Journal: Frontiers in Medicine

Article Title: Altered Differentiation and Inflammation Profiles Contribute to Enhanced Innate Responses in Severe COPD Epithelium to Rhinovirus Infection

doi: 10.3389/fmed.2022.741989

Figure Lengend Snippet: HRV16 growth kinetics, tropism and impact on cell integrity in cultures derived from healthy and severe COPD subjects. (A) Cultures from both groups ( n = 6 per group) in duplicate were infected with HRV16 (MOI = 1) for 6 h at 33°C. At 6, 12, 24, 36, 48, 60, and 72 hpi, virus released from the apical compartments of the transwells were titrated by TCID50 assay for growth kinetics of HRV in WD-PBECs derived from healthy and severe COPD. Data are plotted as log10 mean ± SD. (B) HRV growth kinetics in healthy and COPD WD-PBECs were compared by calculating area under the curve (AUC). (C) To determine HRV tropism, cultured transwells in each group at 24 hpi (n = 6 in triplicate) were stained for β-tubulin (ciliated cells, green) and HRV VP2 protein (red), Muc5Ac (goblet cells, green) and HRV VP2 protein (red) or CC10 (club cells, green) and HRV VP2 protein (red). En face images were taken using SP5 confocal microscopy (magnification 63x with 3.0 digital zoom), scale bar 20 μm. (D) HRV-induced cytopathic effects were monitored under light microscope at each time point. Representative phase contrast images from each group in both mock infected and HRV infected transwells were captured at 24 hpi (magnification 20x), scale bar 200 μm. (E) The impact of HRV infection on tight junction integrity was examined at 24 hpi by TEER and presented as mean ± SD, * P < 0.01 healthy vs. COPD, ## P < 0.01 mock vs. HRV infection.

Article Snippet: Briefly, PBECs obtained from severe COPD patients undergoing lung transplantation or age matched healthy controls (Lonza or PromoCell) were expanded in collagen-coated flasks.

Techniques: Derivative Assay, Infection, Virus, TCID50 Assay, Cell Culture, Staining, Confocal Microscopy, Light Microscopy

Journal: Frontiers in Medicine

Article Title: Altered Differentiation and Inflammation Profiles Contribute to Enhanced Innate Responses in Severe COPD Epithelium to Rhinovirus Infection

doi: 10.3389/fmed.2022.741989

Figure Lengend Snippet: Dysregulated Th1/Th2 cytokine secretion and interferon responses in COPD cultures at baseline and following infection with HRV. At 24 hpi, basolateral medium of HRV- and mock-infected cultures was harvested and levels of Th1 cytokines, (A) IFNγ, (B) TNFα and Th2 cytokines, (C) IL-13, (D) TSLP quantified. Markers of interferon responses (E) IFNβ, (F) IP-10, (G) IL-29 were also analyzed. Values are mean ± SD, n = 7 for healthy WD-PBECs and n = 5 for cultures derived from severe COPD patients. * P < 0.05 and ** P < 0.01 healthy vs. COPD, # P < 0.05, ## P < 0.01 and ### P < 0.001 mock vs. HRV infection.

Article Snippet: Briefly, PBECs obtained from severe COPD patients undergoing lung transplantation or age matched healthy controls (Lonza or PromoCell) were expanded in collagen-coated flasks.

Techniques: Infection, Derivative Assay

Journal: Frontiers in Medicine

Article Title: Altered Differentiation and Inflammation Profiles Contribute to Enhanced Innate Responses in Severe COPD Epithelium to Rhinovirus Infection

doi: 10.3389/fmed.2022.741989

Figure Lengend Snippet: HRV exacerbates the secretion of cytokines/chemokines associated with COPD pathogenesis. At 24 hpi, levels of (A) IL-6, (B) IL-8, (C) GM-CSF, (D) eotaxin 3 and (E) IL-10 in the basolateral medium of HRV- and mock-infected cultures were measured. Values are mean ± SD, n = 7 for healthy WD-PBECs and n = 5 for cultures derived from severe COPD patients. * P < 0.05 and ** P < 0.01 healthy vs. COPD, # P < 0.05 mock vs. HRV infection.

