Assessing customized multivalent chemokine-binding peptide treatment in a murine model of coxsackievirus B3 myocarditis

Figures

• 

  Fig. 1. Characterization of chemokine profiles in a CVB3 infection mouse model. A Schematic representation of the anti-inflammatory strategy: Cardiac CVB3 infection triggers a chemokine network, leading to harmful immune cell infiltration. Targeting disease-specific chemokines with a custom Evasin-based peptide therapeutic (BK1.3) may reduce cardiac inflammation. B Amino acid sequence of the 17-mer synthetic dimeric peptide BK1.3. The sequence is displayed in white capital letters, with a black line indicating the disulfide bond between two cysteine residues. C Male C57BL/6 J mice were infected with CVB3 and euthanized on days 3 and 8. The illustrated organs were collected for analysis. D Chemokine profiling in liver tissue by RT-qPCR at baseline (co: control; n = 3), day 3 (n = 3), and day 8 (n = 3). The heatmap shows mean ΔΔCT values for the indicated chemokines, comparing day 3 vs. control (1st column), day 8 vs. control (2nd column), and day 8 vs. day 3 (3rd column). Red rectangles indicate upregulated mRNA expression, while blue rectangles denote downregulated mRNA expression. E Quantification of mRNA expression levels for chemokines in liver tissue was performed on day 3, relative…

• 

  Fig. 2. Validation of chemokine-mediated chemotaxis inhibition by BK1.3. A A cell migration assay was conducted using THP-1 cells, with CCL8-induced migration to evaluate the anti-chemotactic activity of BK1.3. Representative dose–response curves are shown for BK1.3 (green). The scrambled control peptide, BK1.3-SCR, is referred to as the sham throughout this study (grey). Data are presented as mean ± SD of three technical replicates. The IC50 of BK1.3 was calculated using a 4-parameter log-logistic model fitted to the inhibitor response curve. B An in vivo model was employed to assess the anti-chemotactic activity of BK1.3. Male C57BL/6 J mice were administered 2 mg/kg LPS intranasally to induce acute lung inflammation. Intraperitoneal treatments were given directly after LPS-administration and 9 h later, with mice receiving either the control peptide BK1.3-SCR (n = 12) or peptide BK1.3 (n = 12). Mice were euthanized after 24 h. The final analysis time point was reached by 11 mice in the BK1.3-SCR group and 12 mice in the BK1.3 group, respectively. 1 mouse in the BK1.3-SCR group prematurely met termination criteria and was not further analyzed. Bronchoalveolar lavage fluid (BALF) was…

• 

  Fig. 3. Early systemic compromise and pancreatitis remain unaffected under BK1.3 treatment. A Male C57BL/6 J mice were infected with CVB3. Treatments with sham (scrambled BK1.3-SCR control peptide, n = 23), P672 (n = 12), or BK1.3 (n = 12) began 12 h post-infection and were repeated every 12 h until euthanization on day 3 post-infection. All mice reached the final analysis time point in each group. No mice had to be excluded from the experiment due to prematurely met termination criteria. B Body weight was measured daily (sham, n = 23; P672, n = 12; BK1.3, n = 12). Data are presented as mean ± SD of relative body weight, normalized to baseline values on day 0. Statistical analysis was performed using one-way ANOVA, revealing no significant results. C Body temperature was recorded on day 0 and day 3 (sham, n = 23; P672, n = 12; BK1.3, n = 12). Statistical analysis was performed using two-way ANOVA with Sidak’s post hoc analysis. D Blood glucose levels were assessed on day 3 post-infection (sham, n = 23; P672, n = 12; BK1.3, n = 12). E Free fatty acid levels were measured 3 days post-infection (sham, n = 20; P672, n = 11; BK1.3, n = 12). F Viral load in pancreas tissue was quantified on…

