Neutrophils initiate and exacerbate Stevens–Johnson syndrome and toxic epidermal necrolysis

Manao Kinoshita; Youichi Ogawa; Natsumi Hama; Inkin Ujiie; Akito Hasegawa; Saeko Nakajima; Takashi Nomura; Jun Adachi; Takuya Sato; Schuichi Koizumi; Shinji Shimada; Yasuyuki Fujita; Hayato Takahashi; Yoshiko Mizukawa; Takeshi Tomonaga; Keisuke Nagao; Riichiro Abe; Tatsuyoshi Kawamura

doi:10.1126/scitranslmed.aax2398

. Author manuscript; available in PMC: 2022 Aug 20.

Published in final edited form as: Sci Transl Med. 2021 Jun 30;13(600):eaax2398. doi: 10.1126/scitranslmed.aax2398

Neutrophils initiate and exacerbate Stevens–Johnson syndrome and toxic epidermal necrolysis

Manao Kinoshita ¹, Youichi Ogawa ¹, Natsumi Hama ², Inkin Ujiie ³, Akito Hasegawa ², Saeko Nakajima ⁴, Takashi Nomura ⁴, Jun Adachi ^5,⁶, Takuya Sato ¹, Schuichi Koizumi ⁷, Shinji Shimada ¹, Yasuyuki Fujita ³, Hayato Takahashi ⁸, Yoshiko Mizukawa ⁹, Takeshi Tomonaga ^5,⁶, Keisuke Nagao ¹⁰, Riichiro Abe ², Tatsuyoshi Kawamura ¹

PMCID: PMC9392155 NIHMSID: NIHMS1828912 PMID: 34193610

Abstract

Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN) are life-threatening mucocutaneous adverse drug reactions characterized by massive epidermal detachment. Cytotoxic T cells and associated effector molecules are known to drive SJS/TEN pathophysiology, but the contribution of innate immune responses is not well understood. We describe a mechanism by which neutrophils triggered inflammation during early phases of SJS/TEN. Skin-infiltrating CD8⁺ T cells produced lipocalin-2 in a drug-specific manner, which triggered the formation of neutrophil extracellular traps (NETs) in early lesional skin. Neutrophils undergoing NETosis released LL-37, an antimicrobial peptide, which induced formyl peptide receptor 1 (FPR1) expression by keratinocytes. FPR1 expression caused keratinocytes to be vulnerable to necroptosis that caused further release of LL-37 by necroptotic keratinocytes and induced FPR1 expression on surrounding keratinocytes, likely amplified the necroptotic response. The NETs-necroptosis axis was not observed in less severe cutaneous adverse drug reactions, autoimmune diseases, or neutrophil-associated disorders, suggesting that this was a process specific to SJS/TEN. Initiation and progression of SJS/TEN keratinocyte necroptosis appear to involve a cascade of events mediated by innate and adaptive immune responses, and understanding these responses may contribute to the identification of diagnostic markers or therapeutic targets for these adverse drug reactions.

One Sentence Summary:

Neutrophil extracellular traps induced by drug-specific CD8⁺ T cells initiate SJS/TEN.

Introduction

Severe cutaneous adverse drug reactions (cADRs), including Stevens–Johnson syndrome/toxic epidermal necrolysis (SJS/TEN), drug reactions with eosinophilia and systemic symptoms (DRESS)/drug-induced hypersensitivity syndrome (DIHS), and acute generalized exanthematous pustulosis (AGEP) pose significant challenges in clinical management, in part due to our insufficient understanding of disease pathophysiology and limited treatment modalities (1–3). In particular, SJS/TEN are life-threatening conditions due to the extensive epidermal detachment that occurs during disease progression. SJS and TEN are considered to be a disease spectrum and are distinguished by the body surface area of epidermal detachment as follows: SJS < 10%, SJS/TEN overlap 10%–30%, and TEN > 30% (4). Thereafter, we use ‘SJS/TEN’ as the term including all these three conditions or each specific term to indicate the specific condition. The overall mortality rate in SJS/TEN remains unacceptably high, at approximately 25% (1, 3, 5). Involvement of CD8⁺ T cells in SJS/TEN is well-established (6, 7), for which several pathways of human leukocyte antigen/peptide/T cell receptor interaction have been proposed (2). Several effector molecules have been reported, such as Fas/Fas ligand (FasL) (8), soluble FasL (8, 9), perforin/granzyme B (10), granulysin (7), and interleukin (IL)-15 (11). Given that these molecules relate to cytotoxic CD8⁺ T cells as well as natural killer cells and natural killer T cells, it is generally accepted that SJS/TEN pathogenesis is primarily driven by these cytotoxic cells.

Contribution of innate immunity in SJS/TEN disease pathophysiology remains largely unexplored. Drug-specific T cells are capable of releasing cytokines and chemokines that recruit and activate monocytes, eosinophils, and neutrophils (12). Keratinocyte death in SJS/TEN involves necroptosis, a form of programmed cell death. Necroptosis is mediated by the binding of monocyte-derived annexin A1 to formyl peptide receptor 1 (FPR1), a family of G protein-coupled receptors, expressed on SJS/TEN keratinocyte surfaces (13). Skin-infiltrating CD14⁺CD16⁺ inflammatory monocytes may boost the proliferation and cytotoxicity of CD8⁺ T cells via co-stimulatory molecule interaction (14). Eosinophils can be observed in SJS/TEN skin, but their numbers correlate inversely with SJS/TEN disease severity (15).

Neutrophils are among the first responders during innate immune responses and augment host immunity, including pathological inflammation mediated by T cells in allergic skin reactions such as contact hypersensitivity (16, 17). Skin-infiltrating neutrophils form neutrophil extracellular traps (NETs), a network of extracellular nucleic acids containing surface histones and granular proteins (18, 19) that drive autoimmunity in small vessel vasculitis (20), lupus erythematosus (21–24), and psoriasis (25, 26). Neutrophil contribution to SJS/TEN disease pathophysiology has not been considered to date. Interestingly, the presence of neutrophils has been documented in SJS/TEN skin (27–29). In addition, SJS/TEN patients may exhibit neutropenia (30–32), which increases the risk for life-threatening infections. We hypothesized that neutrophils are aberrantly activated and then displaced from the circulation to enhance subsequent skin inflammation. Herein, we report that circulating SJS/TEN neutrophils were prone to NETosis and subsequently formed NETs in the skin, which triggered a cascade of responses that led to keratinocyte necroptosis. These findings shed light on the immunological events that take place in the early phases of SJS/TEN, highlighting neutrophils as an innate component that contributes to disease pathophysiology.

