Journal: The European Respiratory Journal

Article Title: Particulate matter-related ITIH4 deficiency is associated with an emphysema phenotype of COPD through JNK-dependent and JNK-independent signalling

doi: 10.1183/13993003.01610-2024

Figure Lengend Snippet: Role of reactive oxygen species (ROS) in mediating low-level inter-α-trypsin inhibitor heavy chain 4 (ITIH4) protein expression. a) Secretory ITIH4 levels in bronchial epithelial (BEAS-2B), normal human small airway epithelial cells (HSAEpCs) or COPD HSAEpC cells exposed to irritants, including vehicle control, lipopolysaccharide (LPS) (20 ng·mL −1 ), interleukin (IL)-1β (10 ng·mL −1 ) and hydrogen peroxide (H 2 O 2 ) (100 μM). ITIH4 levels in cells treated with the antioxidant N-acetyl- l -cysteine (NAC; 5 mM). b) Secretory ITIH4 levels in cells subject to 50 μg·mL −1 carbon black (CB), diesel exhaust particles (DEP), urban dust (UD) or vehicle control exposure. c) Levels of normalised ITIH4 gene expression (log 2 FPKM) in lung tissues from 98 patients with COPD ( GSE57148 dataset cohort), compared with the normal group (n=91). d) Histograms comparing ITIH4 expression levels in BEAS-2B cells exposed to irritants, including LPS (20 ng·mL −1 ), IL-1β (10 ng·mL −1 ), 100 μM H 2 O 2 and vehicle control (n=5−8 per group). The gene expression levels were measured using a quantitative real-time reverse transcription (qRT)-PCR assay. The levels of ITIH4 expression were examined in cells subject to 50 μg·mL −1 CB, DEP, UD or vehicle control exposure. e) ITIH4 gene expression levels in normal and COPD HSAEpCs exposed to irritants including LPS, IL-1β, H 2 O 2 and vehicle control. ITIH4 expression levels were measured in normal control and COPD HSAEpCs subject to CB, DEP, UD or vehicle control exposure (n=5−8 per group). f) Effect of 100 μM H 2 O 2 or vehicle with or without 50 μg·mL −1 cycloheximide on ITIH4 in BEAS-2B cells over a duration of 2 h. The half-life (t 1/2 ) of ITIH4 protein in vehicle and H 2 O 2 was analysed (n=5 per group). Relative values are expressed as the fold change calculated by comparison with vehicle control cells. Data are presented as mean± sd of at least five for each independent group. *: p<0.05; **: p<0.01.

Article Snippet: Human small airway epithelial cells (HSAEpCs) from healthy (aged 53–70 years) and COPD (aged 59–67 years) donors (C-12642, PromoCell, Heidelberg, Germany) were cultured as submerged undifferentiated monolayers in PromoCell cell growth medium and passaged fewer than five times to prevent cell senescence.

Techniques: Expressing, Control, Gene Expression, Reverse Transcription, Quantitative RT-PCR, Comparison

Journal: The European Respiratory Journal

Article Title: Particulate matter-related ITIH4 deficiency is associated with an emphysema phenotype of COPD through JNK-dependent and JNK-independent signalling

