Journal: The FASEB Journal

Article Title: Matrix Metalloproteinase‐8 Deficiency Alters the Balance Between Viral Clearance and Immunopathology in H1N1 Infection

doi: 10.1096/fj.202601719RRR

Figure Lengend Snippet: MMP‐8 staining is increased in airway epithelial cells and macrophages and is present in PMNs recruited to the lungs of WT mice infected with H1N1. Lung sections from uninfected control mice and WT mice on Days 3, 7, and 10 after H1N1 infection were analyzed. (A and B) Representative double immunostaining images of MMP‐8 (green) and Pan‐CK (red; marker of lung epithelial cells) (A). MMP‐8 staining was quantified per unit area of bronchial epithelium using MetaMorph software (B). (C and D) Representative double immunostaining images of MMP‐8 (green) and MPO (red; marker of PMNs) (C). The percentage of PMNs staining positively for MMP‐8 was quantified (D). (E and F) Representative double immunostaining images of MMP‐8 (green) and MAC‐3 (red; marker of macrophages) (E). The percentage of macrophages staining positively for MMP‐8 was quantified (F). The lungs of infected mice were also stained with non‐immune isotype‐matched primary antibodies [rabbit (Rb) IgG, murine (Ms) IgG, rat IgG, or goat IgG] and are shown in the lower panels. Nuclei in the sections were counterstained blue using DAPI. Immunostained lung sections were examined with a confocal microscope, and merged images are shown in the right panels. Images shown are representative of sections from 5 to 8 mice per experimental group. No staining was detected in the lungs of infected WT mice stained with a non‐immune primary antibody (lower panels). Boxes in the box‐plots show the median values and 25th and 75th percentiles, and whiskers show the 10th and 90th percentiles. n = 5–8 mice per group. Data were analyzed using a Kruskal–Wallis One‐Way ANOVA test followed by pair‐wise testing with Mann–Whitney U tests. The percentage of PMNs and alveolar macrophages that were positively stained for MMP‐8 was quantified in 10 randomly acquired microscopic fields (at magnification 200×). The bars show the mean ± SD. Data were analyzed using a One‐Way ANOVA test followed by pair‐wise testing with Student's t ‐tests. * p ≤ 0.05; ** p ≤ 0.001 versus uninfected control or the group indicated.

Article Snippet: Primary cells were cultured in Airway Epithelial Cell Basal Medium (ATCC, Manassas, VA).

Techniques: Staining, Infection, Control, Double Immunostaining, Marker, Software, Confocal, Microscopy, MANN-WHITNEY

Journal: The FASEB Journal

Article Title: Matrix Metalloproteinase‐8 Deficiency Alters the Balance Between Viral Clearance and Immunopathology in H1N1 Infection

doi: 10.1096/fj.202601719RRR

Figure Lengend Snippet: H1N1 IAV infection induced greater necroptosis in airway epithelial cells in Mmp‐8 −/− versus WT mice. WT and Mmp‐8 −/− mice were infected with H1N1 via the intranasal route, and at intervals post infection, lungs were inflated and removed. Lung sections were double immunostained with a red fluorophore for a marker of lung epithelial cells (Pan‐CK) and with a green fluorophore for p‐RIP3‐Ser 232 (A) or p‐MLKL‐Ser 345 (C). Lungs were also stained with a non‐immune rabbit (Rb) IgG and a non‐immune mouse (Ms) IgG, as shown (lower panels). DAPI was used to counterstain the nuclei. Lung sections were analyzed using a confocal microscope, and merged images are shown in A and C. The images shown are representative of 5–7 mice per group. In B and D, p‐RIP3‐Ser 232 staining (B) and p‐MLKL‐Ser 345 staining (D) in airway epithelial cells were quantified in 10 randomly acquired images per lung section using MetaMorph software, and the staining signal was normalized to the unit area of bronchial wall evaluated. The bars show the mean ± SD; n = 5–7 mice per group. Data were analyzed using a One‐Way ANOVA test followed by pair‐wise testing with Student's t ‐tests. ** p ≤ 0.001 versus the group indicated. In E–G, WT and Mmp‐8 −/− mice were infected with H1N1 via the intranasal route. On Day 3 p.i., p‐MLKL‐Ser 345 , total MLKL, p‐RIP3‐Ser 232 , total RIP3, and housekeeping control (α‐Tubulin) were quantified in homogenates of lung samples using Western blotting and densitometry. The p‐MLKL‐Ser 345 or p‐RIP3‐Ser 232 signals were normalized to total MLKL or RIP3 levels and expressed as a % of the values for uninfected WT mice. The images shown in (E) are representative of 4 mice per group. (F and G) The bars show means ± SD; n = 6 mice/group. (H and I) H1N1‐infected Mmp‐8 −/− mice were treated with the RIP1 inhibitor Necrostatin‐1 (Nec‐1) or vehicle containing 1% DMSO in saline beginning 24 h after infection. BAL fluid albumin levels (H) and lung viral titers (I) were measured. Data are presented as mean ± SD or box plots as indicated. * p ≤ 0.05; ** p ≤ 0.01 versus the indicated group.

Article Snippet: Primary cells were cultured in Airway Epithelial Cell Basal Medium (ATCC, Manassas, VA).

Techniques: Infection, Marker, Staining, Confocal, Microscopy, Software, Control, Western Blot, Saline

Journal: The FASEB Journal

Article Title: Matrix Metalloproteinase‐8 Deficiency Alters the Balance Between Viral Clearance and Immunopathology in H1N1 Infection

doi: 10.1096/fj.202601719RRR

Figure Lengend Snippet: H1N1 IAV infection induces greater increases in Type I (but not Type II) IFN levels in Mmp‐8 −/− versus WT mice. WT and Mmp‐8 −/− mice were infected with 10 3 PFU H1N1 via the intranasal route. Uninfected mice were used as controls. At intervals post‐infection, lung levels of IFN‐α (A, n = 4–10 mice per group), IFN‐β (B, n = 4–15 mice per group), and IFN‐γ (C, n = 4–10 mice per group) were measured using ELISAs and normalized to lung total protein levels. (D–H) Primary airway epithelial cells isolated from WT and Mmp‐8 −/− mice were treated with 20 ng/mL rhIFN‐β for 24 h. Eif2ak2 ( Pkr ) mRNA expression was measured by RT‐qPCR (D). Representative immunofluorescence images are shown in E and G, and the corresponding p‐RIP3 and p‐MLKL signals are quantified in F and H, respectively. In A–C, boxes in the box‐plots show the medians and 25th and 75th percentiles, and the whiskers show the 10th and 90th percentiles. Data were analyzed using a Kruskal–Wallis One‐Way ANOVA followed by pair‐wise testing with Mann–Whitney U tests. In D–H, data are from 3 independent experiments and are presented as mean ± SD. * p ≤ 0.05; ** p ≤ 0.01 versus the indicated group.

Article Snippet: Primary cells were cultured in Airway Epithelial Cell Basal Medium (ATCC, Manassas, VA).

Techniques: Infection, Isolation, Expressing, Quantitative RT-PCR, Immunofluorescence, MANN-WHITNEY