Results

Neutrophil extracellular trap formation in circulating neutrophils from patients with SJS/TEN

Neutropenia often occurs in patients with SJS/TEN (30–32). Given this observation we examined circulating neutrophils from healthy volunteer (HV) and patients with maculopapular exanthema (MPE; a type of non-severe cADRs), SJS, TEN, and sepsis. Approximately 30% of patients with SJS/TEN also develops sepsis (33), so we focused on non-septic SJS/TEN patients with undetectable levels of serum procalcitonin, a marker for bacterial infections (fig. S1).

Neutrophil preparations isolated from peripheral blood prior to corticosteroid therapy were stained with Giemsa. Compared with normal morphology of neutrophils from HV and MPE patients, those from patients with SJS and sepsis displayed irregular cell borders, stronger cytoplasmic Giemsa staining, and enlarged nuclei, suggestive of altered structural integrity. Neutrophils from TEN patients displayed marked loss of characteristic neutrophil morphology (Fig. 1A).

Fig. 1 — **(A)** Giemsa stain of neutrophils isolated from the peripheral blood of healthy volunteers (HV) and patients with MPE, SJS, TEN, and sepsis. Scale bar = 50 μm. **(B)** Immunofluorescence images of neutrophils from HV and patients stained with LL-37 (green), neutrophil elastase (NE; red), and DAPI (blue). Scale bar = 100 μm. (A) and (B) represent data from individual experiments repeated five times. Representative expression **(C)** and mean fluorescence intensity (MFI) **(D)** of CD11b and CD62L in circulating neutrophils (live CD66b⁺ cells) from HV and SJS/TEN patients using flow cytometry. MFI ratios between HV (n = 6) and SJS/TEN patients (n = 6) were shown in **(D)**. *p < 0.05 compared to HV (Mann–Whitney U test). Patient numbers included in the study are below and their information is described in Table S1: **(A) and (B)** HV #1, MPE #18, SJS #32, TEN #37, Sepsis #2; **(C)** HV #1, SJS #36; **(D)** HV #1, 2, 5, 7, 15, 16, SJS/TEN #35, 36, 37, 38, 40, 41.

NETosis is a process during which neutrophils actively release NETs through cell death or non-cell death mechanisms (18, 34). Given that neutrophils actively form NETs during sepsis, we hypothesized that circulating SJS/TEN neutrophils might also undergo NETosis. NET formation occurred in neutrophils from patients with SJS/TEN and sepsis; however, not in those from HV or MPE patients as evidenced by the release of neutrophil elastase (NE) and LL-37, an antimicrobial peptide enriched in NETs (35). In particular, TEN neutrophils exhibited prominent NET formation, consistent with their morphological changes (Fig. 1, A and B). SJS/TEN neutrophils upregulated CD11b and downregulated CD62L compared with HV neutrophils (Fig. 1, C and D). These data suggest that circulating SJS/TEN neutrophils exhibited an activated phenotype and were prone to undergoing NETosis.

Neutrophilic infiltration in SJS/TEN skin

Enhanced NET formation by circulating SJS/TEN neutrophils prompted us to assess neutrophilia and NET formation in SJS/TEN skin. Neutrophilic skin infiltration is not known to be a hallmark of SJS/TEN at the time of initial diagnosis. Indeed, polymorphonuclear (PMN) cells in hematoxylin-eosin (H&E)-stained SJS/TEN skin sections taken at day 1 after onset were rarely observed, as is the case in H&E-stained HV skin. However, evaluation of those taken at day 3 after onset demonstrated transient increases in PMN numbers, both in the epidermis and dermis (Fig. 2, A and B; fig. S2, A and B). Consistently, in contrast to HV and MPE skin (fig. S2, C and D), a substantial number of myeloperoxidase (MPO)-positive cells were detected in the epidermis and dermis of SJS/TEN skin (Fig. 2A; fig. S2, A and B). Because MPO can also stain positive in monocytes in SJS/TEN skin (14), we further stained skin sections for CD66b, a marker for activated neutrophils (36, 37). Fewer numbers of CD66b-positive cells than MPO-positive cells were observed, suggesting that both neutrophils (MPO⁺CD66b⁺) and monocytes (MPO⁺CD66b⁻) infiltrated SJS/TEN skin lesions (Fig. 2, A and C; fig. S2, A and B). In line with a previous study (14), CD14⁺CD16⁺ inflammatory monocytes also accumulated in the dermo–epidermal junction together with CD66b⁺CD16⁺ neutrophils (fig. S2E).

Fig. 2 — **(A)** H&E stain (first and second lines), MPO stain (third line), and CD66b stain (fourth line) of skin specimens from HV and SJS/TEN patients (on days 1, 3, and 5 after onset). Patient number correspond to cases is listed in Table S1 and is noted to the right of the days after onset. Panels on the first and second lines are identical, but the panels on the second line have had their color lightened and PMN cells are indicated by black triangles. Scale bar = 100 μm. **(B)** Number of epidermal and dermal PMN cells identified in H&E-stained skin specimens taken from HV (n = 9) and SJS/TEN patients 1 (n = 6), 3 (n = 26), and 5 (n = 9) days following onset. *p < 0.05 and **p < 0.01 (Kruskal-Wallis test). **(C)** Number of neutrophils (CD66b⁺MPO⁺) and monocytes (CD66b⁻MPO⁺) that infiltrated epidermis and dermis was determined using HV (n = 9) and SJS/TEN skin specimens taken from 1 (n = 6), 3 (n = 26), and 5 (n = 9) days following onset. *p < 0.05 and **p < 0.01 compared to each CD66b⁺MPO⁺ column. ^##p < 0.01 compared to each CD66b⁻MPO⁺ column (Kruskal-Wallis test). Patient information is described in Table S1.

In contrast to the morphological findings in H&E-stained specimens, immunohistochemistry (IHC) revealed increased numbers of epidermis-infiltrating MPO⁺CD66b⁺ neutrophils throughout the course of disease (Fig. 2, A to C; fig. S2, A and B), suggesting that IHC detected neutrophils that could not be readily distinguished by morphology, possibly due to their alteration of cellular morphology in the epidermis.

In line with the above observations, CD11b⁺ myeloid cells with a robust CD66b⁺ neutrophil fraction were identified in the blister fluids from a patient with TEN, but not in those from suction blisters generated in a HV (fig. S2F). Thus, a systematic time-point morphological analysis, together with IHC for neutrophil markers and flow cytometry, demonstrated that neutrophils infiltrated the skin of non-septic SJS/TEN patients. Neutrophilic infiltration in very early SJS/TEN skin prior to the occurrence of epidermal damage suggested that neutrophil recruitment was unlikely to be secondary to skin-associated microbes.

