2026/05/07 by Ryo Hasegawa, Shigeharu Ueki, Misaki Arima +16 · 1 voice
Medicine · #Asthma and respiratory diseases #Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis #Sinusitis and nasal conditions
paper · doi:10.1111/all.70381
openalex publication_date 2026/05/07 · openalex created_date 2026/05/08 · openalex updated_date 2026/08/02
Eosinophilic chronic rhinosinusitis (ECRS) represents a refractory endotype of chronic rhinosinusitis with nasal polyps (CRSwNP), characterized by marked eosinophilic infiltration, type 2 inflammation, and excessive fibrin deposition within nasal polyp tissue [1, 2]. Although dysregulated coagulation and impaired fibrinolysis are recognized features of nasal polyps [2], the upstream mechanisms linking eosinophilic inflammation to local fibrin accumulation remain incompletely understood. In particular, the contribution of complement activation to eosinophil-associated procoagulant responses in ECRS has not been fully clarified. We analyzed sinonasal tissues from patients with ECRS, non-ECRS CRSwNP, and control uncinate tissue to explore the relationship between complement activation, eosinophilic inflammation and fibrin deposition (Table S1). Nasal polyps from patients with ECRS exhibited significantly elevated levels of thrombin/anti-thrombin complex (TATc), indicating enhanced local thrombin generation (Figure 1A). In parallel, complement activation products C3a and C5a were markedly increased in nasal polyps compared with uncinate tissue, with C5a levels significantly higher in ECRS than in non-ECRS disease (Figure 1B,C). Importantly, tissue levels of both C3a and C5a showed significant positive correlations with TATc, suggesting a close coupling between complement activation and coagulation activity in ECRS nasal tissue (Figure 1D,E). To assess eosinophilic inflammation, we examined galectin-10, the Charcot–Leyden crystal–forming protein [3]. Galectin-10 levels were substantially elevated in ECRS nasal polyps (Figure 1F) and correlated with TATc, C3a, and C5a (Figure 1G–I). In addition, IL-5 protein levels were increased in ECRS tissue and showed significant correlations with complement components and galectin-10 (Figure S1). In nasal polyps (Table S2), mRNA expression of type 2 cytokines was significantly higher in ECRS than in non-ECRS, whereas IFN-γ expression was unchanged (Figure S2), and galectin-10 mRNA levels correlated with type 2 cytokine expression (Figure S3). These findings indicate that complement activation, eosinophil activation, and type 2 cytokine signaling converge within the ECRS nasal polyp microenvironment. Histological analyses revealed that eosinophils within ECRS nasal polyps frequently exhibited cytolytic morphology consistent with eosinophil extracellular trap–forming cell death (EETosis) [4]. EETotic eosinophils, eosinophil granule proteins, and extracellular DNA structures were observed in close spatial proximity to fibrin deposits (Figure S4), suggesting that EETosis remnants may contribute to fibrin accumulation in vivo. To examine whether complement signals can enhance eosinophil activation under type 2 conditions, we examined the effects of complement components on human eosinophils in vitro. IL-5 was included as a priming cytokine because of its central role in eosinophil biology. Complement components alone induced minimal eosinophil cell death; however, co-stimulation with C5a and IL-5 triggered NADPH oxidase-dependent (diphenyleneiodonium chloride–inhibitable) EETosis (Figure 2A–C). C3a in combination with IL-5 also increased EETosis, although the effect was less pronounced than that observed with C5a + IL-5 (Figure 2A,C). This C5a + IL-5–induced response was dependent on extracellular albumin/serum conditions and was not inhibited by dexamethasone (Figure S5), indicating a steroid-resistant eosinophil activation pathway. Morphological analyses, including ultrastructural examination, identified characteristic features of EETosis, including plasma and nuclear membrane disintegration, chromatin decondensation, and extracellular release of intact eosinophil granules (Figure 2C, Figure S5, Movie 1). In contrast, these features were not observed in control conditions or with C5a + IL-5 + DPI (Figure 2C). Functionally, eosinophils undergoing C5a- and IL-5–induced EETosis acquired a procoagulant phenotype. Phosphatidylserine (PS) exposure provides a catalytic surface for the assembly of coagulation enzyme complexes [5]; in these eosinophils, PS was exposed on vesicular debris and disrupted plasma membranes (Figure 2D). In addition, C5a and IL-5 increased tissue factor mRNA expression in eosinophils (Figure 2E). These findings are