Mitigated viral myocarditis in A/J mice by the immunoproteasome inhibitor ONX 0914 depends on inhibition of systemic inflammatory responses in CoxsackievirusB3 infection

Figures

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  Fig. 1. Phenotype of CV infection in ip-deficient LMP7−/− A/J mice. a LMP7+/+ (wild-type littermate controls, n = 9) and age and gender matched LMP7−/− mice (n = 12) were infected with 104 pfu of CV Nancy and were analysed on day 8 postinfection (p.i.). b Survival and c body weight was monitored daily. d Formalin fixated and paraffin embedded heart tissue sections were stained using hematoxylin and eosin and myocarditis severity was assessed. e Representative micrographs of haematoxylin and eosin stains are depicted for each group. f For further quantification of immune cell infiltration into the heart tissue, infiltrating immune cells per total heart were quantified by flow cytometry. g Virus concentration in heart tissue was assessed by plaque assay

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  Fig. 2. Peripheral immune cell composition in LMP7−/− A/J mice during CV infection. a At day 8 p.i., blood was drawn for a peripheral blood count. b, c At the same time point, spleen tissue was homogenized and single cell suspensions were subjected to immune cell quantification by flow cytometry (n = 6 LMP7+/+ and n = 9 LMP7−/− mice)

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  Fig. 3. Proteasome inhibition and effects on the expression levels of proteasome subunits by short time ONX 0914 treatment. A/J mice were treated with the vehicle Captisol® or 10 mg/kg BW of the ip inhibitor ONX 0914. After 3 and 24 h, bone marrow cells (BMC) and spleen tissue were removed and protein lysates were subjected to Western blot analysis (n = 1 per time point and treatment). Proteasome composition in each sample was analysed using primary antibodies against the α4 and α6 subunits expressed in all proteasome isoforms and the proteolytically active immunoproteasome subunits LMP7 and LMP2 and their respective standard proteasome counterparts β5 and β1. The additional upwards shifted bands indicate a binding and inactivation by ONX 0914 of the respective proteasomal subunits. Actin was used to indicate protein loading

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  Fig. 4. Immune cell phenotyping in naive ip-deficient LMP7−/− A/J mice and ONX 0914 treated A/J mice. a Immune cell composition of LMP7+/+ and age and gender matched LMP7−/− littermates (n = 6 per group) was analysed in the bone marrow, spleen and peripheral blood by flow cytometry. Bone marrow was taken from 2 femurs and 2 tibias of each mouse and total cells were quantified. b Similarly, in A/J mice treated with vehicle (n = 4) or 10 mg/kg BW ONX 0914 (n = 4), immune cell compartments were harvested 18 h later. Immune cells were quantified by flow cytometry in the bone marrow of 2 femurs and 2 tibias, spleen and peripheral blood. The variability between the absolute immune cell abundancies, e.g., for Ly6Chigh monocytes in bone marrow from WT mice in a and sham-treated WT mice in b, may be attributed due to heterogenous experimental conditions and slightly diverse counting approaches of the two experimenters for a and b. A direct comparison between the absolute cell counts in (a) and (b) is not valid

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  Fig. 5. Biphasic course of CV infection in A/J mice. A/J mice were infected with CV Nancy. Mice were analysed on day 3/4 and day 6 after infection for a blood glucose levels using a clinically approved glucose meter and virus concentration in b liver and c heart tissue by plaque assay

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  Fig. 6. Impact of ONX 0914 treatment on viral myocarditis after systemic inflammation has been established. a Age and gender matched A/J mice were infected with CV Nancy. Starting on day 3, CV infected mice were either treated with the vehicle Captisol® or with 10 mg/kg BW ONX 0914 daily (n = 10 per group) and mice were analysed on day 8. b Body weight was monitored daily. c, d Formalin fixated and paraffin embedded heart tissue sections were stained with hematoxylin and eosin and myocarditis severity was assessed. Representative micrographs of the hematoxylin and eosin stained heart tissue sections are depicted d. e, f Immune cell infiltration into the heart was quantified by flow cytometry. Chemotactic g and h pro-inflammatory cytokine mRNA expression in cardiac tissue on day 8 after infection, as determined by qPCR in both groups (n = 10) and normalized to vehicle (n = 5) or ONX 0914-treated control groups (n = 6). Virus concentration was quantified by plaque assay (I) and viral genome was analyzed by qPCR j. Data is summarized as mean ± SEM

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  Fig. 7. Impact of ONX 0914 treatment on T cell responses. Age and gender matched A/J mice were infected with CV Nancy. Starting on day 3, CV infected mice were either treated with the vehicle Captisol® or with 10 mg/kg BW ONX 0914 daily (n = 10 per group) and mice were analysed on day 8. The data reflect cardiac a and splenic b mRNA expression levels, as determined by qPCR in each group (n = 10) and normalized to vehicle ( = 5) and nONX 0914-treated control groups (n = 6). Data is summarized as mean ± SEM