NETosis in SJS/TEN skin

We next examined whether skin-infiltrating SJS/TEN neutrophils formed NETs. Strikingly, staining for NE and LL-37 in SJS/TEN skin was detected as early as day 1 after onset, which intensified over time. The morphological changes of NE-positive neutrophils, together with LL-37 staining that extended beyond neutrophil cell bodies into the stroma, suggested that neutrophils had undergone NETosis (Fig. 3A and fig. S3A; isotype-matched control IgG staining). Furthermore, NETotic neutrophils expressing LL-37 were already present in the unaffected peri-lesional epidermis (fig. S3B).

Staining for citrullination of histone H3, which occurs as a consequence of NET formation (38), demonstrated positivity in SJS/TEN skin (Fig. 3B). Furthermore, electron microscopy analysis revealed nuclear chromatin decondensation with disintegration of the nuclear membrane into numerous small vesicles, vacuolization, and disruption of the plasma membrane, which are characteristics of NETosis, in both epidermal and dermal neutrophils of patients with SJS (Fig. 3C) (39). Moreover, double-stranded (ds) DNA and LL-37 levels in the blister fluids from patients with SJS/TEN were higher than those from patients with traumatic blisters, insect bites, bullous pemphigoid (BP), pemphigus vulgaris (PV), burns, or herpes zoster (HZ) (Fig. 3, D and E). MPO-DNA complex levels in the blister fluids from patients with SJS/TEN also trended to be higher than those of patients with other blistering disorders (fig. S3C). Collectively, these data demonstrated that skin-infiltrating neutrophils undergo NETosis in early phases of SJS/TEN.

Aberrant NETosis in SJS/TEN

Given then occurrence of NETosis in circulation and skin of SJS/TEN, we hypothesized that sera and blister fluids contain soluble factors that trigger NETosis. HV neutrophils were exposed to sera from other HV and TEN patients, or phorbol 12-myristate 13-acetate (PMA), a known NET inducer (38, 39). TEN sera, but not HV sera, induced morphological changes, LL-37 and NE release, and histone H3 citrullination in HV neutrophils (Fig. 4, A and B). dsDNA (Fig. 4C) and LL-37 (Fig. 4D) levels were higher in the culture supernatants of SJS/TEN sera-exposed HV neutrophils compared with those exposed to sera from HV and non-SJS/TEN cADR patients such as MPE, erythema multiforme major (EMM), fixed drug eruption (FDE), AGEP, and DRESS/DIHS. Similarly, blister fluid from SJS/TEN patients, but not those from patients with traumatic blisters, insect bites, BP, PV, burns, and HZ, triggered the release of dsDNA from HV neutrophils into culture supernatants (Fig. 4E). These data indicated that soluble factors that trigger NETosis were enriched in the sera and blister fluids of SJS/TEN patients.

Moreover, higher concentrations of dsDNA (Fig. 4F), LL-37 (Fig. 4G), and MPO-DNA complex (Fig. 4H) were detected in the sera from SJS/TEN patients as compared to those from a variety of autoimmune or inflammatory skin diseases, highlighting NETosis as a feature most prominently observed in SJS/TEN.

Induction of neutrophil extracellular traps by lipocalin-2 and LL-37

We next sought to understand the mechanisms by which NETosis was triggered in SJS/TEN. To define the cellular source of NET inducers, peripheral blood mononuclear cells (PBMCs) and granulocytes were isolated from recovered SJS/TEN patients and then cultured with or without causative drugs. Culture supernatants were collected, to which HV neutrophils were exposed. Culture supernatants from drug-exposed PBMCs, but not those from granulocytes, triggered dsDNA release from HV neutrophils (Fig. 5A), demonstrating that NET inducers were produced by cells in the PBMC fraction in a manner that was dependent on causative drugs.

Fig. 5 — **(A)** Supernatants were collected from drug-exposed or unexposed PBMCs or granulocytes isolated from recovered SJS/TEN patients. HV neutrophils were incubated with these supernatants for 3 h. Then, the released NET-related dsDNA in the culture supernatant was measured by PicoGreen^®. The mean value is indicated by a black line. *p < 0.05 compared to drug-unexposed PBMCs (Wilcoxon matched-pairs signed rank test). **(B)** HV neutrophils were incubated with recombinant LCN-2 (30 μg/mL), protein S100-A7 (S100-A7; 5 μg/mL), IL-36γ (50 nM), calmodulin-like protein 5 (CALML5; 1 μg/mL), annexin A3 (1 μg/mL), galectin-7 (1 μg/mL), LL-37 (25 μg/mL) and a combination of LCN-2 (30 μg/mL) and LL-37 (25 μg/mL) for 3 h, fixed, and then immunostained with antibodies against LL-37 (green) and NE (red). DNA (blue) was stained with DAPI. Results are representative of experiments repeated three times. Scale bar = 50 μm. **(C)** HV neutrophils (n = 4) were incubated with the noted reagents at the indicated concentrations for 3 h. dsDNA levels in the culture supernatant were quantified by PicoGreen^®. **p < 0.01 compared to untreated (Kruskal-Wallis test). **(D)** HV neutrophils were incubated with the noted concentration of LCN-2 and/or LL-37 for 1 h, and ROS production was measured. The dashed line is data from HV neutrophils pretreated for 1 h with an ROS production inhibitor (Diphenyleneiodonium chloride; DPI 0.1 μM) and then stimulated with both LCN-2 (30 μg/mL) and/or LL-37 (25 μg/mL). Results are representative of experiments repeated four times. Scale bar = 100 μm. **(E)** HV neutrophils were preincubated with anti-LCN-2 and/or anti-LL-37 antibody (10 μg/mL) or control mouse IgG (n = 7) for 30 min, and then exposed to 10% sera from SJS/TEN patients (n = 7). dsDNA levels in the culture supernatant were measured by PicoGreen^®. Sera were obtained from SJS/TEN patients prior to systemic treatment. **p < 0.01, *p < 0.05 compared to control IgG (Kruskal-Wallis test). Results are representative of three replications of the procedure (A, C, and E). Patient numbers included in the study is below and their information is described in Table S1: **(A)** SJS/TEN #1, 14, 17, 32, 34, 35, 36; **(E)** SJS/TEN #1, 2, 4, 8, 25, 27, 30.