doi: 10.1183/13993003.01610-2024

Figure Lengend Snippet: Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) protects epithelial cells from particulate matter with aerodynamic diameter <2.5 µm (PM 2.5 )-induced and reactive oxygen species (ROS)-induced apoptosis. a) Cleaved caspase-3 levels in the airway or alveolar epithelium of normal control participants or patients with COPD. Cleaved caspase-3 levels in lung tissue sections from patients with COPD (n=16) and normal controls (n=20) were analysed through immunohistochemical (IHC) staining. An enlarged view of two rectangular regions in the airway and alveolar epithelium is shown in the right panel. Airway epithelial cells are represented by pink dashed lines. Staining signals were observed under a conventional light microscope. Scale bar=200 μm. Original magnification ×200. The IHC scores of cleaved caspase-3 levels in the airway or alveolar areas in COPD patients were compared with that in normal controls. b) ROS accumulation levels in control and N-acetyl- l -cysteine (NAC)-treated bronchial epithelial cells (BEAS-2B) exposed to 0–100 μg·mL −1 PM 2.5 (n=5−8 per group). Moreover, ROS levels were measured in normal human small airway epithelial cells (HSAEpCs) and COPD HSAEpCs exposed to 0–50 μg·mL −1 PM 2.5 (n=5–9 per group). c) Secreted ITIH4 levels in control and NAC-treated BEAS-2B, normal and COPD HSAEpCs exposed to PM 2.5 (n=5–8 per group). d) Apoptotic cell population (%) in si-ITIH4-knockdown and ITIH4-overexpressing BEAS-2B cells in comparison with scramble si-RNA and pcDNA vector control cells exposed to 20 μg·mL −1 PM 2.5 , respectively. Apoptosis was measured by knockdown and overexpression of ITIH4 in HSAEpCs and COPD HSAEpCs exposed to 20 μg·mL −1 PM 2.5 , respectively (n=5–9 per group). e) Apoptosis was analysed in ITIH4-knockdown and ITIH4-overexpressing cells exposed to hydrogen peroxide (H 2 O 2 ) as indicated (n=5–8 per group). Relative values are expressed as the fold change calculated through comparison with vehicle-treated si-scramble or pcDNA control cells. Data are presented as mean± sd of at least five for each independent group. *: p<0.05; **: p<0.01.

Article Snippet: Human small airway epithelial cells (HSAEpCs) from healthy (aged 53–70 years) and COPD (aged 59–67 years) donors (C-12642, PromoCell, Heidelberg, Germany) were cultured as submerged undifferentiated monolayers in PromoCell cell growth medium and passaged fewer than five times to prevent cell senescence.

Techniques: Control, Immunohistochemical staining, Immunohistochemistry, Staining, Light Microscopy, Knockdown, Comparison, Plasmid Preparation, Over Expression

Journal: The European Respiratory Journal

Article Title: Particulate matter-related ITIH4 deficiency is associated with an emphysema phenotype of COPD through JNK-dependent and JNK-independent signalling

doi: 10.1183/13993003.01610-2024

Figure Lengend Snippet: Overexpression of inter-α-trypsin inhibitor heavy chain 4 (ITIH4) inhibits oxidative-stress-induced activation of c-Jun N-terminal kinase (JNK) signalling and reduction of β-catenin. a) Levels of ITIH4, β-catenin, p-JNK and JNK in bronchial epithelial (BEAS-2B) cells exposed to lipopolysaccharide (LPS) (20 ng·mL −1 ), interleukin (IL)-1β (10 ng·mL −1 ) or hydrogen peroxide (H 2 O 2 ) (100–300 μM), examined through an immunoblot analysis (n=6 per group). b) Levels of ITIH4, β-catenin, p-JNK and JNK in BEAS-2B cells exposed to 50–100 μg·mL −1 carbon black (CB), diesel exhaust particles (DEP), urban dust (UD) and vehicle control (ctrl) were measured. Moreover, levels of ITIH4, β-catenin, p-JNK and JNK were determined in c) bronchial epithelial (BEAS-2B) cells and d) human small airway epithelial cells (HSAEpCs) exposed to 50–100 μg·mL −1 DEP and DEP-derived particulate matter with aerodynamic diameter <2.5 µm (PM 2.5 ) (n=6 per group). e) The effects of exposure to 100 μM H 2 O 2 -mediated expression of ITIH4, β-catenin, p-JNK and JNK proteins were assessed in BEAS-2B cells transfected with ITIH4-overexpression vector or empty vector control (EV) (n=6–7 per group). f) By contrast, the effects of exposure to H 2 O 2 -mediated β-catenin expression and JNK activation were evaluated in BEAS-2B cells transfected with siRNA control or si-ITIH4 (n=6 per group). Levels of p-JNK and JNK were measured in cells exposed to stimuli for 2 h. Levels of ITIH4, β-catenin and actin were measured in cells treated with irritants for 24 h. Relative values are expressed as the fold change calculated through comparison with vehicle-treated cells. Data are presented as mean± sd of at least five for each independent group. *: p<0.05; **: p<0.01.

