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Background: Massive eosinophil infiltration into the esophagus is associated with subepithelial fibrosis and esophageal stricture in patients with eosinophilic esophagitis (EoE). However, the pathogenesis of esophageal fibrosis remains unclear. Objective: We sought to elucidate the cellular and molecular mechanisms underlying the induction of esophageal fibrosis. Methods: We established a murine model of EoE accompanied by fibrotic responses following long-term intranasal administration of house dust mite antigen. Using this murine model, we investigated the characteristics of immune cells infiltrating the fibrotic region of the inflamed esophagus using flow cytometry and histological analyses. We also analyzed the local inflammatory sites in the esophagus of patients with EoE using single-cell RNA sequencing, flow cytometry, and immunohistochemistry. Results: Enhanced infiltration of both amphiregulin-producing and IL-5-producing TH2 cells was detected in the fibrotic area of the esophagus in mice subjected to repeated house dust mite exposure. Deletion of amphiregulin in CD4+ T cells ameliorates esophageal fibrosis. An analysis of human esophageal biopsy samples showed that the infiltration of amphiregulin-producing CD4+ T cells was higher in patients with EoE than in control patients. Furthermore, the number of infiltrated amphiregulin-producing CD4+ T cells was associated with the degree of esophageal fibrosis in patients with EoE. Conclusions: Amphiregulin, produced by TH2 cells, contributes to esophageal fibrosis in EoE and may be a therapeutic target.
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OBJECTIVES: During fasting, liver pivotally regulates blood glucose levels through glycogenolysis and gluconeogenesis. Kidney also produces glucose through gluconeogenesis. Gluconeogenic genes are transactivated by fasting, but their expression patterns are chronologically different between the two organs. We find that renal gluconeogenic gene expressions are positively correlated with the blood ß-hydroxybutyrate concentration. Thus, we herein aim to investigate the regulatory mechanism and its physiological implications. METHODS: Gluconeogenic gene expressions in liver and kidney were examined in hyperketogenic mice such as high-fat diet (HFD)-fed and ketogenic diet-fed mice, and in hypoketogenic PPARα knockout (PPARα-/-) mice. Renal gluconeogenesis was evaluated by rise in glycemia after glutamine loading in vivo. Functional roles of ß-hydroxybutyrate in the regulation of renal gluconeogenesis were investigated by metabolome analysis and RNA-seq analysis of proximal tubule cells. RESULTS: Renal gluconeogenic genes were transactivated concurrently with blood ß-hydroxybutyrate uprise under ketogenic states, but the increase was blunted in hypoketogenic PPARα-/- mice. Administration of 1,3-butandiol, a ketone diester, transactivated renal gluconeogenic gene expression in fasted PPARα-/- mice. In addition, HFD-fed mice showed fasting hyperglycemia along with upregulated renal gluconeogenic gene expression, which was blunted in HFD-fed PPARα-/- mice. In vitro experiments and metabolome analysis in renal tubular cells showed that ß-hydroxybutyrate directly promotes glucose and NH3 production through transactivating gluconeogenic genes. In addition, RNA-seq analysis revealed that ß-hydroxybutyrate-induced transactivation of Pck1 was mediated by C/EBPß. CONCLUSIONS: Our findings demonstrate that ß-hydroxybutyrate mediates hepato-renal interaction to maintain homeostatic regulation of blood glucose and systemic acid-base balance through renal gluconeogenesis regulation.