We recently performed mass spectrometric analysis for molecules that were triggered by causative drug-exposure in SJS/TEN PBMCs, identifying lipocalin-2 (LCN-2), LL-37, S100-A7, IL-36γ, calmodulin-like protein 5 (CALML5), annexin A3, and galectin-7 (40). To determine if these molecules were capable of inducing NETosis, HV neutrophils were exposed to these recombinant proteins. Interestingly, only LCN-2 and to a lesser extent, LL-37, induced NETosis. Additionally, the combination of LCN-2 and LL-37 augmented NETosis (Fig. 5B). NET induction was confirmed by dsDNA release. Only LCN-2, but not the other five recombinant proteins triggered dsDNA release from HV neutrophils in a dose-dependent manner. This analysis did not include data of LL-37, because LL-37 is a cationic protein that binds to dsDNA (41). Thus, a LL-37/dsDNA complex was formed in the culture supernatants, leading to the difficultly of correct dsDNA detection (Fig. 5C).

NET formation depends on the generation of reactive oxygen species (ROS) by NADPH oxidase (38, 39, 42). Both LCN-2 and LL-37 induced ROS generation in HV neutrophils in a dose-dependent manner (fig. S3D: left and middle panels). Furthermore, the combination of LCN-2 and LL-37 increased ROS production, which was restored by a ROS scavenger (fig. S3D: right panel) as well as a ROS production inhibitor (Fig. 5D). SJS/TEN sera-induced NETs levels in HV neutrophils were lowered by 46.7%, 43.8%, and 67.1% after preincubation of SJS/TEN sera with anti-LCN-2 antibody, anti-LL-37 antibody, or a combination of both antibodies, respectively (Fig. 5E). Taken together, these observations indicate that LCN-2 and LL-37 secreted by causative drug-primed PBMCs induced NETs via ROS generation and that the presence of both molecules augmented NET formation.

Lipocalin-2 production by drug-specific CD8⁺ T cells in early phases of SJS/TEN

We next sought to identify LCN-2-secreting cells in SJS/TEN PBMCs. CD4⁺ T cells, CD8⁺ T cells, and histocompatibility locus antigen (HLA)-DR⁺ antigen-presenting cells (APCs) were isolated from peripheral blood of recovered SJS/TEN patients and each leukocyte subset was cultured with or without causative drugs, or with phytohemagglutinin (PHA). Strikingly, upregulation of LCN-2 at both mRNA and protein levels were observed only in CD8⁺ T cells that were cultured in the presence of APCs and the causative drug (Fig. 6, A and B). In stark contrast, LCN-2 was undetected in the supernatants of MPE and DRESS/DIHS leukocyte subsets under any conditions (fig. S4A). Interestingly, PHA alone failed to induce LCN-2 mRNA and protein upregulation any of the leukocyte subsets from all diseases (Fig. 6, A and B; fig. S4A). However, PHA induced the production of the cytotoxic molecules perforin, granzyme B, and granulysin by SJS/TEN CD4⁺ and CD8⁺ T cells even in the absence of APCs and causative drugs, comparable to or exceeding that of CD8⁺ T cells stimulated with APCs and causative drugs (fig. S4B). These results demonstrated that the production of perforin, granzyme B, and granulysin by both CD4⁺ and CD8⁺ T cells could be solely induced by PHA, suggesting that the production of these cytotoxic molecules occurred as a result of T-cell activation, whereas LCN-2 production by CD8⁺ SJS/TEN T cells was dependent on causative drugs and antigen presentation.

Similar to serum dsDNA, LL-37, and MPO-DNA complex levels (Fig. 4, F to H), serum LCN-2 levels were increased in SJS/TEN sera, but not in those from patients with non-SJS/TEN cADRs and a variety of autoimmune or inflammatory skin diseases, except for generalized pustular psoriasis (GPP) patients (Fig. 6C), whose serum dsDNA and LL-37 levels were much lower than those of SJS/TEN patients (Fig. 4, F and G). The elevated serum LCN-2 levels in SJS/TEN patients prior to receiving systemic treatments were rapidly downregulated after initiating systemic treatments (Fig. 6C).

In the paraffin sections, LCN-2 (red staining)-producing CD8⁺ T cells (green staining) were absent from HV and MPE patient skin (Fig. 6D: left panels). In contrast, at day 1 after SJS/TEN onset, these cells had infiltrated the dermo–epidermal junction along with MPO⁺ neutrophils and monocytes (blue staining) and were particularly abundant in the perivascular dermis (Fig. 6D: day 1), further infiltrated the epidermis at later time points (Fig. 6D: days 3 and 5). In the frozen sections, LCN-2 (white staining) was detected not only in the CD8⁺ T cells (green staining) but also in the CD66b⁺ neutrophils (red staining) (fig. S5A). Interestingly, circulating neutrophils isolated from HV did not release LCN-2 in a steady state condition. In contrast, neutrophils released LCN-2 in response to PMA in a dose-dependent manner (fig. S5B).

Collectively, these data imply that CD8⁺ T cells produced LCN-2 upon stimulation with the causative drug and antigen presentation, a phenomenon that was specific to SJS/TEN among the studied diseases. Furthermore, LCN-2-producing CD8⁺ T cells infiltrated SJS/TEN skin in close proximity to neutrophils, suggesting a possible triggering of ROS production and subsequent NET formation in neutrophils by LCN-2. Finally, NETotic neutrophils in turn released LCN-2.

FPR1 induction in keratinocytes by LL-37 and subsequent necroptosis by annexin A1

Monocyte-derived annexin A1 induces necroptosis in SJS/TEN keratinocytes by binding to FPR1 expressed on the cell surface (13). Consistently, annexin A1 was detected in sera (fig. S6A), blister fluids (Fig. 7A), and in monocytes isolated from the blister fluids (fig. S6B), of SJS/TEN patients. FPR1 was readily detected on SJS/TEN keratinocytes but not on HV or MPE keratinocytes (Fig. S6, C and D). To examine if NETs were involved in inducing FPR1 expression in keratinocytes, we cultured normal human epidermal keratinocytes (NHEK) with known protein components of NETs (35). Strikingly, FPR1, but not FPR2, which is another annexin A1 receptor (43), was induced only by LL-37 in a dose-dependent manner (Fig. 7, B and C; fig. S6E). The annexin A1 peptide, Ac2–26, did not induce FPR1 expression (Fig. 7D first through fourth columns; fig. S6F). These data demonstrated that the NET component LL-37 induced FPR1 expression in keratinocytes that likely rendered them susceptible to annexin A1-mediated necroptosis.