consistent with the possibility that complement-activated eosinophils may be involved in fibrin deposition through both cell death–associated phospholipid exposure and active initiation of the coagulation cascade (Figure 2F). In summary, our findings support the presence of a complement–eosinophil–coagulation axis that may contribute to fibrin-rich nasal polyp formation in ECRS. Complement–IL-5 co-stimulation promotes steroid-resistant eosinophil EETosis and procoagulant features, providing a mechanistic framework for the persistence and refractoriness of ECRS nasal polyps. Clinical reductions in polyp size with anti–IL-5Rα therapy are often limited, likely reflecting both the multifactorial and biologically heterogeneous nature of CRSwNP, in which treatment responses may differ across endotypes. In this context, our results highlight eosinophil-associated fibrin deposition as a potentially relevant component of the ECRS/CRSwNP tissue environment. Conceptualization: Shigeharu Ueki, Yoshimasa Imoto. Methodology: Ryo Hasegawa, Misaki Arima, Shohei Nishiyama, Keinosuke Hizuka, Yoshimasa Imoto, Shigeharu Ueki. Investigation: Ryo Hasegawa, Misaki Arima, Shohei Nishiyama, Keinosuke Hizuka, Kohei Tatsumi, Yoshimasa Imoto. Clinical sample collection: Yoshimasa Imoto, Ayako Sawai, Anna Shimizu, Yuki Sonoda, Eiichi Kato, Masanori Kidoguchi, Masafumi Sakashita, Shigeharu Fujieda. Data curation and formal analysis: Ryo Hasegawa, Yoshimasa Imoto, Yohei Sato, Keinosuke Hizuka, Kohei Tatsumi, Shigeharu Ueki. Supervision: Shigeharu Ueki, Yoshimasa Imoto, Shigeharu Fujieda, Atsushi Kato, Robert P. Schleimer. Funding acquisition: Shigeharu Ueki, Yoshimasa Imoto, Masanori Kidoguchi. Writing – original draft: Ryo Hasegawa, Yoshimasa Imoto, Shigeharu Ueki. Writing – review and editing: Shigeharu Ueki, Yoshimasa Imoto, Atsushi Kato, Bruce K. Tan, Robert C. Kern, Robert P. Schleimer. All authors approved the final version of the manuscript. We are grateful to Ms. Hiroko Tsuchiya and Ms. Noriko Tan for skillful technical assistance, and Satomi Misawa, from Academiart LCC. for creating schematic drawing. Declaration of Generative AI in Scientific Writing: During the preparation of this work, the authors used ChatGPT (OpenAI, San Francisco, CA, USA) for English language editing. After using this tool, the authors reviewed and edited the content as needed and take full responsibility for the final version of the manuscript. This work was supported by JSPS KAKENHI Grant Number (JP17K11355, 21K09558 to YI, 20K08794, 21K08434, 20H03832, 21K07833, and 24K11593 to SU, 20K22998, 21KK0287 to MK) and MSD Life Science Foundation, Public Interest Incorporated Foundation and Naito Foundation to YI. The study was also funded in part by Research Grants on Allergic Disease and Immunology from the Japan Agency for Medical Research and Development JP25ek0410138 and MHLW 202213003A to SU, JP22ek0410077 to MK, and JP23jf0126005 to SF. The study was conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Fukui (approval no. 20180023) and the Institutional Review Board of Akita University (approval no. 994). Written informed consent was obtained from all participants. Shigeharu Ueki has received honoraria from AstraZeneca, GSK and Sanofi and grants from AstraZeneca, VIB, Cytrill, and Maruho Co. Ltd. Atsushi Kato has served on an advisory board for AstraZeneca. Atsushi Kato also has received research grants from Regeneron and AstraZeneca. Bruce K. Tan has received research grants from Sanofi. Bruce K. Tan also reports personal fees from Sanofi/Regeneron as speaker's bureau and has served on an advisory board for Sanofi/Regeneron. Robert C. Kern reports consulting fees from Lyra Therapeutics, GSK, and Sanofi/Regeneron. Robert P. Schleimer reports consulting fees from Allakos. Robert P. Schleimer also has royalty rights to Siglec-8 and Siglec-8–ligand related patents licensed by Johns Hopkins to Allakos Inc. Shigeharu Fujieda reports personal fees from Sanofi as speaker's bureau. Shigeharu Fujieda has also served on an advisory board for AstraZeneca, GSK, and Sanofi. Yoshimasa Imoto reports personal fees from GSK and Sanofi as speaker's bureau. The rest of the authors declare that they have no relevant conflicts of interest. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Movie 1 Time-lapse imaging of eosinophil extracellular trap formation in vitro. Human eosinophils were stimulated with C5a (100 ng/mL) in combination with IL-5 (10 ng/mL) in RPMI 1640 medium containing 0.3% BSA. Time-lapse imaging demonstrates progressive morphological changes leading to eosinophil extracellular trap formation. Red arrowheads indicate nuclear membrane disintegration. Table S1: all70381-sup-0002-Supinfo.docx. Table S2: all70381-sup-0002-Supinfo.docx. Figure S1:. Levels of IL-5 and its correlation with TATc, C3a, C5a, and galectin-10 in nasal tissue. (A) NPs from patients with ECRS showed higher IL-5 levels than those of UT and non-ECRS NP tissue (●; UT, ■; non-ECRS NP tissue, ▲; ECRS NP tissue). Correlations between IL-5 levels and (B) TATc, (C) C3a, (D) C5a, and (E) galectin-10 in nasal tissue (n = 55). The levels of all proteins in nasal tissue extract were measured by ELISA. The concentrations of all proteins were normalized to the concentration of total protein. Correlations were assessed using a Spearman's rank correlation test. *p < 0.05, *p < 0.01, ***p < 0.001, and ****p < 0.0001. Figure S2: Nasal tissue samples were immediately stabilized in RNAlater (Invitrogen, Valencia, CA), and total RNA was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Bethlehem, PA, USA) with on-column DNase I treatment according to the manufacturer's instructions. RNA quality was assessed using an Agilent 2100 Bioanalyzer with RNA 6000 Nano chips, and samples with an RNA integrity number (RIN) >7.0 were used. Complementary DNA was synthesized using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Quantitative real-time PCR was performed using TaqMan assays on an Applied Biosystems StepOnePlus Real-Time PCR System. TaqMan probes for IL4 (Hs00174122), IL5 (Hs01548712), IL13 (Hs00174379), IFNG (Hs00989291), tissue factor (F3; Hs01076029), galectin-10 (CLC; Hs00171342), and GAPDH were obtained from Thermo Fisher Scientific. Gene expression levels were normalized to GAPDH expression using standard curve–based quantification. Each reaction contained cDNA equivalent to 10 ng total RNA. *p < 0.05, ****p < 0.0001. (■; non-ECRS NP tissue, ▲; ECRS NP tissue, n = 67). NPs: Nasal Polyps; ECRS: Eosinophilic Chronic Rhinosinusitis Figure S3: Correlations between mRNA gene expression levels of galectin-10 and (A) IL-4, (B) IL-5, and (C) IL-13 levels in nasal tissue (n = 67). mRNA expression was normalized to the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Correlations were assessed using Spearman's rank correlation test. ****p < 0.0001. Figure S4: H&E (A, B) and immunofluorescence staining (C, D) of an identical nasal polyp section of a patient with ECRS. Deparaffinized sections were treated with 0.1% proteinase K at room temperature for 6 min for antigen retrieval. The samples were incubated for 30 min at 37°C with the following primary antibodies as follows: rabbit anti-human MBP (5 μg/mL; BMK-13, MCA5751, Bio-Rad, Hercules, CA) and mouse anti-human fibrin beta-chain antibody (1:100; REF 350, BIO MEDICA, Windsor, Nova Scotia, Canada). Subsequently, the samples were incubated for 30 min at room temperature with Alexa-488-conjugated goat anti-mouse IgG (1:200; A11001, Invitrogen, Carlsbad, CA), Alexa-594 goat antirabbit IgG antibody (1:200; A11072, Invitrogen, Carlsbad, CA), and Hoechst 33342 (1:5000; H3570, Invitrogen, Carlsbad, CA). Images were obtained using an LSM 980 confocal microscope (Carl Zeiss, Oberkochen, Germany). Coverslips were then removed, and the samples were stained with hematoxylin–eosin (HE). The boxed area in A and C (original magnification, ×200)is magnified in B and C (original magnification, ×1000). Samples were immunostained for eosinophil granular protein MBP (red), fibrin (green), and DNA (blue). EETs: eosinophil extracellular traps. Cfegs: Cell-free eosinophil granules. Scale bars indicate 50 μm. (E) Line-scan analysis of fluorescence intensity along the yellow line indicated in panel D. The fluorescence intensity profiles of MBP, fibrin, and DNA are plotted along the selected axis, demonstrating spatial co-localization of MBP and fibrin signals. Figure S5:. (A) Eosinophils were stimulated with vehicle (Cont) or 100 ng/mL C5a and 10 ng/mL IL-5 in RPMI 1640 medium containing 0.3% BSA. Morphological changes and ETs over time were visualized using Diff-Quik (upper panels) and immunofluorescence staining for citrullinated histone H3 (green) and DNA (blue) (lower panels, ×400). Scale bars indicate 20 μm. (B) Eosinophils were stimulated with 100 ng/mL C5a and 10 ng/mL IL-5, with or without dexamethasone at the specified concentrations, in 0.3% BSA-RPMI medium for 3 h. To quantify cell death and ETs, the SYTOX fluorescence intensity was measured (n = 3, mean ± SD). **p < 0.01, vs. control. (C) Eosinophils were stimulated with C3a and C5a, with or without 10 ng/mL IL-5, in RPMI 1640 medium supplemented with 10% FCS. The SYTOX fluorescence intensity was measured at indicated time points (n = 3, mean ± SD). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.