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Gluconeogénesis , Cuerpos Cetónicos , Riñón , Hígado , Ratones Endogámicos C57BL , Ratones Noqueados , Animales , Ratones , Cuerpos Cetónicos/metabolismo , Hígado/metabolismo , Masculino , Riñón/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Dieta Alta en Grasa , PPAR alfa/metabolismo , PPAR alfa/genética , Glucemia/metabolismo , Dieta CetogénicaRESUMEN
PURPOSE: Auto-antibodies (auto-abs) to type I interferons (IFNs) have been identified in patients with life-threatening coronavirus disease 2019 (COVID-19), suggesting that the presence of auto-abs may be a risk factor for disease severity. We therefore investigated the mechanism underlying COVID-19 exacerbation induced by auto-abs to type I IFNs. METHODS: We evaluated plasma from 123 patients with COVID-19 to measure auto-abs to type I IFNs. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood mononuclear cells from the patients with auto-abs and conducted epitope mapping of the auto-abs. RESULTS: Three of 19 severe and 4 of 42 critical COVID-19 patients had neutralizing auto-abs to type I IFNs. Patients with auto-abs to type I IFNs showed no characteristic clinical features. scRNA-seq from 38 patients with COVID-19 revealed that IFN signaling in conventional dendritic cells and canonical monocytes was attenuated, and SARS-CoV-2-specific BCR repertoires were decreased in patients with auto-abs. Furthermore, auto-abs to IFN-α2 from COVID-19 patients with auto-abs recognized characteristic epitopes of IFN-α2, which binds to the receptor. CONCLUSION: Auto-abs to type I IFN found in COVID-19 patients inhibited IFN signaling in dendritic cells and monocytes by blocking the binding of type I IFN to its receptor. The failure to properly induce production of an antibody to SARS-CoV-2 may be a causative factor of COVID-19 severity.
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Autoanticuerpos , COVID-19 , Interferón Tipo I , Células Mieloides , Femenino , Humanos , Masculino , Autoanticuerpos/inmunología , Autoanticuerpos/sangre , COVID-19/inmunología , Células Dendríticas/inmunología , Interferón Tipo I/inmunología , Interferón Tipo I/metabolismo , Células Mieloides/inmunología , SARS-CoV-2/inmunología , Índice de Severidad de la Enfermedad , Transducción de Señal/inmunologíaRESUMEN
OBJECTIVES: IgG4-related disease (IgG4-RD) is a systemic autoimmune disease with an unknown etiology, affecting single/multiple organ(s). Pathological findings include the infiltration of IgG4-producing plasma cells, obliterative phlebitis, and storiform fibrosis. Although immunological studies have shed light on the dysregulation of lymphocytes in IgG4-RD pathogenesis, the role of non-immune cells remains unclear. This study aimed to investigate the demographics and characteristics of non-immune cells in IgG4-RD and explore potential biomarkers derived from non-immune cells in the sera. METHODS: We conducted single-cell RNA sequence (scRNA-seq) on non-immune cells isolated from submandibular glands of IgG4-RD patients. We focused on fibroblasts expressing collagen type XV and confirmed the presence of those fibroblasts using immunohistochemistry. Additionally, we measured the levels of collagen type XV in the sera of IgG4-RD patients. RESULTS: The scRNA-seq analysis revealed several distinct clusters consisting of fibroblasts, endothelial cells, ductal cells, and muscle cells. Differential gene expression analysis showed upregulation of COL15A1 in IgG4-RD fibroblasts compared to control subjects. Notably, COL15A1-positive fibroblasts exhibited a distinct transcriptome compared to COL15A1-negative counterparts. Immunohistochemical analysis confirmed a significant presence of collagen type XV-positive fibroblasts in IgG4-RD patients. Furthermore, immune-suppressive therapy in active IgG4-RD patients resulted in decreased serum levels of collagen type XV. CONCLUSIONS: Our findings suggest that collagen type XV-producing fibroblasts may represent a disease-characterizing non-immune cell population in IgG4-RD and hold potential as a disease-monitoring marker.
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Enfermedad Relacionada con Inmunoglobulina G4 , Humanos , Enfermedad Relacionada con Inmunoglobulina G4/genética , Enfermedad Relacionada con Inmunoglobulina G4/patología , Glándula Submandibular/patología , Células Endoteliales/patología , Fibroblastos/patología , Colágeno , Análisis de Secuencia de ARNRESUMEN
Uncontrolled type 2 immunity by type 2 helper T (Th2) cells causes intractable allergic diseases; however, whether the interaction of CD4+ T cells shapes the pathophysiology of allergic diseases remains unclear. We identified a subset of Th2 cells that produced the serine proteases granzyme A and B early in differentiation. Granzymes cleave protease-activated receptor (Par)-1 and induce phosphorylation of p38 mitogen-activated protein kinase (MAPK), resulting in the enhanced production of IL-5 and IL-13 in both mouse and human Th2 cells. Ubiquitin-specific protease 7 (USP7) regulates IL-4-induced phosphorylation of STAT3, resulting in granzyme production during Th2 cell differentiation. Genetic deletion of Usp7 or Gzma and pharmacological blockade of granzyme B ameliorated allergic airway inflammation. Furthermore, PAR-1+ and granzyme+ Th2 cells were colocalized in nasal polyps from patients with eosinophilic chronic rhinosinusitis. Thus, the USP7-STAT3-granzymes-Par-1 pathway is a potential therapeutic target for intractable allergic diseases.
