Inflammatory Responses Potentiate GAS M Protein Induced Cardiac Damage in an Experimental Model of Rheumatic Heart Disease

Figure 1. BPTx promotes development of a Th1 response. BPTx upregulates antigen specific IFN‐γ production. Splenocytes were harvested 35 days after initial and three booster injection with GAS rM5 and re‐stimulated with GAS rM5. IFN‐γ producing T‐cells were assessed by Flow cytometry and the production of IL‐1β, IL‐6 and IFN‐γ in 72‐h culture supernatants and serum were determined by ELISA. Significantly higher levels of IFN‐γ producing CD4+ T‐cells were observed in splenocytes from rats injected with GAS rM5/CFA/BPTx re‐stimulated with GAS rM5 compared to spleen cells from GAS rM5/CFA/BPToxoid and PBS rM5/CFA/BPTx (A and B). There are elevated levels of IL‐1β, IL‐6, and IFN‐γ in supernatants from cultured splenocytes (C–E) and serum (F–H) in rats injected with GAS rM5/CFA/BPTx compared to GAS rM5/CFA/BPToxoid and PBS/CFA/BPTx. Data are from single experiment with age‐matched rats injected with GAS rM5 (n = 4/5). Statistics: mean ± SEM (n = 5); (B–D) One‐way ANOVA with Tukey's post hoc multiple comparison test; *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 2. BPTx induced responses lead to cardiac functional impairment, increased inflammation and production of tissue cross‐reactive antibodies. ECG was performed pre and post GAS rM5 injection to assess conduction abnormalities or functional changes in the heart. Rats injected with GAS rM5/CFA/BPTx showed prolonged P‐R interval compared to pre‐injection. No ECG changes were observed pre and post injection of GAS rM5/CFA/BPToxoid and PBS/CFA/BPTx (A). Cardiac tissues were stained with H&E to assess the infiltration of mononuclear cells. Significantly higher infiltration with mononuclear cells were observed in the myocardium and the valvular tissues of rats injected with GAS rM5/CFA/BPTx (E and H) compared to GAS rM5/CFA/BPToxoid (D and G) and PBS/CFA/BPTx (C and F). Carditis cores (B) which quantify mononuclear cell infiltration were significantly higher in tissues from rats injected with GAS rM5/CFA/BPTx (E, H) compared to GAS rM5/CFA/BPToxoid (D and G) and PBS/CFA/BPTx (C and F). Furthermore, elevated levels of cross‐reactive antibodies against cardiac myosin (I), laminin (J), tropomyosin (K), and keratin (L) were observed in serum from rats injected with GAS rM5/CFA/BPTx (E and H)…

Figure 3. BPTx is required for the induction of inflammatory responses. ECG was performed pre and post injection of GAS rM5/CFA and PBS/CFA and two different concentration (0.3 and 0.6 µg) of BPTx, to determine the minimal concentration of BPTx required to induce conduction abnormalities in the heart. Rats injected with GAS rM5/CFA/0.6 µg BPTx showed prolonged P‐R interval compared to rats injected with GAS rM5/CFA/0.3 µg BPTx and GAS rM5/CFA alone or PBS/CFA/0.6 µg BPTx (A). Levels of anti‐BPTx antibodies after injection of rats with GAS rM5/CFA/0.6 µg BPTx, GAS rM5/CFA/0.3 µg BPTx, GAS rM5/CFA, and PBS/CFA/0.6 µg BPTx (B). We also analyzed key cytokines circulating in the serum. Significantly higher levels of circulating IL‐1β (C), IL‐6 (D), IFN‐γ (E), and IL‐17A (F) in rats injected with GAS rM5/CFA/0.6 µg BPTx. This group also showed prolonged P‐R interval compared to rats injected with GAS rM5/CFA/0.3 µg BPTx, GAS rM5/CFA alone or PBS/CFA/0.6 µg BPTx. Data are from single experiment with age‐matched rats injected with GAS rM5 (n = 4). Statistics: mean ± SEM (n = 4); (A,C–F) One‐way ANOVA with Tukey's post hoc multiple comparison test; (B) t test; *p < 0.05, **p < 0.01, ***p < 0.001…