Article Snippet: Briefly, PBECs obtained from severe COPD patients undergoing lung transplantation or age matched healthy controls (Lonza or PromoCell) were expanded in collagen-coated flasks.

Techniques: Infection, Derivative Assay

Journal: Clinical and Experimental Immunology

Article Title: Airway epithelial cells enhance the immunogenicity of human myeloid dendritic cells under steady state

doi: 10.1111/cei.12983

Figure Lengend Snippet: Airway epithelial cells (AECs) alter the expression of chemokine and S100 protein genes in myeloid dendritic cells (mDCs). Bar graphs depict the level of chemokines and S100 A8 proteins secreted by mDC ± primary bronchial epithelial cells (PBECs) as determined by multiplexing. Data are mean ± standard error (s.e.) of five different subjects.

Article Snippet: The B‐ALI‐certified PBECs from healthy subjects are obtained from Lonza Inc., and differentiation to ALI is performed following Lonza's protocol.

Techniques: Expressing, Multiplexing

Journal: Clinical and Experimental Immunology

Article Title: Airway epithelial cells enhance the immunogenicity of human myeloid dendritic cells under steady state

doi: 10.1111/cei.12983

Figure Lengend Snippet: Myeloid dendritic cells (mDCs) cultured with primary bronchial epithelial cells (PBECs) display enhanced gene expression of pathogen recognition receptor (PRRs). Box‐plots depict the gene expression changes in PRRs in mDCs after culture with PBECs as determined by NanoString. (a) Toll‐like receptor (TLR)‐2; (b) TLR‐4; (c) TLR‐8; (d) myeloid differentiation primary response gene 88 (MyD88); (e) interleukin‐1 receptor‐associated kinase 1 (IRAK‐1); (f) Nod‐like receptor family CARD domain containing 5 (NLRC5); (g) triggering receptor expressed on myeloid cells 2 (TREM‐2); (h) Nod‐like receptor (NLR)P3; (i) caspace 1 (CASP‐1); (j) interleukin (IL)‐1β; (k) NOD2. Data are mean ± standard deviation (s.d.) of five different subjects.

Article Snippet: The B‐ALI‐certified PBECs from healthy subjects are obtained from Lonza Inc., and differentiation to ALI is performed following Lonza's protocol.

Techniques: Cell Culture, Gene Expression, Standard Deviation

Journal: Clinical and Experimental Immunology

Article Title: Airway epithelial cells enhance the immunogenicity of human myeloid dendritic cells under steady state

doi: 10.1111/cei.12983

Figure Lengend Snippet: Increased Toll‐like receptor (TLR) and Nod‐like receptor (NLR) expression and response of myeloid dendritic cells (mDCs) cultured with primary bronchial epithelial cells (PBECs). Figures depict the expression and response of TLRs in mDCs cultured with PBECs for 24 h. (a) Histograms depict the level of expression of TLRs and NLRs on mDCs with and without culture with PBECs, as determined by flow cytometry. Data are representative of six different subjects. (b) Bar graphs depict the secretion of tumour necrosis factor (TNF)‐α, interleukin (IL)‐1β, IL‐6 and IL‐10 by lipopolysaccharide (LPS)‐activated mDCs cultured with and without PBECs. (c) Bar graphs depict the secretion of TNF‐α, IL‐1β, IL‐6 and IL‐10 by LPS‐activated CD1c mDCs cultured with and without PBECs. Data are mean ± standard error (s.e.) of five different subjects. [Colour figure can be viewed at wileyonlinelibrary.com]

Article Snippet: The B‐ALI‐certified PBECs from healthy subjects are obtained from Lonza Inc., and differentiation to ALI is performed following Lonza's protocol.

Techniques: Expressing, Cell Culture, Flow Cytometry