Journal: The Journal of Experimental Medicine

Article Title: A stem-loop RNA RIG-I agonist protects against acute and chronic SARS-CoV-2 infection in mice

doi: 10.1084/jem.20211818

Figure Lengend Snippet: SLR14 does not significantly elicit IFN-III responses in the respiratory tract. (A–C) Experimental scheme. K18-hACE2 mice were i.v. administered with 15 µg SLR14 or vehicle. 3 h after injection, BALF and lung tissues were collected for IFN-λ ELISA (B) and RT-qPCR (C), respectively. (D and E) Experimental scheme. Ifnar −/− mice were intratracheally administered with 10 11 genome copies of AAV9-hACE2 and allowed to rest for 2 wk before intranasal infection with 10 6 PFU SARS-CoV-2 (2019n-CoV/USA_WA1/2020). 15 µg SLR14 or vehicle were i.v. administered at 4 h after infection. Lung tissues were collected for virological analysis at 4 DPI. Measurement of vRNA at 4 DPI by RT-qPCR using the CDCN2 primer-probe set (E). Mean ± SEM; statistical significance was calculated by two-way ANOVA followed by Bonferroni correction (B and C) or one-way ANOVA followed by Tukey correction (E); ****, P ≤ 0.0001. Data are representative of two independent experiments.

Article Snippet: Concentration of IFN-λ in BAL fluid was determined by ELISA (DY1789B; R&D Systems) according to manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Infection

Journal: The Journal of Experimental Medicine

Article Title: A stem-loop RNA RIG-I agonist protects against acute and chronic SARS-CoV-2 infection in mice

doi: 10.1084/jem.20211818

Figure Lengend Snippet: SLR14-mediated disease prevention and antiviral control rely on IFN-I signaling. (A and B) Experimental scheme. K18-hACE2 mice were i.v. administered with 15 µg SLR14 or vehicle. 3 h after injection, BALF and lung tissues were collected for IFN-I ELISA (A) and RT-qPCR (B), respectively. (C–M) Experimental scheme. K18-hACE2 mice were intranasally infected with 10 3 PFU SARS-CoV-2 (2019n-CoV/USA_WA1/2020). 2 h before infection, 15 µg SLR14 or vehicle was i.v. administered. 24 h before SLR14 injection, half of the SLR14-treated mice were additionally given 2 mg anti-IFNAR antibodies. Weight loss and survival were monitored daily up to 14 DPI. In a separate cohort, lung and trachea tissues were collected for virological analysis 3, 6, and 8 DPI. Nasal washes and brain tissues were collected for virological analysis at 8 DPI. (C–E) Weight loss and survival of K18-hACE2 mice treated with vehicle + PBS, SLR14 + PBS, or SLR14 + αIFNAR from 1 to 14 DPI. (F–H) Measurement of vRNA in the lung parenchyma 3, 6, and 8 DPI by RT-qPCR using the CDCN2 primer-probe set. (I–K) Measurement of vRNA in the trachea 3, 6, and 8 DPI by RT-qPCR using the CDCN2 primer-probe set. (L and M) Measurement of vRNA in the nasal wash (L) or the brain (M) 8 DPI by RT-qPCR using the CDCN2 primer-probe set. (N) The experimental scheme was similar to that of , with the exception that mice were infected with a sublethal dose of SARS-CoV-2. Sera were then collected from survivor mice 14 DPI and used for anti–SARS-CoV-2 S1 IgG measurement by ELISA. Mean ± SEM; statistical significance was calculated by two-way ANOVA followed by Bonferroni correction (A and B), log-rank Mantel–Cox test (E), or one-way ANOVA followed by Tukey correction (F–M); *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. Data are pooled from or representative of two independent experiments.

Article Snippet: Concentration of IFN-λ in BAL fluid was determined by ELISA (DY1789B; R&D Systems) according to manufacturer’s instructions.

Techniques: Injection, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Infection

Journal: American Journal of Respiratory and Critical Care Medicine

Article Title: Ezrin, a Membrane Cytoskeleton Cross-Linker Protein, as a Marker of Epithelial Damage in Asthma

doi: 10.1164/rccm.201802-0373OC

Figure Lengend Snippet: Ezrin expression and epithelial cell–cell adhesion were decreased in an ovalbumin (OVA)-treated allergic mouse model of asthma and restored by anti–IL-13 treatment. (A) Hematoxylin and eosin (H&E) staining of lung tissue in “asthma mice” (black arrows indicate bronchial epithelial cells). Representative image of E-cadherin and ZO-1 immunostaining (black arrows in the middle and bottom panels indicate their expression on the bronchial epithelial cells) was examined in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and was analyzed by Image-Pro Plus 6.0. Scale bars, 50 μm. (B) Epithelial cell–cell adherence was determined by electron microscopy (scale bars, 1 μm; white arrow). (C) Immunohistochemical analysis of ezrin expression in saline-exposed control mice (control), OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13) (original magnification, ×400; scale bar = 100 μm; black arrow) and scored (right graph). (D) The concentrations of ezrin in BAL fluid (BALF) of OVA-treated mice (OVA), OVA + anti-IgG antibody–treated mice (anti-IgG), and OVA + anti–IL-13 antibody–treated mice (anti–IL-13), and IL-13 of asthma mice and controls were measured using an ELISA. The data are presented as mean ± SEM and were analyzed by Student’s t test (control group, n = 8–15; asthma group; n = 8–17). The correlation between ezrin and IL-13 in BALF of mice was analyzed by Pearson’s correlation test. ns = not significant. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with respective controls.