Figure 4. BPTx induced inflammatory changes lead to collagen deposition and production of host proteins cross‐reactive antibodies. Mononuclear cell infiltration into the myocardium and valvular tissue (A) represented by carditis score. The proportion of collagen deposition observed in valvular tissue (B). Significantly higher infiltration of mononuclear cells and collagen deposition were observed in cardiac tissue from rats injected with GAS rM5/CFA/0.6 µg BPTx (F, J, N) compared to rats injected with GAS rM5/CFA/0.3 µg BPTx (E, I, M) and GAS rM5/CFA alone (D, H, L) or PBS/CFA/0.6 µg BPTx (C, G, K). Furthermore, significantly higher levels of cross‐reactive antibodies against host proteins cardiac myosin (O), laminin (P), tropomyosin (Q), and keratin (R) were observed serum from rats injected with GAS rM5/CFA/0.6 µg BPTx compared to rats injected with GAS rM5/CFA/0.3 µg BPTx and GAS rM5/CFA alone or PBS/CFA/0.6 µg BPTx. Images magnified at ×200 and scale bar represent 50 µm. Data are from single experiment with age‐matched rats injected with GAS rM5 (n = 4). Statistics: mean ± SEM (n = 4); (A and B) t test; (O–R) One‐way ANOVA with Tukey's post hoc multiple comparison test; ns –not…

Figure 5. BPTx induces increased expression of adhesion molecules and infiltration of mononuclear cells into the cardiac tissue. Myocardial tissue was stained to detect expression of the endothelial cell adhesion molecule VCAM‐1. Percentage of VCAM‐1 expression in the myocardium (A). Representative images of myocardium highlighting increased expression of VCAM‐1 (B‐E) in rats injected with GAS rM5/CFA/0.6 µg BPTx (E) compared to rats injected with GAS rM5/CFA/0.3 µg BPTx (D) and GAS rM5/CFA alone (C) and PBS/CFA/0.6 µg of BPTx (B). T‐cells and macrophages in the mononuclear cells infiltrate were characterized by staining with pan T‐cell antibody against CD3+ and CD68+ respectively (F and O). A significantly higher infiltration of CD3+ T‐cells and CD68+ macrophages were observed in myocardium and valvular tissues of rats injected with GAS rM5/CFA/0.6 µg BPTx (J, N, S, W) compared to rats injected with GAS rM5/CFA/0.3 µg BPTx (I, M, R, V) and GAS rM5/CFA alone (H, L, Q, U) and PBS/CFA/0.6 µg BPTx (G, K, P, T). Images magnified at ×200, snippets magnified at ×400, and scale bar represents 50 µm. Data are from single experiment with age‐matched rats injected with GAS rM5 (n = 4). Statistics:…

Figure 6. BPTx induces infiltration of IFN‐γ+ and IL‐17A+ T‐cells into the cardiac tissue. Cumulative number of infiltrating IFN‐γ+ (A) and IL‐17A+ T‐cells (J) in myocardial and valvular tissue. Significantly higher levels of IFN‐γ+ and IL‐17A+ cells were observed in and representative images of IFN‐γ+ and IL‐17A+ T‐cells in myocardial and valvular tissue (I and R). A significantly higher infiltration of IFN‐γ+ and IL‐17A+ T‐cells were observed in the myocardium and valvular tissues of rats injected with GAS rM5/CFA/0.6 µg BPTx (E, I, N, R) compared to rats injected with GAS rM5/CFA/0.3 µg BPTx (D, H, M, Q) and GAS rM5/CFA alone (C, G L, P) or PBS/CFA/0.6 µg BPTx (B, F, K, O). Images magnified at ×200, snippets magnified at ×400, and scale bar represents 50 µm. Data are from single experiment with age‐matched rats injected with GAS rM5 (n = 4). Statistics: mean ± SEM (n = 4); (A and J) t test; **p < 0.01 and ***p < 0.001.