• 

  Fig. 4. Regardless of P672 or BK1.3. treatment, CVB3 causes severe hepatic damage with predominantly myeloid immune cell infiltration. Male C57BL/6 J mice were infected with CVB3 and treated with sham (scrambled BK1.3-SCR control peptide, n = 23), P672 (n = 12) or BK1.3 (n = 12) starting 12 h post-infection until euthanization on day 3 post-infection. All mice reached the final analysis time point in each group. No mice had to be withdrawn from the experiment due to prematurely met termination criteria. A Serum enzyme activities of alkaline phosphatase (AP), aspartate-aminotransferase (ASAT) and alanine-aminotransferase (ALAT) were determined on day 3 post infection (sham, n = 23; P672, n = 12; BK1.3, n = 12). B Viral load in liver tissue was quantified on day 3 post-infection using a Plaque Assay (sham, n = 22; P672, n = 12; BK1.3, n = 12). C Semiquantitative scoring (0–3) of liver necrosis in hematoxylin and eosin-stained liver tissue sections (day 3) was performed by a pathologist (sham, n = 23; P672, n = 12; BK1.3, n = 12). D Similarly, semiquantitative scoring (0–3) of liver inflammation was carried out. E Representative micrographs of liver tissue section stained with HE and…

• 

  Fig. 5. Cardiac function is compromised early during CVB3 infection, but BK1.3 treatment does not exacerbate this deterioration. Male C57BL/6 J mice were intraperitoneally infected with CVB3. Treatment with sham (scrambled BK1.3-SCR control peptide, n = 23), P672 (n = 12), or BK1.3 (n = 12) began 12 h post-infection and was administered every 12 h until euthanasia on day 3 post-infection. All mice reached the final analysis time point in each group. No mice had to be withdrawn from the experiment due to prematurely met termination criteria. Echocardiography was performed at baseline and on day 3 post-infection. A Viral load in heart tissue on day 3 post-infection was assessed via Plaque Assays (sham, n = 21; P672, n = 12; BK1.3, n = 12). B Histological samples were stained with hematoxylin and eosin. Myocarditis severity was evaluated semi-quantitatively by a pathologist (sham, n = 23; P672, n = 12; BK1.3, n = 12). Data for (A/B) were analyzed using one-way ANOVA, with no significant differences observed. C, D Left ventricular (LV) end-diastolic volume, ejection fraction, cardiac output, and systolic wall motion velocity (S’) were measured at baseline and on day 3 post-infection using…

• 

  Fig. 6. BK1.3 treatment does not affect immune cell abundance and activation in the spleen during CVB3 infection. C57BL/6 J mice were intraperitoneally infected with CVB3 and treated as described above. A Absolute immune cell counts per mg of splenic tissue were measured for the following lymphoid subpopulations: CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and B cells (sham, n = 23; P672, n = 12; BK1.3, n = 12). B Immune cell activation was assessed in these lymphoid subpopulations by evaluating the expression of activation markers CD44 and CD69. Data are presented as the proportion of activation marker-positive cells relative to the corresponding immune cell subpopulation. C Absolute immune cell counts per mg of splenic tissue were measured for the following myeloid subpopulations: CD11c+ macrophages, CD11c− macrophages, and monocytes (sham, n = 23; P672, n = 12; BK1.3, n = 12). D Immune cell activation was assessed in these myeloid subpopulations by evaluating the expression of activation markers CD86, CD169, MHCII, and Ly6C. Data are presented as the proportion of activation marker-positive cells relative to the corresponding immune cell subpopulation. Data for (A-D) were…

• 

  Fig. 7. Prolonged BK1.3 treatment during the subacute phase of CVB3 infection. A Male C57BL/6 J mice were intraperitoneally infected with CVB3. Treatment with sham (scrambled BK1.3-SCR control peptide, n = 18), or BK1.3 (n = 18) began 12 h post-infection and was administered every 12 h until euthanasia on day 8 post-infection. This final analysis time point was reached by 8 mice in the sham group and 4 mice in the BK1.3 group, respectively. 10 mice in the sham and 14 mice in the BK1.3 group prematurely met termination criteria and were not further analyzed. B Body weight of the mice was measured daily (sham, n = 18; BK1.3, n = 18). Data are shown as mean ± SD relative body weight, normalized to body weight measured at baseline on day 0. Data were analyzed using paired 2-way ANOVA, revealing no differences between the treatment groups. C Survival of the mice from baseline until day 8 post-infection was assessed using the Log-rank (Mantel-Cox) test, which yielded a p-value of 0.10 (sham, n = 18; BK1.3, n = 18). D Blood glucose levels were determined on day 8 post-infection (sham, n = 8; BK1.3, n = 4), with data from uninfected animals (n = 10) also presented. E Free fatty acid levels were…