Article Snippet: The levels of IL-4, IL-5, IL-13 (R&D Systems), and ezrin in mouse BAL fluid (BALF; CSB-EL007914MO; Cusabio) and human serum ezrin (SEB297Hu; Cloud Clone Corp.), IL-13, periostin, and IgE were measured by ELISA kit, according to the manufacturer’s instructions.

Techniques: Expressing, Staining, Immunostaining, Saline, Control, Electron Microscopy, Immunohistochemical staining, Enzyme-linked Immunosorbent Assay

Journal: Physiological Reports

Article Title: CFTR dysfunction increases endoglin and TGF‐ β signaling in airway epithelia

doi: 10.14814/phy2.13977

Figure Lengend Snippet: Increased endoglin in CF bronchoalveolar lavage fluid ( BAL ) fluid. Quantitative analysis by human CD 105 ELISA kit (Cat.# EK 0644, Boster Immuno‐Leader Biotechnology) indicates a fivefold increase in endoglin in severe CF ( n = 14) compared to non‐ CF samples (* P < 0.05, n = 5; A ). Immunohistochemistry on paraffin‐embedded lung sections shows increased ENG (brown staining) in CF lungs with severe disease as compared to non‐ CF (100x; primary Ab: Rabbit polyclonal anti‐human ENG , 1:50 dilution, Santa Cruz, Calibration bar = 250 μ m) (B).

Article Snippet: Quantitative analysis of ENG levels was measured in BAL fluid of CF and non‐CF subjects by following manufacturer's protocol using the human CD105 ELISA kit (Boster Biological Technology, Cat#EK0644).

Techniques: Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: JCI Insight

Article Title: Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.88947

Figure Lengend Snippet: (A) Macrophage TGF-β1 production. Mice were challenged with IT bleomycin, and bronchoalveolar lavage (BAL) was performed 24 hours later. Macrophages were separated by adherence to cell culture plates and then cultured for 24 hours in vitro. Total TGF-β1 was quantitated in the cell media by ELISA. Data are presented as box-and-whisker Tukey plots; n = 7 WT and HPS2/CCR2–/–, n = 6 WT/CCR2–/–, n = 5 HPS2, and n = 4/group for HPS1, HPS1/CCR2–/–, and HPS2/TG+. Comparisons were conducted by 2-way ANOVA with Bonferroni’s post-hoc test, *P < 0.05 vs. WT and CCR2–/–, **P < 0.05 vs. HPS2/TG+. (B) MCP-1 augments TGF-β production by WT macrophages. Macrophages were isolated by BAL from unchallenged WT mice and cultured with recombinant MCP-1 (10 ng/ml) or vehicle for 60 minutes. Total TGF-β1 was quantitated in the cell media by ELISA. Data are presented as box-and-whisker Tukey plots; n = 6/group. Comparisons between groups were conducted by Mann-Whitney U analysis, *P < 0.05.

Article Snippet: Cytokine levels were measured in cell culture media supernatant or BAL fluid by ELISA (R&D Systems : MCP-1, #MJE00; MCP-5, #MCC120; MIP-1α, # MMA00; GM-CSF, #MGM00; RANTES, #MMR00; TGFβ1, MB100B; and Abcam: MCP-3, #ab205571; and M-CSF, #ab155457).

Techniques: Cell Culture, In Vitro, Enzyme-linked Immunosorbent Assay, Whisker Assay, Isolation, Recombinant, MANN-WHITNEY