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Hipersensibilidad , Células Th2 , Humanos , Animales , Ratones , Granzimas/genética , Granzimas/metabolismo , Interleucina-5/metabolismo , Peptidasa Específica de Ubiquitina 7/metabolismo , Inflamación/metabolismo , Diferenciación Celular , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Allergic diseases arise from a complex interplay between immune system and environmental factors. A link between the pathogenesis of allergic diseases and type 2 immune responses has become evident, with conventional and pathogenic type 2 helper T (Th2) cells involved in both. Recently, there has been a significant development in therapeutic agents for allergic diseases: IL-5 and IL-5 receptor antagonists, Janus kinase (JAK) inhibitors, and sublingual immunotherapy (SLIT). Mepolizumab, an IL-5, and Benralizumab, an IL-5 receptor antagonist, modulate eosinophilic inflammation mediated by IL-5-producing Th2 cells. Delgocitinib shows that JAK-associated signaling is essential for the inflammatory reaction in atopic dermatitis, one of the common allergic diseases. SLIT has a significant effect on allergic rhinitis by reducing pathogenic Th2 cell numbers. More recently, novel molecules that are involved in pathogenic Th2 cell-mediated allergic diseases have been identified. These include calcitonin gene-related peptide (CGRP), reactive oxygen species (ROS) scavenging machinery regulated by the Txnip-Nrf2-Blvrb axis, and myosin light chain 9 (Myl9), which interacts with CD69. This review provides an updated view of the recent research on treatment of allergic diseases and their cause: conventional and pathogenic Th2 cells.
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Dermatitis Atópica , Hipersensibilidad , Humanos , Citocinas , Interleucina-5/uso terapéutico , Hipersensibilidad/tratamiento farmacológico , Células Th2RESUMEN
Tumor-specific CD8+ T cells play a pivotal role in antitumor immunity and are a key target of immunotherapeutic approaches. Intratumoral CD8+ T cells are heterogeneous; Tcf1+ stemlike CD8+ T cells give rise to their cytotoxic progeny-Tim-3+ terminally differentiated CD8+ T cells. However, where and how this differentiation process occurs has not been elucidated. We herein show that terminally differentiated CD8+ T cells can be generated within tumor-draining lymph nodes (TDLN) and that CD69 expression on tumor-specific CD8+ T cells controls its differentiation process through regulating the expression of the transcription factor TOX. In TDLNs, CD69 deficiency diminished TOX expression in tumor-specific CD8+ T cells, and consequently promoted generation of functional terminally differentiated CD8+ T cells. Anti-CD69 administration promoted the generation of terminally differentiated CD8+ T cells, and the combined use of anti-CD69 and anti-programmed cell death protein 1 (PD-1) showed an efficient antitumor effect. Thus, CD69 is an attractive target for cancer immunotherapy that synergizes with immune checkpoint blockade.
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Linfocitos T CD8-positivos , Neoplasias , Humanos , Neoplasias/terapia , Neoplasias/patología , Diferenciación Celular , Ganglios LinfáticosRESUMEN
BACKGROUND: Chronic rhinosinusitis is a common disease of the nasal cavity and is classified into two major endotypes, which are neutrophilic and eosinophilic. Some patients with neutrophilic and eosinophilic chronic rhinosinusitis are refractory to treatment, and the mechanism of drug resistance is not completely understood. METHODS: Nasal polyp samples were collected from patients with non-eosinophilic chronic rhinosinusitis (nECRS) and eosinophilic chronic rhinosinusitis (ECRS). Transcriptomic and proteomic analyses were performed simultaneously. Gene Ontology (GO) analysis was conducted to extract genes involved in drug resistance. Then, GO analysis results were validated via real-time polymerase chain reaction and immunohistochemistry analysis. RESULTS: The nasal polyps of patients with ECRS were enriched with 110 factors in the genes and 112 in the proteins, unlike in those of patients with nECRS. GO analysis on the combined results of both showed that the factors involved in extracellular transportation were enriched. Our analysis focused on multidrug resistance protein 1-5 (MRP1-5). Real-time polymerase chain reaction revealed that the MRP4 expression was significantly upregulated in ECRS polyps. Immunohistochemical staining showed that the MRP3 and MRP4 expressions significantly increased in nECRS and ECRS, respectively. MRP3 and MRP4 expressions were positively correlated with the number of neutrophil and eosinophil infiltrates in polyps and associated with the tendency to relapse in patients with ECRS. CONCLUSIONS: MRP is associated with treatment resistance and is expressed in nasal polyps. The expression pattern had different features based on chronic rhinosinusitis endotype. Therefore, drug resistance factors can be associated with therapeutic outcomes.