• 

  Fig. 8. BK1.3 treatment facilitates restoration of liver function with anti-inflammatory effects. Male C57BL/6 J mice were infected with CVB3 and treated with sham (scrambled BK1.3-SCR control peptide, n = 18), or BK1.3 (n = 18) until euthanization on day 8 post-infection. This final analysis time point was reached by 8 mice in the sham group and 4 mice in the BK1.3 group, respectively. 10 mice in the sham and 14 mice in the BK1.3 group prematurely met termination criteria and were not further analyzed. A Viral load in liver tissue on day 8 post-infection was investigated using a Plaque Assay (sham, n = 8; BK1.3, n = 4). B Serum enzyme activities of alkaline phosphatase (AP), aspartate-aminotransferase (ASAT), and alanine-aminotransferase (ALAT) were determined on day 8 post-infection (sham, n = 8; BK1.3, n = 4). C Serum albumin levels were measured on day 8 post-infection (sham, n = 8; BK1.3, n = 4). D Liver tissue sections were obtained from animals sacrificed on day 8 post-infection, stained with HE, and semi-quantitatively scored (0–3) for liver necrosis (sham, n = 8; BK1.3, n = 4). E Representative micrographs of liver tissue sections stained with HE and immunohistochemistry against…

• 

  Fig. 9. Impact of Evasin treatment on liver metabolism during CVB3 infection. A Principal component analysis (PCA) of the liver proteome from uninfected mice (control) and CVB3-infected mice treated with either sham (scrambled BK1.3-SCR control peptide), the Evasin protein P672 or the peptide BK1.3, and euthanized on days 3 and 8 post-infection. B Pearson correlation analysis matrix of the liver proteome across the different experimental groups. The matrix illustrates the degree of similarity between the proteomic profiles. Hierarchical cluster analysis of all proteins and all samples normalized so that for each protein the mean is 0 and the standard deviation is 1. Red and blue colors indicate increased and decreased abundance of the respective protein in each sample compared to the overall mean. There is a perfect separation between the livers of control and infected (early vs. subacute) animals. C Hepatokin1 modeling of liver metabolic processes based on individual blood glucose and fatty acid levels, illustrating the impact of CVB3 infection and Evasin treatments on hepatic metabolism. Metabolic functions are depicted in individual metabolic state. The data in panel C were analyzed…

• 

  Fig. 10. BK1.3 treatment reduces cardiac myeloid immune cell infiltration and helps preserve heart function. Male C57BL/6 J mice were infected with CVB3 intraperitoneally and treatment with sham (scrambled BK1.3-SCR control peptide, n = 18), or BK1.3 (n = 18) began 12 h post-infection and was repeated every 12 h until euthanization on day 8 post-infection. The final analysis time point was reached by 8 mice in the sham group and 4 mice in the BK1.3 group, respectively. 10 mice in the sham and 14 mice in the BK1.3 group prematurely met termination criteria and were not further analyzed. A Virus load in heart tissue on day 8 post-infection was analyzed using Plaque Assays (sham, n = 8; BK1.3, n = 4). B Semiquantitative myocarditis score ranging from 0 to 4 was assigned to cardiac HE-stained tissue sections (sham, n = 8; BK1.3, n = 4). C Immune cell infiltration into heart tissue on day 8 post-infection was investigated using multicolor flow cytometry. Absolute immune cell counts per mg of heart tissue were quantified for total myeloid cells, monocytes, and macrophages (sham, n = 8; BK1.3, n = 3). D Similarly, absolute immune cell counts per mg of heart tissue were quantified for the…