**(A)** Blister fluid was collected from patients with traumatic blisters (n = 5), insect bites (n = 3), BP (n = 3), PV (n = 1), burns (n = 3), HZ (n = 1), and SJS/TEN (n = 9). SJS/TEN sera were obtained prior to receiving systemic treatment. Annexin A1 levels were determined by ELISA. **p < 0.01 compared to traumatic blisters (Kruskal-Wallis test). **(B)** NHEK (n = 3) were incubated with the noted reagents at the indicated concentrations (LL-37: 25 μg/mL) for 24 h, and the expression of *FPR1* mRNA was analyzed by quantitative RT-PCR. Values are normalized to the untreated control. **p < 0.01 compared to untreated control (Kruskal-Wallis test). **(C)** NHEK (n = 5) were incubated with LL-37 (25 μg/mL) for 24 h, and the expression of *FPR1* and *FPR2* was analyzed by quantitative RT-PCR. Values are normalized to the untreated control. *p < 0.05 compared to untreated control (Kruskal-Wallis test). **(D)** Western blot analysis of FPR1 protein in NHEK. NHEK were pretreated with WRW4 (FPR2 inhibitor; 10 μM), KN-62 (P2X₇-R inhibitor; 5 μM) or AG-1478 (EGFR inhibitor; 1 μM) for 30 min, and then incubated with LL-37 (25 μg/mL) and/or Ac2–26 (50 ng/mL) for 24 h. **(E)** Western blot analysis of RIP3 and phosphorylated RIP3 (pRIP3). NHEK were treated with LL-37 (25 μg/mL) and/or Ac2–26 (50 ng/mL) for 24 h. **(F) and (G)** NHEK (n = 5) were pretreated for 30 min with the indicated inhibitors (50 μM zVAD or 50 μM Nec-1) and/or antagonists (10 μM WRW4, 5 μM KN-62, or 1 μM AG-1478), and then exposed to a combination of LL-37 (25 μg/mL) and Ac2–26 (50 ng/mL) for 30 h. Cytotoxicity was determined with the trypan blue staining in **(F)** and LDH assay in **(G)**. **p < 0.01. ^#p < 0.05, ^##p < 0.01 compared to co-application of LL-37 and Ac2–26. N.S.; not significant (Kruskal-Wallis test). **(H)** Effects of antagonists to LL-37 receptors on LL-37 (25 μg/mL)-induced *FPR1* mRNA in NHEK. NHEK were pretreated with WRW4 (FPR2 inhibitor; 10 μM), KN-62 (P2X₇-R inhibitor; 5 μM) or AG-1478 (EGFR inhibitor; 1 μM) for 30 min, and then incubated with LL-37 (25 μg/mL) for 24 h. *p < 0.05 compared to LL-37 alone (Kruskal-Wallis test). Values are normalized to untreated controls. All results in Fig. 7 are representative of three replications of the procedure. Information of patients analyzed in **(A)** is described in Table S2.

Necroptosis is mediated by the phosphorylation of RIP3 (pRIP3) (44). To further solidify the roles of LL-37 and annexin A1 in inducing necroptosis, their effects on pRIP3 induction and subsequent cytotoxicity were examined in NHEK. LL-37 and Ac2–26 individually failed to induce pRIP3, but the combination of LL-37 and Ac2–26 induced pRIP3 (Fig. 7E; fig. S6, G and H). This resulted in enhanced cell death, as determined by trypan blue staining (Fig. 7F; first and second columns) and lactate dehydrogenase (LDH) release assay (Fig. 7G; first and second columns). To further examine the relative roles of apoptosis and necroptosis in mediating cell death, NHEK were pretreated with an apoptosis inhibitor (zVAD), a necroptosis inhibitor (Nec), or both inhibitors, followed by treatment with LL-37 and Ac2–26. Single treatment with Nec suppressed NHEK cell death, whereas zVAD treatment trended towards reduction. Pre-treatment with both zVAD and Nec reduced cell death most effectively (Fig. 7, F and G). Thus, keratinocyte cell death induced by the combination of LL-37 and annexin A1 is primarily mediated by necroptosis, with some contributions via the apoptotic pathway.

Three receptors for LL-37 have been identified to date, including FPR2 (45), P2X₇ receptor (P2X₇-R) (46), and epidermal growth factor receptor (EGFR) (47). To investigate which of these receptors were responsible for LL-37-mediated FPR1 upregulation and subsequent necroptosis, NHEK were pretreated with an inhibitor for each receptor. P2X₇-R inhibitor (KN-62), but not FPR2 and EGFR inhibitors (WRW4 and AG-1478, respectively), led to a reduction of FPR1 at the mRNA (Fig. 7H) and protein (Fig. 7D; fig. S6F) levels, and suppressed LL-37 and annexin A1-mediated cell death (Fig. 7G; gray columns) in NHEK.

Taken together, the NET component LL-37 induced FPR1 expression by keratinocytes through P2X₇-R signaling, followed by the triggering of necroptosis mediated by annexin A1 binding to FPR1.

LL-37 secretion by damaged SJS/TEN keratinocytes

LL-37 is a NET component that triggered necroptosis in early SJS/TEN phases by inducing FPR1 on keratinocytes, but LL-37 can also be released from injured keratinocytes (48). We hypothesized that initial epidermal damage may further induce LL-37 released by keratinocytes, which may amplify the necroptosis process. Serum levels of LCN-2 and dsDNA rapidly decreased upon therapeutic intervention (Fig. 6C; Fig. S7A), but decreases in LL-37 were delayed, with high serum levels that persisted until days 5–7 (Fig. 8A). To examine if keratinocytes could be sources of LL-37, we stained SJS/TEN skin sections. Indeed, LL-37 was detected in SJS/TEN keratinocytes and intensified throughout the course of the disease (Fig. 8B and fig. S7B; isotype-matched control IgG), suggesting that keratinocytes may represent LL-37 sources at later phases of SJS/TEN. To validate this observation in vitro, NHEK were exposed to LL-37 and Ac2–26, as this combination induced LL-37 secretion into the culture supernatants (Fig. 8C). The increases in LL-37 likely represented de novo-synthesis because neither exogenous LL-37 nor Ac2–26 individually induced this response, and the combination of the two also induced cathelicidin upregulation in NHEK (fig. S7C). These data suggested that LL-37 released by necroptotic keratinocytes may further induce FPR1 on adjacent keratinocytes, potentially enhancing the LL-37-FPR1-annexin A1 axis during SJS/TEN disease progression.