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Pólipos Nasales , Rinitis , Humanos , Rinitis/complicaciones , Pólipos Nasales/metabolismo , Proteómica , Eosinófilos/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Enfermedad CrónicaRESUMEN
Tertiary lymphoid structures (TLSs) are ectopic lymphoid tissues that drive antigen-specific immune responses at sites of chronic inflammation. Unlike secondary lymphoid organs such as lymph nodes, TLSs lack capsules and have their own unique characteristics and functions. The presumed influence of TLSs on the disease course has led to widespread interest in obtaining a better understanding of their biology and function. Studies using single-cell analyses have suggested heterogeneity in TLS composition and phenotype, and consequently, functional correlates with disease progression are sometimes conflicting. The presence of TLSs correlates with a favourable disease course in cancer and infection. Conversely, in autoimmune diseases and chronic age-related inflammatory diseases including chronic kidney disease, the presence of TLSs is associated with a more severe disease course. However, the detailed mechanisms that underlie these clinical associations are not fully understood. To what extent the mechanisms of TLS development and maturation are shared across organs and diseases is also still obscure. Improved understanding of TLS development and function at the cellular and molecular levels may enable the exploitation of these structures to improve therapies for chronic diseases, including chronic kidney disease.
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Neoplasias , Insuficiencia Renal Crónica , Estructuras Linfoides Terciarias , Humanos , Estructuras Linfoides Terciarias/genética , Estructuras Linfoides Terciarias/patología , Progresión de la Enfermedad , Enfermedad CrónicaRESUMEN
CD4+ memory T cells are central to long-lasting protective immunity and are involved in shaping the pathophysiology of chronic inflammation. While metabolic reprogramming is critical for the generation of memory T cells, the mechanisms controlling the redox metabolism in memory T cell formation remain unclear. We found that reactive oxygen species (ROS) metabolism changed dramatically in T helper-2 (Th2) cells during the contraction phase in the process of memory T cell formation. Thioredoxin-interacting protein (Txnip), a regulator of oxidoreductase, regulated apoptosis by scavenging ROS via the nuclear factor erythroid 2-related factor 2 (Nrf2)-biliverdin reductase B (Blvrb) pathway. Txnip regulated the pathology of chronic airway inflammation in the lung by controlling the generation of allergen-specific pathogenic memory Th2 cells in vivo. Thus, the Txnip-Nrf2-Blvrb axis directs ROS metabolic reprogramming in Th2 cells and is a potential therapeutic target for intractable chronic inflammatory diseases.
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Células T de Memoria , Factor 2 Relacionado con NF-E2 , Humanos , Especies Reactivas de Oxígeno/metabolismo , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Oxidación-Reducción , Inflamación , Tiorredoxinas/genética , Tiorredoxinas/metabolismoAsunto(s)
Pólipos Nasales , Rinitis , Sinusitis , Humanos , Eosinófilos , Células Asesinas Naturales , Enfermedad CrónicaRESUMEN
Allergic conjunctivitis is a chronic inflammatory disease that is characterized by severe itch in the conjunctiva, but how neuro-immune interactions shape the pathogenesis of severe itch remains unclear. We identified a subset of memory-type pathogenic Th2 cells that preferentially expressed Il1rl1-encoding ST2 and Calca-encoding calcitonin-gene-related peptide (CGRP) in the inflammatory conjunctiva using a single-cell analysis. The IL-33-ST2 axis in memory Th2 cells controlled the axonal elongation of the peripheral sensory C-fiber and the induction of severe itch. Pharmacological blockade and genetic deletion of CGRP signaling in vivo attenuated scratching behavior. The analysis of giant papillae from patients with severe allergic conjunctivitis revealed ectopic lymphoid structure formation with the accumulation of IL-33-producing epithelial cells and CGRP-producing pathogenic CD4+ T cells accompanied by peripheral nerve elongation. Thus, the IL-33-ST2-CGRP axis directs severe itch with neuro-reconstruction in the inflammatory conjunctiva and is a potential therapeutic target for severe itch in allergic conjunctivitis.
