Journal: Science Advances

Article Title: Influenza A virus infection induces immune dysregulation in the placenta and fetus without vertical transmission in nonhuman primates

doi: 10.1126/sciadv.aee9861

Figure Lengend Snippet: ( A ) The volcano plot shows log 2 fold change versus –log 10 ( P value) for FLU ( N = 11) versus CTRL ( N = 7). Red dots mark significant genes with |log 2 fold change| (|log 2 FC|) ≥ 1; blue dots mark genes with |log 2 FC| < 1 but P < 0.05; gold dots indicate nonsignificant genes with |log 2 FC| ≥ 1; black dots indicate nonsignificant genes with |log 2 FC| <1. ( B ) Gene set enrichment analysis of significant GO terms ( P < 0.05) displays the normalized enrichment score (NES) for each category; false discovery rate (FDR) values reflect correction for multiple testing ( C ) Heatmap of normalized gene counts from placental total RNA-seq comparing FLU-exposed ( N = 11) and CTRL ( N = 7) cohorts. Each column represents an individual placental sample, and each row represents a single gene. Yellow-to-blue color intensity reflects the gene-level z -score, calculated using the mean expression value across all animals for each gene. Unsupervised hierarchical clustering was performed on the top 500 differentially expressed genes identified in FLU placentas, using Euclidean distance and Ward’s linkage method. ( D ) Pearson correlation analysis between a select panel of 64 placental genes (listed in figs. S8 and S9) and key metrics of maternal IAV disease severity: maximum clinical score, lung viral load, lung IFN-α concentration, lung IFN-β concentration, and lung IL-6 concentration. Cytokine concentrations and viral load were log 10 -transformed before analysis. The Pearson correlation coefficient ( R ) is plotted for each gene-metric pair. Each dot/triangle represents a correlation that was run between a maternal disease indicator (specified by the column) and the normalized gene count of a given gene using FLU animals. The number of FLU animals used to run each correlation is consistent for each column, and the N is shown at the bottom of the column. A total of 320 distinct correlations were run. Significant correlations (unadjusted P < 0.05) are shown as triangles, and nonsignificant correlations (unadjusted P ≥ 0.05) are shown as dots. Genes with statistically significant correlations are labeled.

Article Snippet: Type I IFNs (IFN-α and IFN-β) were quantified using the cynomolgus IFN-β and cynomolgus/rhesus IFN-α enzyme-linked immunosorbent assay (ELISA) kits (#46415-1 and #46100-1, respectively, PBL Biosciences, Menlo Park, CA, USA).

Techniques: Analysis, RNA Sequencing, Expressing, Concentration Assay, Transformation Assay, Labeling

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: ELISA kits to quantify murine IFN‐β were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: Infection, Isolation, Expressing, Quantitative RT-PCR, Immunofluorescence, 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: Active MMP‐8 cleaves IL‐1β and IL‐6 in vitro. Recombinant human (rh) pro‐MMP‐8 was activated, and its activity was confirmed in each experiment using a quenched fluorogenic substrate that is susceptible to cleavage by MMP‐8, as described in the . Recombinant human cytokines, including 1000 nM IFN‐α2 (A), 1000 nM IFN‐β (B), 1100 nM IL‐1β (C), and 900 nM IL‐6 (D), were incubated with or without 300 nM active rhMMP‐8 for 18 h, with or without a non‐selective metalloproteinase inhibitor (1,10‐phenanthroline; 1,10 phen), which inhibits MMP‐8. The reaction products were separated on Tris‐tricine gels and visualized with silver staining. The arrows indicate the bands corresponding to rhMMP‐8 and the intact cytokines. The long arrows indicate the cleavage products of IFN‐α2, IFN‐β, and IL‐6 (in A, B, and D). MMP‐8 completely degraded IL‐1β; no degradation products were detected (C). The addition of 1,10‐phenanthroline blocked the cleavage or degradation of the four cytokines that was mediated by active MMP‐8.

Article Snippet: ELISA kits to quantify murine IFN‐β were purchased from PBL Assay Science (Piscataway, NJ).

Techniques: In Vitro, Recombinant, Activity Assay, Incubation, Silver Staining

Journal: mLife

Article Title: z‐VAD‐FMK triggers macrophage necroptosis during mycobacterial infection via a non‐canonical off‐target mechanism

doi: 10.1002/mlf2.70101

Figure Lengend Snippet: Mtb‐related necroptosis relies on Toll‐like receptor (TLR) – tumor necrosis factor (TNF) signaling as well as type I interferon (IFN) signaling. (A) Effects of various gene deficiencies on Mtb‐related macrophage necroptosis based on the cell viability assay in the presence of Mtb + z‐VAD. BMDMs from wild‐type (WT) and indicated knockout mice were infected with Mtb at an MOI of 20 in the presence of z‐VAD, as in Figure , and cell viability was assayed at 24 hpi by measuring the ATP level. Detailed data are shown in Figure . (B, C) Effects of genetic deficiency on TNFα secretion by Mtb‐infected macrophages. BMDMs were infected with Mtb at indicated MOI and the cell culture supernatant was harvested at 24 hpi and analyzed by Enzyme‐Linked Immunosorbent Assay (ELISA). (D) Effects of Ifnar1 deficiency on Mtb‐related necroptosis activation. BMDMs were treated as in (A) and cells were harvested at indicated time for Western blot. (E, F) Effects of blockade of type I IFN signaling on macrophage necroptosis. Immunoblots of cell lysates (E) and cell viability measured by the ATP assay (F) are shown. BMDMs were pretreated with the indicated antibody at 20 μg/ml and compounds of Z or Z and N 2 h before infection and the treatment was continued till harvest at 10 hpi (E) or at 24 hpi (F). Data are means ± SD of two (B) or three technical replicates (C, F) and representative of two experiments. D, DMSO; Z, z‐VAD; N, Nec‐1; ND, not detected; ns, not significant; * p < 0.05; and ** p < 0.01 (two‐tailed unpaired Student's t test).

Article Snippet: Mouse IFN‐αA (#12100‐1) and mouse IFN‐β (#12405‐1) were from PBL Assay Science.

Techniques: Cell Viability Assay, Knock-Out, Infection, Cell Culture, Enzyme-linked Immunosorbent Assay, Activation Assay, Western Blot, ATP Assay, Two Tailed Test