**(A)** Serum LL-37 levels were determined by ELISA using sera from HV (n = 16) and patients with MPE (n = 32), EMM (n = 15), FDE (n = 8), AGEP (n = 7), DRESS/DIHS (n = 19), and SJS/TEN: before treatment (n = 29), 2–4 days following initiation of treatment (n = 18), 5–7 days following initiation of treatment (n = 19), 8–10 days following initiation of treatment (n = 6), and more than 11 days (n = 10) after initiation of treatment. **p < 0.01 compared to HV and MPE (Kruskal-Wallis test). **(B)** Immunohistochemistry of skin specimens with antibody against FPR1 in HV and patients with psoriasis, MPE, and SJS (on days 1, 3 and 5 following onset). Scale bar, 100 μm. **(C)** NHEK (n = 5) were treated with Ac2–26 (50 ng/mL) and/or LL-37 for 24 h, and then released LL-37 in the culture supernatant were quantified by ELISA. **p < 0.01 compared to untreated controls (Kruskal-Wallis test). All results in Fig. 8 are representative of three replications of the procedure. Information of patients analyzed in **(A)** is described in Table S1. Patient numbers included in the study is below and their information is described in Table S1: **(B)** Healthy-1 #3, Healthy-2 #4, Psoriasis #5, MPE-1 #8, MPE-2 #9, MPE-3 #13, SJS Day 1 #33, Day 3 #12, Day 5 #36.

Discussion

In the SJS/TEN skin, NETs induced by LCN-2 initiate keratinocyte necroptosis in the early phase, leading to an amplification of subsequent necroptotic response in the later phase (fig. S8). CD8⁺ T cells, which were primed by causative drugs and APCs to produce LCN-2, co-infiltrated early SJS/TEN skin with neutrophils. LCN-2 triggered neutrophils to undergo NETosis and LL-37 production. LL-37 appears to be involved in two phases of disease progression. In the early phase, LL-37 amplified NET formation through a paracrine effect, acting on the keratinocytes to upregulate FPR1, rendering keratinocytes vulnerable to necroptosis mediated by monocyte-derived annexin A1. Upon the triggering of necroptosis, keratinocytes also produced LL-37, further leading the upregulation of FPR1 by surrounding keratinocytes. This likely amplifies the necroptotic process, potentially representing a mechanism for disease progression in later phases of SJS/TEN.

Research into NETs began with the discovery that PMA and IL-8 induced NET formation in neutrophils (49). It is now well established that NETs trigger or exacerbate autoimmunity. IL-8, enriched in SJS/TEN sera but not in MPE and DRESS/DIHS sera (11), induces NET formation as well as neutrophil activation, leading to enhanced migratory capacity (50, 51). We found that circulating SJS/TEN neutrophils were activated and prone to undergoing NETosis, which may explain the underlying mechanism of neutropenia observed in SJS/TEN patients (30–32). In addition to IL-8, we have identified that two NET inducers, LCN-2 and LL-37, were also enriched in SJS/TEN sera.

Although neutrophils have not received much focus in SJS/TEN, the documentation of neutrophilic skin infiltration (27–29), together with the neutropenia that is observed in the peripheral blood of the patients (30–32), prompted us to focus on neutrophils. Neutrophilic infiltration is, however, not a prominent histological feature of SJS/TEN. Indeed, it took multiple sections to identify them in H&E sections from SJS/TEN patients, particularly in the very early skin lesions prior to epidermal detachment. This may not only be due to their scarcity, but due to their altered morphology and rapid NETosis upon entering skin. The latter possibility was supported by the identification of MPO⁺CD66b⁺ or NE⁺ neutrophils and abundant NETosis, as determined by positive staining of molecular components of NETs. Neutrophilic infiltration could be triggered in response to microbes in the presence of severely compromised epidermis, as observed in burn patients (52, 53). However, the detection of neutrophils and NETs in the very early stages of SJS/TEN prior to epidermal detachment suggested that this event was not secondary to microbial exposure. Indeed, prominent neutrophilic infiltration can be observed in diseases such as GPP or AGEP in the absence of compromised epidermis. Interestingly, the presence of NETosis has been not been studied in these neutrophilic dermatoses. It would be of interest to compare the immune cascades that we described in SJS/TEN to such conditions, which may enable a better understanding of distinct neutrophilic or tissue responses in the presence of drugs and specific T cell subsets, potentially further providing insight on why a given drug leads to distinct types of cADRs with different severities.

LCN-2 was first identified as a component of neutrophil granules (54). It is also reported to be expressed in the kidneys, prostate, and epithelia of the respiratory and alimentary tracts (55). LCN-2 binds to the bacterial iron-binding receptor to limit bacterial accessibility to host-derived iron, thereby suppressing growth (56). Detection of urinary LCN-2 is clinically employed to diagnose acute kidney injury (57). Similarly, LCN-2 was detectable in SJS/TEN sera, and we identified CD8⁺ T cells and NETotic neutrophils to be sources of this protein. LCN-2 was detectable in CD8⁺ T cells from both formalin-fixed paraffin-embedded and unfixed-frozen SJS/TEN skin sections but was detectable in neutrophils only in the latter. In humans, LCN-2 is known to exist in three forms: 25 kDa monomers, 45 kDa dimers, and the 135 kDa LCN-2/matrix metalloproteinase-9 complex (54). Activated neutrophils release dimeric LCN-2, whereas injured kidney tubular cells release monomeric LCN-2 (58). Therefore, different clones have been known to display differences in LCN-2 detection patterns (59). Recognition of a specific form of LCN-2 by the antibodies used in the study may explain the differential detection of LCN-2 in CD8⁺ T cells but not in neutrophils.

Three pivotal immune cells were highlighted in this study: CD8⁺ T cells, neutrophils, and monocytes, all of which were involved in the ultimate formation of NETs. LL-37 chemoattracts T cells, neutrophils, and monocytes through FPR2 (45, 60). Similarly, both TNF-α (61–63) and IL-15 (64–66), which are SJS/TEN-related effector molecules, enhance chemotaxis and activation of T cells, neutrophils, and monocytes. Both activated monocytes and neutrophils have been reported to secrete annexin A1 (67), but we found that monocytes were the primary source of annexin A1 that could trigger necroptosis of keratinocytes in SJS/TEN. Taken together, the pathophysiology of SJS/TEN appears to be mediated by the orchestration of innate and adaptive immune cells, which provides additional insights into a disease that was thought to be mediated primarily by CD8⁺ T cells.

The production of perforin, granzyme B, and granulysin was triggered independently of antigens via PHA stimulation, but LCN-2 production by CD8⁺ T cells was induced only in the presence of causative drugs and APCs. In this respect, the study has limitations in the evaluation of specific characteristic of LCN-2-producing CD8⁺ T cells, thereby warranting further analyses regarding their phenotype and the precise mechanism that triggers LCN-2 production.