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Conjuntivitis Alérgica , Neuropéptidos , Humanos , Interleucina-33/genética , Proteína 1 Similar al Receptor de Interleucina-1/genética , Péptido Relacionado con Gen de Calcitonina , Conjuntivitis Alérgica/patología , Células Th2 , Calcitonina , Prurito/patología , Conjuntiva/patología , NeuronasRESUMEN
The liver stores glycogen and releases glucose into the blood upon increased energy demand. Group 2 innate lymphoid cells (ILC2) in adipose and pancreatic tissues are known for their involvement in glucose homeostasis, but the metabolic contribution of liver ILC2s has not been studied in detail. Here we show that liver ILC2s are directly involved in the regulation of blood glucose levels. Mechanistically, interleukin (IL)-33 treatment induces IL-13 production in liver ILC2s, while directly suppressing gluconeogenesis in a specific Hnf4a/G6pc-high primary hepatocyte cluster via Stat3. These hepatocytes significantly interact with liver ILC2s via IL-13/IL-13 receptor signaling. The results of transcriptional complex analysis and GATA3-ChIP-seq, ATAC-seq, and scRNA-seq trajectory analyses establish a positive regulatory role for the transcription factor GATA3 in IL-13 production by liver ILC2s, while AP-1 family members are shown to suppress IL-13 release. Thus, we identify a regulatory role and molecular mechanism by which liver ILC2s contribute to glucose homeostasis.
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Gluconeogénesis , Interleucina-13 , Glucemia/metabolismo , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Glucógeno/metabolismo , Inmunidad Innata , Interleucina-13/metabolismo , Hígado/metabolismo , Linfocitos/metabolismo , Receptores de Interleucina-13/metabolismo , Factor de Transcripción AP-1/metabolismoRESUMEN
Type 2 helper T (Th2) cells, a subset of CD4+ T cells, play an important role in the host defense against pathogens and allergens by producing Th2 cytokines, such as interleukin-4 (IL-4), IL-5, and IL-13, to trigger inflammatory responses. Emerging evidence reveals that Th2 cells also contribute to the repair of injured tissues after inflammatory reactions. However, when the tissue repair process becomes chronic, excessive, or uncontrolled, pathological fibrosis is induced, leading to organ failure and death. Thus, proper control of Th2 cells is needed for complete tissue repair without the induction of fibrosis. Recently, the existence of pathogenic Th2 (Tpath2) cells has been revealed. Tpath2 cells produce large amounts of Th2 cytokines and induce type 2 inflammation when activated by antigen exposure or tissue injury. In recent studies, Tpath2 cells are suggested to play a central role in the induction of type 2 inflammation whereas the role of Tpath2 cells in tissue repair and fibrosis has been less reported in comparison to conventional Th2 cells. In this review, we discuss the roles of conventional Th2 cells and pathogenic Th2 cells in the sequence of tissue inflammation, repair, and fibrosis.
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Citocinas , Células Th2 , Alérgenos , Fibrosis , Humanos , InflamaciónRESUMEN
The mortality of coronavirus disease 2019 (COVID-19) is strongly correlated with pulmonary vascular pathology accompanied by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection-triggered immune dysregulation and aberrant activation of platelets. We combined histological analyses using field emission scanning electron microscopy with energy-dispersive X-ray spectroscopy analyses of the lungs from autopsy samples and single-cell RNA sequencing of peripheral blood mononuclear cells to investigate the pathogenesis of vasculitis and immunothrombosis in COVID-19. We found that SARS-CoV-2 accumulated in the pulmonary vessels, causing exudative vasculitis accompanied by the emergence of thrombospondin-1-expressing noncanonical monocytes and the formation of myosin light chain 9 (Myl9)-containing microthrombi in the lung of COVID-19 patients with fatal disease. The amount of plasma Myl9 in COVID-19 was correlated with the clinical severity, and measuring plasma Myl9 together with other markers allowed us to predict the severity of the disease more accurately. This study provides detailed insight into the pathogenesis of vasculitis and immunothrombosis, which may lead to optimal medical treatment for COVID-19.
