAL

水疱性類天疱瘡を始めとする類天疱瘡疾患は、そう痒を伴う全身性の緊満性水疱を特徴とし、真皮表皮接合部に局在する蛋白に対する病原性自己抗体を持つ自己免疫性表皮下水疱症である。これらの疾患で認められる病原性自己抗体は、HLA型や制御性T細胞の機能不全などを引き起こす遺伝的背景に、外傷や薬剤摂取などの後天的なイベントが加わって獲得されているようであるが、その機序はまだよくわかっていない。獲得された病原性自己抗体は自己抗原に結合し、補体を活性化し、真皮表皮接合部へ炎症細胞を浸潤させ、様々な活性化プロテアーゼを集積させる。それらプロテアーゼが真皮表皮間接着分子を分解し、真皮表皮間接着を脆弱にさせ、表皮下水疱を引き起こすことが、本疾患の主な発症機序のひとつであると考えられている。また、いくつかの類天疱瘡疾患では、こうした補体活性化経路を経ない非炎症性の水疱形成機序が確認されている。類天疱瘡疾患の罹患患者の大半を占める高齢者において、標準治療である内服または外用ステロイドは免疫抑制等の副作用がしばしば問題となる。より安全で有効な新規治療法を開発するために、類天疱瘡疾患の発症機序の詳細な理解が求められている。(著者抄録)

水疱性類天疱瘡は、そう痒を伴う全身の緊満性水疱を特徴とする自己免疫性水疱症である。標準治療は内服または外用ステロイドであるが、罹患患者の大半を占める高齢者における免疫抑制等の副作用や、標準治療に反応しづらい難治例が臨床上しばしば問題となる。このために、水疱性類天疱瘡の詳細な発症機序の理解と、それに基づいた新規治療法が求められている。これまでに水疱性類天疱瘡の発症には、表皮真皮間接着因子である17型コラーゲンに対する自己抗体が、補体やプロテアーゼを活性化することが重要であると考えられてきた。しかし近年、われわれを含む複数のグループが、自己抗体は17型コラーゲンを内在化させ表皮細胞の接着力を減弱させていることを明らかにした。本稿では、近年明らかになった水疱性類天疱瘡における自己抗原内在化の機序をまとめ、その疾患における意義を考察する。(著者抄録)

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Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Presentation type：Poster presentation  

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Presentation type：Oral presentation (general)