Early diagnosis and intervention are crucial to the successful management of SJS/TEN. Currently, the diagnosis of SJS/TEN requires a collective approach, taking into account physical signs, including fever, erosive mucous membrane lesions, and cutaneous atypical targetoid lesions, as well as the histological identification of keratinocyte cell death in skin biopsy specimen. However, very early phases of SJS/TEN often poses a diagnostic challenge. As for treatment, there have been only two randomized clinical studies (68, 69), and no internationally unified consensus for the management of SJS/TEN exists to date. Systemic corticosteroids are the first line of therapy based on Japanese guidelines, but its validity is not accepted globally (70). Thus, tools that enable early diagnosis and targeted therapies are still needed in order to improve SJS/TEN prognosis. The detection of LCN-2, LL-37, and annexin A1, and their involvement in SJS/TEN pathophysiology described herein suggest that they may be useful as diagnostic markers and may also be therapeutic targets. Given the involvement of neutrophils, granulocytapheresis might be a reasonable strategy in early phases of SJS/TEN. Establishment of these approaches will require systematic clinical studies in the future.

In conclusion, we propose an additional mechanism that underlies SJS/TEN onset and progression, wherein the causative drugs triggered the orchestration of CD8⁺ T cells, neutrophils, and monocytes mediated keratinocyte necroptosis through a pathway that was centered on NETosis. These findings shed light on the intricate communication of adaptive and innate immune cells in SJS/TEN pathophysiology and provide a foundation for the development of early diagnostic and therapeutic strategies, which may lead to an improved prognosis for SJS/TEN.

Materials and Methods

Study design

This study was approved by the Institutional Review Board of Yamanashi University Hospital, Niigata University Hospital, Hokkaido University Hospital, Keio University Hospital, Kyoto University Hospital, Kyorin University Hospital, and Sapporo City General Hospital. We obtained skin specimens, serum, and blister fluids from patients with SJS/TEN as well as individuals less severe cutaneous adverse drug reactions, autoimmune diseases, neutrophil-associated disorders, and healthy volunteers following informed consent from all participants. Details of sample sizes and experimental replicates are provided in each figure legend. Detailed patient information is reported in the Table S1 (HV; n = 16, SJS/TEN; n = 51, MPE; n = 32, DRESS/DIHS; n = 19, EM major; n = 15, FDE; n = 8, AGEP; n = 7, GVHD; n = 5, SLE; n = 8, BP/anti-p200 pemphigoid; n = 62, PV; n = 12, MMP; n = 10, EBA; n = 2, Psoriasis vulgaris; n = 7, GPP; n = 5, Sepsis; n = 3, Burn; n = 3, PG; n = 2) and Table S2 (SJS/TEN; n = 9, Traumatic blister; n = 5, insect bite; n = 3, BP; n = 3, PV; n = 1, Dermal burn; n = 3, Herpes zoster; n = 3). We defined the day when the patient realized the first skin involvement as day 1. Causative drugs were determined based on a reasonable review of the medication history, the stimulation index of lymphocyte transformation test, patch test positivity, and the algorithm for drug causality for epidermal necrolysis (ALDEN) score.

Cell separation procedures and cultures

Neutrophils were isolated from whole blood using Mono-Poly resolving medium (DS Pharma Biomedical), as described previously (71). We seeded neutrophils in an 8-well chamber slide (Lab-Tek) in 400 μL of serum-free medium, RPMI-1640, at a density of 10⁵ cells/well. PBMCs were isolated from whole blood using Ficoll-Paque PLUS (GE Healthcare). CD4, CD8, HLA-DR, and CD14 positive cells were isolated from PBMCs using CD4, CD8, HLA-DR, and Monocyte Isolation Kits (Miltenyi Biotec) per the manufacturer’s protocol. NHEK was purchased from Kurabo and cultured on EpiLife medium supplemented with insulin (10 mg/mL), rhEGF (epidermal growth factor, 0.1 ng/mL), hydrocortisone (0.5 mg/mL), gentamicin (50 mg/mL), amphotericin B (50 ng/ml), and bovine pituitary extract (0.4% v/v); EpiLife-KG2 medium, all from Kurabo) in a humidified atmosphere with 5% CO₂ at 37°C. Monocytes in blister fluids were isolated from whole blister fluids using a Monocyte Isolation Kit (Miltenyi Biotec) per the manufacturer’s protocol.

Visualization of NETs by fluorescence microscopy

Neutrophils were cultured with the indicated 10% serum, PMA (25 nM) or the noted reagents in an 8-well chamber slide (Lab-Tek). After incubation for 3 h, they were fixed with 4% paraformaldehyde, washed three times with PBS, blocked with 5% goat serum for 1 h at room temperature and then incubated in antibody solutions overnight at 4°C. We used the following antibodies: anti-neutrophil elastase (1:200; Merck), anti-LL-37 (1:200; Hycult) and anti-histone H3 (citrulline R2 + R8 + R17; 1:200; Abcam) antibodies. Samples were washed three times and then incubated for 1 h at room temperature with the following secondary antibodies: Alexa Fluor 488- and Alexa Fluor 546-conjugated anti-mouse and rabbit IgG, respectively. All samples were mounted with VECTASHIELD Mounting Medium including DAPI (Vector Lab). Immunofluorescent images were obtained using a confocal laser microscope (Fluoview3000; Olympus).

NET quantification

Isolated neutrophils were suspended in serum-free medium, RPMI-1640, and then incubated for 3 h at 37°C with stimulation by the noted 10% serum, 10% blister fluid, PMA (25 nM), or noted reagents. For NET quantification, the cells were treated with micrococcal nuclease (Takara) at a concentration of 0.1 U/mL for 15 min at 37°C and then centrifuged for 5 min at 300 × g. Subsequently, the supernatants were transferred into a 96-well flat-bottom plate (100 μL/well) in duplicate. PicoGreen^® (Invitrogen), a specific DNA-binding fluorescent dye (diluted 1:200 in 10 nM Tris/1 mM EDTA buffer), was added (50 μL/sample). We determined NETs by spectrofluorometric analysis at an excitation wavelength of 484 nm and an emission wavelength of 520 nm using an automated plate reader (SpectraMAX Gemini EM, Molecular Devices). NETs were quantified by fluorescence intensity analyses. dsDNA concentration was determined by a standard curve generated by the addition of increasing amounts of recombinant dsDNA of a known concentration.

Enzyme-linked immunosorbent assay

The concentration of human LL-37 (Cusabio), LCN-2 (Sigma), annexin A1 (Abcam), procalcitonin (Abcam), perforin (Mabtech), granzyme B (Mabtech), and granulysin (Boster) were measured by ELISA in each patient’s sera and blister fluids or culture supernatants per the manufacturer’s instructions.

