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: Nature

Article Title: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

doi: 10.1038/s41586-022-04543-1

Figure Lengend Snippet: a , The design of synthesized SP9 with biotin or CPPs conjugated to the N terminus and Cy3 to the C terminus, respectively. Biotin–SP9–Cy3 was bound to streptavidin-conjugated C2 or CRM197. b , Diagram of the analysis of cumulated Cy3 intensities within individual HAE cells. c , Confocal collapse (projected) images of fixed HAE cells that were treated with SP9–Cy3 or SP9–Cy3 conjugated to bacterial toxins (C2, CRM197) or CPPs. Scale bar, 10 µm. The experiment was independently repeated twice with air–liquid interface (ALI) cultures from different donors with similar results. d , Quantitative analysis of intracellular Cy3 fluorescence for each peptide in MUC5AC + HAE cells. Box plots and data points are shown for n cells (indicated below each box plot). Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by post hoc Dunnett’s test; *** P < 0.0001. e , Schematic of the peptide application and sample collection from HAE cells maintained under ALI conditions. f , Representative western blot immunofluorescence images for MUC5AC on an apical surface of untreated HAE cells (control), HAE cells treated with 10 μM of SP9–Cy3, or 10 μM of either SP9–Cy3 or P9–Cy3 conjugated to CPPs for 30 min before stimulation. Basl., MUC5AC secretion during a 30 min period before stimulation (baseline). Exp., MUC5AC secreted within a 30 min experimental period with (IL-13 + ATP) or without (IL-13) stimulation of HAE cells with 100 µM ATP. Cells were treated with IL-13 to induce mucous metaplasia. All of the original blots are shown in Supplementary Fig. . g , h , The ratio of baseline to reference wash secretion (fold increase in baseline secretion over reference secretion) ( g ) and the ratio of experimental to baseline (fold increase in stimulated secretion over baseline secretion) ( h ) for each condition in f . The numbers below the box plots indicate n for each condition, representing individual ALI cultures derived from four donors for each condition. Statistical analysis was performed using two-way ANOVA followed by post hoc Dunnett’s test; * P = 0.013.

Article Snippet: Primary HAE cells from several donors were obtained from Promocell at passage 2 or isolated from fresh tissues that were obtained during tumour resections or lung transplantation with fully consent of patients (Ethics approval: ethics committee Medical School Hannover, project no. 2701-2015).

Techniques: Synthesized, Fluorescence, Western Blot, Immunofluorescence, Derivative Assay

Journal: Nature

Article Title: Inhibition of calcium-triggered secretion by hydrocarbon-stapled peptides

doi: 10.1038/s41586-022-04543-1

Figure Lengend Snippet: a , Representative confocal images (z-sections) of fixed HAE cells treated with SP9-Cy3 or SP9-Cy3 conjugated to CPPs or biotin. Biotin-SP9-Cy3 was bound to streptavidin-conjugated C2 or CRM197. The experiment was repeated twice with ALI cultures from different donors with similar results. Scale bar, 10 µm. b , The diagram illustrates the analysis of intracellular localization of MUC5AC, Cy3, and DAPI in airway secretory cells. Fluorescence intensities of DAPI, AlexaFluor 488 (MUC5AC) and Cy3 were analysed within individual MUC5AC + cells at each z-section, normalized and fluorescence intensity traces calculated along the basolateral to apical cell axis. c , Representative western blot immunofluorescence images for MUC5AC on apical surface of untreated HAE cells (control 1 and 2) or HAE cells treated with 100 μM SP9-Cy3, PEN-SP9-Cy3, TAT-SP9-Cy3, PEN-P9-Cy3, or TAT-P9-Cy3 for 24 h before stimulation. Wash represents MUC5AC accumulated during culture and before start of experiment. Baseline represents unstimulated MUC5AC secretion during a 15 min period after removal of accumulated MUC5AC and experimental represents MUC5AC secreted within 15 min of stimulation with (ATP) or without (no ATP) 100 µM ATP. Lysate represents MUC5AC within HAE cells at the end of the experiment. Cells were treated with IL-13 to induce mucous metaplasia. All original blots are shown in Supplementary Fig . d , Box plots and data points show the ratio of experimental / baseline secretion (fold increase of stimulated secretion over baseline secretion) following 24 h preincubation with 100 μM of the respective peptides. Numbers below box-plots indicate n for each condition, representing individual ALI cultures derived from 4 donors for each condition. * p = 0.046 for HAE cells treated with 100 μM PEN-SP9-Cy3, and p = 0.016 for HAE cells treated with 100 μM TAT-SP9-Cy3, assessed by two-way ANOVA followed by post-hoc Dunnett`s test.

Article Snippet: Primary HAE cells from several donors were obtained from Promocell at passage 2 or isolated from fresh tissues that were obtained during tumour resections or lung transplantation with fully consent of patients (Ethics approval: ethics committee Medical School Hannover, project no. 2701-2015).

Techniques: Fluorescence, Western Blot, Immunofluorescence, Derivative Assay