Article Snippet: Human small airway epithelial cells (HSAEpCs) from healthy (aged 53–70 years) and COPD (aged 59–67 years) donors (C-12642, PromoCell, Heidelberg, Germany) were cultured as submerged undifferentiated monolayers in PromoCell cell growth medium and passaged fewer than five times to prevent cell senescence.

Techniques: Over Expression, Activation Assay, Western Blot, Control, Derivative Assay, Expressing, Transfection, Plasmid Preparation, Comparison

Journal: bioRxiv

Article Title: Oncogenic Stress is a Novel Immunogenic Signal Driven by the Unfolded Protein Response and Detected by Neutrophils

doi: 10.1101/2025.01.17.633444

Figure Lengend Snippet: a. Anatomopathological characterization of healthy (MMTV-Neu-) and preneoplastic stage (MMTV-Neu+) tissues by Hematoxylin and Eosin staining on Formalin-Fixed Paraffin-Embedded (FFPE) slides. b., c. Violin plots representing ssGSEA enrichment score of the Neu /ERBB2 Signaling Pathway ( b. ) and the UPR ( c. ) signatures in healthy (n=3) and preneoplastic (n=4) epithelial cells in the MMTV-Neu mouse model. d. Violin plot representing the expression of cxcl1 in healthy (n=3) and preneoplastic (n=4) epithelial cells in the MMTV-Neu mouse model. e. Representative Ly6G immunofluorescence staining on FFPE healthy and preneoplastic tissues (left panel). Box plot representing the number of neutrophils per mm 2 in healthy (n=6) and preneoplastic (n=5) tissues (right panel) in the MMTV-Neu mouse model. f. Principal Component Analysis of luminal epithelial cells from previous wild-type (WT) or pre-cancer stages annotation. WT-like, Stage I and Stage II were manually annotated in BLG-Cre, BRCA1 f/f , p53 +/- mouse model. g. Dot plot representing ssGSEA enrichment scores of ER stress and UPR response signature in luminal epithelial cells from WT, WT-like, Stage I, and Stage II in the BLG-Cre, BRCA1 f/f , p53 +/- mouse model. h. Box plot representing the frequency of neutrophil among immune cells in healthy (n=16) and preneoplastic (n=34) tissues in the BLG-Cre, BRCA1f/f, p53+/-mouse model, assessed by flow cytometry. Data information: ( b-d ) Welch’s t test (*p<0.05; **p<0.01). ( e, h ) Two-tailed Wilcoxon test. Shown is the median ± SEM. (**p<0.01).

Article Snippet: Human mammary epithelial cells (HMEC - Promocell, ref#C-12650) were immortalized with by transduction with a retroviral vector pBABE-puro containing the Human Telomerase Reverse Transcriptase (hTERT - Addgene).

Techniques: Staining, Formalin-fixed Paraffin-Embedded, Expressing, Immunofluorescence, Flow Cytometry, Two Tailed Test

Journal: bioRxiv

Article Title: Oncogenic Stress is a Novel Immunogenic Signal Driven by the Unfolded Protein Response and Detected by Neutrophils

doi: 10.1101/2025.01.17.633444

Figure Lengend Snippet: a. Violin plot representing the expression of neu/erbb2/her2 in healthy (n=3) and preneoplastic (n=4) epithelial cells in the MMTV-Neu mouse model. b. Box plot representing the frequency of immune cells among viable cells in healthy (n=16) and preneoplastic stage (n=34) tissues in BLG-Cre, BRCA1 f/f , p53 +/- mouse model assessed by flow cytometry. Data information: ( a ) Welch’s t test (****p<0.0001) ( b ) Two-tailed Wilcoxon test. Shown is the median ± SEM. (**p<0.01).

Article Snippet: Human mammary epithelial cells (HMEC - Promocell, ref#C-12650) were immortalized with by transduction with a retroviral vector pBABE-puro containing the Human Telomerase Reverse Transcriptase (hTERT - Addgene).

Techniques: Expressing, Flow Cytometry, Two Tailed Test