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COVID-19 , Pulmón , Cadenas Ligeras de Miosina , SARS-CoV-2 , Índice de Severidad de la Enfermedad , Tromboinflamación , Vasculitis , COVID-19/sangre , COVID-19/complicaciones , COVID-19/patología , Humanos , Leucocitos Mononucleares , Pulmón/irrigación sanguínea , Pulmón/metabolismo , Pulmón/patología , Pulmón/virología , Cadenas Ligeras de Miosina/sangre , RNA-Seq , SARS-CoV-2/aislamiento & purificación , Análisis de la Célula Individual , Espectrometría por Rayos X , Tromboinflamación/patología , Tromboinflamación/virología , Vasculitis/patología , Vasculitis/virologíaRESUMEN
BACKGROUND: Allergic rhinitis is a growing problem worldwide. Currently the only treatment that can modify the disease is antigen-specific immunotherapy, but its mechanism of action is not fully understood. OBJECTIVE: We comprehensively investigated the role and changes of antigen-specific T cells before and after sublingual immunotherapy (SLIT) for Japanese cedar pollinosis. METHODS: We cultured peripheral blood mononuclear cells obtained both before and 1 year after initiating SLIT and used a combination of single-cell RNA sequencing and repertoire sequencing. To investigate biomarkers, we used cells from patients participating a phase 2/3 trial of SLIT tablets for Japanese cedar pollinosis and cells from outpatients with good and poor response. RESULTS: Antigen-stimulated culturing after SLIT led to clonal expansion of TH2 and regulatory T cells, and most of these CD4+ T cells retained their CDR3 regions before and after treatment, indicating antigen-specific clonal responses and differentiation resulting from SLIT. However, SLIT reduced the number of clonal functional TH2 cells but increased the trans-type TH2 cell population that expresses musculin (MSC), TGF-ß, and IL-2. Trajectory analysis suggested that SLIT induced clonal differentiation of the trans-type TH2 cells differentiated into regulatory T cells. Using real-time PCR, we found that the MSC levels increased in the active SLIT group and those with good response after 1 year of treatment. CONCLUSION: The combination of single-cell RNA sequencing and repertoire analysis helped reveal part of the underlying mechanism: SLIT promotes the expression of MSC on pathogenic TH2 cells and suppresses their function. MSC may be a potential biomarker of SLIT for allergic rhinitis.
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Cryptomeria , Rinitis Alérgica Estacional , Rinitis Alérgica , Inmunoterapia Sublingual , Alérgenos , Biomarcadores , Humanos , Factores Inmunológicos , Interleucina-2 , Leucocitos Mononucleares , Rinitis Alérgica/metabolismo , Rinitis Alérgica/terapia , Rinitis Alérgica Estacional/terapia , Inmunoterapia Sublingual/métodos , Factor de Crecimiento Transformador betaAsunto(s)
Hipersensibilidad , Células Th2 , Citocinas , Humanos , Memoria Inmunológica , InflamaciónRESUMEN
In bronchial asthma patients, mucous cell metaplasia (MCM) and fibrosis occur in the bronchial epithelium and interstitium, respectively. The mucus and collagen fibers are identified by Periodic acid-Schiff stain (PAS) or Sirius red stain on optical microscopy. On a scanning electron microscope (SEM) observation, formalin-fixed-paraffin-embedded specimens have high insulation, thereby attenuating the scattered electron signals leading to insufficient contrast. Moreover, there were no staining methods for SEM observation, which characterizes the changes in epithelium and interstitium by enhancing the scattered electrons. In this study, we established a method of coating osmium thin film on pathological tissue specimens using plasma chemical vapor deposition technology. This method ensured the intensity of scattered electron signals and enabled SEM observation. Furthermore, we found that morphological changes in MCM and interstitial fibrosis could be characterized by Grocott stain, which we optimized to evaluate pathological remodeling in bronchial asthma. Using these techniques, we compared asthma-induced mice with Amphiregulin (Areg) knockout mice, and found that Areg induce MCM, but the production of Grocott-stain-positive substrate in the interstitium is Areg-independent. The method developed in this study provides an understanding of the pathological spatial information linked to the ultrastructural changes in cells and interstitium due to disease-related signaling abnormalities.