Journal: EMBO Molecular Medicine
Article Title: The immunoproteasome‐specific inhibitor ONX 0914 reverses susceptibility to acute viral myocarditis
Figure Lengend Snippet: Impact of ONX 0914 on CVB 3 infection in C57 BL /6 mice Starting one day prior to CVB3 (Nancy) inoculation, C57BL/6 mice were treated daily until day 10 p.i. with either ONX 0914 or vehicle and further three times a week until day 28 p.i. Survival (A) as well as body weight (B) was monitored for 28 days. Average percentages of body weight loss relative to initial weight ± SEM are shown (vehicle n = 8, ONX 0914 n = 8). On day 8 p.i., where inflammation of the myocardium is expected to be fully developed, animals were sacrificed (C), and heart tissue was analyzed. Paraffin‐embedded heart sections were stained using hematoxylin and eosin. A representative image for each group is shown. Extent of myocardial infiltration was scored microscopically, and percentage of inflamed area was assessed and calculated using ImageJ (vehicle n = 13, ONX 0914 n = 11). Total heart tissue mRNA was isolated (D), reverse transcribed and cytokine/chemokine‐specific gene expression was determined by TaqMan qPCR (vehicle n = 10, ONX 0914 n = 10). Virus load (E) was determined by standard plaque assay assessing the amount of infectious virus particles (vehicle n = 13, ONX 0914 n = 10). IFN‐β serum levels (F) during early infection (day 2 p.i.) were determined by ELISA (control=uninfected animals, vehicle n = 7, ONX 0914 n = 7). CVB3‐neutralizing antibody titers in the serum (G) were determined after 8 and 28 days post CVB3 infection (day 8 vehicle n = 13, ONX 0914 n = 10, day 28 vehicle n = 8, ONX 0914 n = 7). To investigate the impact of ONX 0914 treatment on developing an immunological memory status, all mice were challenged with a second CVB3 infection on day 28 after the initial one. Animals received no further ONX 0914 treatment. Proportional body weight loss on day 8 after second challenge relative to the weight on day 28 after the initial challenge (H). Viral load of heart tissue (I) was determined by plaque assay after 8 days. Additionally, paraffin‐embedded heart tissue sections were prepared and Masson's trichrome staining was performed (J). Representative images for each group are shown (one experiment was carried out; vehicle n = 8, ONX 0914 n = 6). Data information: Data are mean ± SEM. Scale bars, 60 μm. P ‐values are indicated in each graph. Survival curves were estimated from the Kaplan–Meier procedure with the log‐rank (Mantel–Cox). For body weight course, two‐way ANOVA was used. Otherwise, unpaired t ‐test was conducted.
Article Snippet: These studies are based on the highly regulated immunoproteasome function of controlling cytokine production and antigen presentation (Muchamuel et al , ; Basler et al , , ; Groettrup et al , ; Kalim et al , ; Mundt et al , ), which is in turn intensively influenced through its inhibition.
Techniques: Infection, Mouse Assay, Staining, Isolation, Expressing, Real-time Polymerase Chain Reaction, Plaque Assay, Enzyme-linked Immunosorbent Assay