Drug stimulation

PBMCs obtained from recovered SJS/TEN patients were exposed to causative drugs for 4 days to allow for the proliferation of drug-specific T cells in PBMCs (Fig. 5A). As a control, granulocytes were also obtained, exposed to causative drugs for 3 h, and then the supernatants were collected (Fig. 5A). Similarly, CD4⁺ T cells, CD8⁺ T cells, and HLA-DR⁺ cells obtained from patients who had recovered from SJS/TEN, MPE, and DRESS/DIHS were co-cultured as indicated at a ratio of 1000:1 or 100:1 and exposed to each causative drug for 4 days (Fig. 6, A and B, fig. S4, A and B). On day 4, the identical causative drugs were re-added to the culture for 1 day, and then supernatants were collected (Fig. 6, A and B, fig. S4, A and B). During the course of the experiments, we determined optimal drug concentrations based on data from the lymphocyte transformation test. PBMCs were incubated with several concentrations of causative drugs for 4 days, and then their proliferation was analyzed by MMT assay (Trevigen). The findings are expressed as stimulation indexes (SI) (ΔOD₅₇₀ in cultures with drug)/(ΔDOD₅₇₀ in cultures without drug) as described previously (72). The concentration with the highest SI was chosen for the experiments.

Cytotoxic assays

NHEK was pretreated with AG1478 (1 μM), KN-62 (5 μM), WRW4 (10 μM), zVAD (50 μM) or Nec-1 (50 μM) for 30 min, and then exposed to LL-37 (25 μg/mL) and/or Ac2–26 (50 ng/mL). After 30 h, cytotoxicity was analyzed with trypan blue staining (Gibco) and lactate dehydrogenase (LDH) assay (Abcam).

Statistical analysis

Unpaired and paired Mann–Whitney U test or Wilcoxon matched-pairs signed rank test was used for comparison of two groups. Kruskal-Wallis test was applied for multiple comparisons. The differences were considered to be significant when the p value was less than 5%. We did not carry out the calculation of sample size and test for outliers. No exclusion criteria were pre-determined.

Supplementary Material

Sup material

fig. S1 Serum procalcitonin levels in SJS/TEN patients.

fig. S2 Presence of neutrophils in SJS/TEN skin.

fig. S3 Accumulation of NETotic neutrophils in SJS/TEN skin.

fig. S4 LCN-2 production by drug-exposed CD8⁺ T cells in early phases of SJS/TEN.

fig. S5 NETotic neutrophils produce LCN-2.

fig. S6 FPR1 induction on keratinocytes by LL-37, followed by necroptosis of FPR1-expressing keratinocytes by annexin A1.

fig. S7 LL-37 secretion by damaged keratinocytes.

fig. S8 Schematic diagram of study findings.

NIHMS1828912-supplement-Sup_material.pdf^{(46.6MB, pdf)}

table

Table S1. Patient list (serum).

Table S2. Patient list (blister fluids).

NIHMS1828912-supplement-table.pdf^{(360.9KB, pdf)}

Acknowledgments:

We thank all the participants. We also thank Miyuki Ogino for technical assistance.

Funding:

This study was partly supported by MEXTKAKENHI Grant Number 18K16022. K.N. is supported by the Intramural Research Program, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health.

Footnotes

Competing interests: The authors declare that they have no competing interests.

Data and materials availability: All data associated with this study are present in the paper or Supplementary Materials. All materials used in this study are commercially available.

Supplementary Materials

Supplementary Materials and Methods

References and Notes:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Sup material

fig. S1 Serum procalcitonin levels in SJS/TEN patients.

fig. S2 Presence of neutrophils in SJS/TEN skin.

fig. S3 Accumulation of NETotic neutrophils in SJS/TEN skin.

fig. S4 LCN-2 production by drug-exposed CD8⁺ T cells in early phases of SJS/TEN.

fig. S5 NETotic neutrophils produce LCN-2.

fig. S6 FPR1 induction on keratinocytes by LL-37, followed by necroptosis of FPR1-expressing keratinocytes by annexin A1.

fig. S7 LL-37 secretion by damaged keratinocytes.

fig. S8 Schematic diagram of study findings.

NIHMS1828912-supplement-Sup_material.pdf^{(46.6MB, pdf)}

table

Table S1. Patient list (serum).

Table S2. Patient list (blister fluids).

NIHMS1828912-supplement-table.pdf^{(360.9KB, pdf)}

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PERMALINK

Neutrophils initiate and exacerbate Stevens–Johnson syndrome and toxic epidermal necrolysis

Manao Kinoshita

Youichi Ogawa

Natsumi Hama

Inkin Ujiie

Akito Hasegawa

Saeko Nakajima

Takashi Nomura

Jun Adachi

Takuya Sato

Schuichi Koizumi

Shinji Shimada

Yasuyuki Fujita

Hayato Takahashi

Yoshiko Mizukawa

Takeshi Tomonaga

Keisuke Nagao

Riichiro Abe

Tatsuyoshi Kawamura

Abstract

One Sentence Summary:

Introduction

Results

Neutrophil extracellular trap formation in circulating neutrophils from patients with SJS/TEN

Fig. 1. NET formation in circulating SJS/TEN neutrophils.

Neutrophilic infiltration in SJS/TEN skin

Fig. 2. Presence of neutrophils in SJS/TEN skin.

NETosis in SJS/TEN skin

Fig. 3. Accumulation of NETotic neutrophils in SJS/TEN skin.

Aberrant NETosis in SJS/TEN

Fig. 4. NETs in patients with SJS/TEN, non-SJS/TEN cutaneous adverse drug reactions, autoimmune diseases, and neutrophil-associated diseases.

Induction of neutrophil extracellular traps by lipocalin-2 and LL-37

Fig. 5. LCN-2 and LL-37 released from drug-exposed PBMCs induced NETs.

Lipocalin-2 production by drug-specific CD8+ T cells in early phases of SJS/TEN

Fig. 6. LCN-2 production by drug-exposed CD8+ T cells in early phases of SJS/TEN.

FPR1 induction in keratinocytes by LL-37 and subsequent necroptosis by annexin A1

Fig. 7. FPR1 induction on keratinocytes by LL-37, followed by necroptosis of FPR1-expressing keratinocytes by annexin A1.

LL-37 secretion by damaged SJS/TEN keratinocytes

Fig. 8. LL-37 secretion by damaged keratinocytes.

Discussion

Materials and Methods

Study design

Cell separation procedures and cultures

Visualization of NETs by fluorescence microscopy

NET quantification

Enzyme-linked immunosorbent assay

Drug stimulation

Cytotoxic assays

Statistical analysis

Supplementary Material

Acknowledgments:

Funding:

Footnotes

References and Notes:

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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Links to NCBI Databases

Lipocalin-2 production by drug-specific CD8⁺ T cells in early phases of SJS/TEN

Fig. 6. LCN-2 production by drug-exposed CD8⁺ T cells in early phases of SJS/TEN.