Journal: JCI Insight

Article Title: Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.88947

Figure Lengend Snippet: (A–C) Mice deficient in myeloid TGF-β1 (LysM.Cre/TGFb1f/f; denoted WT/TGF-βΔMye) were studied in comparison to LysM.Cre– littermate controls (LysM.Cre–/TGFb1f/f; denoted WT). (A) Macrophages were isolated by bronchoalveolar lavage (BAL), and total TGF-β1 was measured in the cell culture media by ELISA after 24 hours in culture (mean ± SEM); n = 3/group; *P < 0.05 by Mann-Whitney U analysis. (B) BAL cell counts from unchallenged mice; n = 5 WT and n = 7 WT/ TGF-βΔMye, P = NS by Mann-Whitney U analysis. (C) Total TGF-β1 in lung homogenates from unchallenged mice. Data are presented as box-and-whisker Tukey plots; n = 4/group, P = NS by Mann-Whitney U analysis. (D–H) TGF-βΔMye mice were bred onto the HPS1 background to generate HPS1/TGF-βΔMye mice or HPS1/Cre– littermate controls (denoted HPS1). (D) Total TGF-β1 was determined by ELISA in BAL fluid from unchallenged mice; n = 15 WT/TGF-βΔMye, n = 8 HPS1/TGF-βΔMye, n = 7 WT controls, and n = 11 HPS1 controls. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. WT, **P < 0.05 vs. HPS1/TGF-βΔMye. (E) Representative H&E histologic images (original magnification, ×10) of lung sections at 7 days after IT bleomycin. (F) Lung collagen content of the left lung in unchallenged mice and at 7 days after bleomycin (mean ± SEM). For unchallenged groups, n = 6. For bleomycin-challenged groups, n = 3 WT, n = 6 WT/TGF-βΔMye, n = 21 HPS1, and n = 15 HPS1/TGF-βΔMye mice. Comparisons between bleomycin-challenged groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. other bleomycin-challenged groups. (G) Fibrosis scoring on trichrome-stained lung tissue (mean ± SEM); n = 3/group for WT and n = 6/group for HPS1, *P < 0.05. (H) TUNEL+ alveolar epithelial cells in lung sections from mice 24 hours after bleomycin challenge (mean ± SEM); n = 10 WT/LysM.Cre– (WT) and n = 12 for other groups. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. WT, **P < 0.05 vs. HPS1/ TGF-βΔMye.

Article Snippet: Cytokine levels were measured in cell culture media supernatant or BAL fluid by ELISA (R&D Systems : MCP-1, #MJE00; MCP-5, #MCC120; MIP-1α, # MMA00; GM-CSF, #MGM00; RANTES, #MMR00; TGFβ1, MB100B; and Abcam: MCP-3, #ab205571; and M-CSF, #ab155457).

Techniques: Comparison, Isolation, Cell Culture, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY, Whisker Assay, Staining, TUNEL Assay

Journal: JCI Insight

Article Title: Epithelial-macrophage interactions determine pulmonary fibrosis susceptibility in Hermansky-Pudlak syndrome

doi: 10.1172/jci.insight.88947

Figure Lengend Snippet: HPS1 mice were bred to homozygosity with SPC.Cre+/TGFBR2f/f mice (denoted HPS1/TGFBR2ΔAEC) and studied in comparison to HPS1/SPC.Cre– littermate controls (denoted HPS1). (A–D) Representative histologic images of lung sections from mice at 7 days after bleomycin. (A and C) H&E images (original magnification, ×10). (B and D) Trichrome images (original magnification, ×20). (E) Lung collagen content of left lung in unchallenged mice or at 7 days after bleomycin (mean ± SEM); n = 5 WT unchallenged, n = 4 WT bleomycin, n = 5 WT/TGFBR2 ΔAEC; n = 13 HPS1/TGFBR2ΔAEC; and n = 18 HPS1. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.01 vs. unchallenged groups and WT bleomycin groups, **P < 0.05 vs. HPS1/ TGFBR2ΔAEC. (F) Fibrosis scoring on trichrome-stained lung tissue. Data are presented as box-and-whisker Tukey plots; n = 6 WT/TGFBR2ΔAEC, n = 5 HPS1/ TGFBR2ΔAEC, n = 4 WT/Cre– littermate controls, and n = 6 HPS1 controls, *P < 0.05 vs. all other groups. (G) TUNEL+ alveolar epithelial cells (AECs) in lung sections from mice 24 hours after bleomycin challenge; n = 10 HPS1/TGFBR2ΔAEC and n = 6 HPS1. Comparison between groups was assessed using Mann-Whitney U analysis, *P < 0.01. (H) Total TGF-β1 in BAL from unchallenged mice quantitated by ELISA; n = 14 HPS1/TGFBR2ΔAEC and n = 13 Cre– controls; Mann-Whitney U analysis, *P < 0.001. (I) MCP-1 production from type II AECs isolated from unchallenged mice and cultured for 24 hours; n = 9 WT/TGFBR2 ΔAEC and HPS1/TGFBR2ΔAEC and n = 6/group for WT and SPC.Cre– littermate controls. Comparisons between groups were conducted by Kruskal-Wallis test with Dunn’s multiple comparisons post-test, *P < 0.05 vs. WT, **P < 0.01 vs. HPS1/TGFBR2ΔAEC. (J) MCP-1 production from WT type II AECs after exposure to TGF-β. WT AECs were cultured for 24 hours in the presence of 20 ng/ml TGF-β or vehicle control, and MCP-1 levels were measured in the cell culture media by ELISA (mean ± SEM); n = 4 WT + vehicle and n = 8 WT + TGF-β, *P < 0.001.

Article Snippet: Cytokine levels were measured in cell culture media supernatant or BAL fluid by ELISA (R&D Systems : MCP-1, #MJE00; MCP-5, #MCC120; MIP-1α, # MMA00; GM-CSF, #MGM00; RANTES, #MMR00; TGFβ1, MB100B; and Abcam: MCP-3, #ab205571; and M-CSF, #ab155457).

Techniques: Comparison, Staining, Whisker Assay, TUNEL Assay, MANN-WHITNEY, Enzyme-linked Immunosorbent Assay, Isolation, Cell Culture