Characterization of an experimental model to determine streptococcal M protein–induced autoimmune cardiac and neurobehavioral abnormalities

Figures

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  Figure 1. Antibodies generated following exposure to streptococcal recombinant proteins react with different host proteins. Indirect ELISAs were performed in duplicates to determine host protein cross‐reactivity of rat sera from GAS rM5 (closed squares), SDSE Stg480 antigen–injected rats (closed triangles) and PBS controls (closed circles). Significantly (P < 0.01) higher titers of (a) serum IgG reactivity were observed in rats injected with GAS rM5 and SDSE Stg480 with corresponding antigens and cross‐reactivity against host proteins including (b) cardiac myosin, (c) tropomyosin, (d) laminin, collagens (e) I and (f) IV, (g) DR1 and (h) DR2, (i) tubulin and (j) lysogangliosideGM1 than in control rats injected with PBS. Data are from one of two experiments with age‐matched rats injected with GAS rM5 (n = 5) and SDSE Stg480 (n = 6). Error bars represent standard errors of the mean. **P < 0.01, ***P < 0.001, by one‐way ANOVA with Tukey’s post hoc multiple comparison test. DR, dopamine receptor; GAS, group A streptococcus; Ig, immunoglobulin; PBS, phosphate‐buffered saline; SDSE, Streptococcus dysgalactiae subspecies equisimilis.

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  Figure 2. Electrocardiographic and histological changes demonstrate functional impairment and inflammatory changes in the cardiac tissue of rats exposed to streptococcal recombinant proteins. (a) Prolongation of P–R intervals was observed in rats injected with streptococcal recombinant proteins compared with controls rats. (b) Carditis scores were significantly higher in rats injected with GAS rM5 (closed squares) and SDSE Stg480 (closed triangles) than control rats (closed circles). Characteristic mononuclear cell infiltrations (arrows) were observed in the (d, e) myocardium and (g, h) valvular tissues of rats injected with GAS rM5 and SDSE Stg480 compared with (c, f) rats injected with PBS. Data are from one of two experiments with age‐matched rats injected with GAS rM5 (n = 5) and SDSE Stg480 (n = 6). **P < 0.01, ***P < 0.001, by one‐way ANOVA and the Mann–Whitney U‐test. Bars = 50 μm. GAS, group A streptococcus; PBS, phosphate‐buffered saline; SDSE, Streptococcus dysgalactiae subspecies equisimilis. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Figure 3. T cells and macrophages infiltrate into cardiac tissue of rats injected with streptococcal recombinant proteins. Rat hearts were stained using antibodies against CD3 to detect infiltrating T cells and CD68 to detect macrophages (arrows). Rats injected with GAS rM5 and SDSE M protein had significantly increased infiltration of CD3+ T cells and CD68+ macrophages. By contrast, control rats injected with PBS had no evidence of infiltration of CD3+ T cells or macrophages. Representative microscopic images of myocardium and valves from an age‐matched rat injected with GAS rM5 and SDSE Stg480. Both CD3+ T cells and CD68+ macrophages were observed in all cardiac tissues collected from three individual age‐matched rats injected with GAS rM5 (n = 3) and SDSE Stg480 (n = 3). Bars = 50 μm. GAS, group A streptococcus; PBS, phosphate‐buffered saline; SDSE, Streptococcus dysgalactiae subspecies equisimilis. [Colour figure can be viewed at wileyonlinelibrary.com]

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  Figure 4. Neurobehavioral abnormalities in rats following exposure to streptococcal recombinant proteins. A series of behavioral experiments including (a) food manipulation, (b) beam walking, (c) marble burying, (d) grooming and (e) light/dark box test (Supplementary figure 2) were performed to assess the neurobehavioral abnormalities (both motor and obsessive–compulsive behavior) in rats before (closed circles) and after (closed squares) injection with streptococcal recombinant proteins. Following injection with streptococcal M proteins, there was impairment in (a) food manipulation and (b) gait and balance compared with controls rats injected with PBS. In addition, rats injected with streptococcal recombinant proteins (c) buried higher number of marbles than control rats and (d) spent more time in induced grooming, compared with controls rats. Data are from one of two experiments with age‐matched rats injected with GAS rM5 (n = 5) and SDSE Stg480 (n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, by two‐way ANOVA with Tukey’s post hoc multiple comparison test. GAS, group A streptococcus; PBS, phosphate‐buffered saline; SDSE, Streptococcus dysgalactiae subspecies equisimilis.

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  Figure 5. Serum IgG from rats injected with streptococcal recombinant proteins binds to neuronal tissue. Representative images of the striatum and cortex demonstrate binding of serum IgG from rats injected with streptococcal recombinant proteins. Significantly higher serum IgG binding was observed in the striatum and cortex incubated with serum from rats injected with GAS rM5 and SDSE Stg480 than that from rats with PBS. (a) Arrows show IgG binding. (b) IgG‐binding scores were significantly higher in serum from Lewis rats injected with GAS rM5 (closed triangles) and SDSE stg480 (closed diamonds) than from control rats with PBS (closed squares) and PBS (closed circles). Mean number of positive cells in three different sections of the striatum and cortex were detected in 0.1 mm2 at 20× magnification. Data are from one of the two experiments with age‐matched rats injected with GAS rM5 (n = 5) and SDSE Stg480 (n = 6). ***P < 0.001, ****P < 0.0001 by one‐way ANOVA with Tukey’s post hoc multiple comparison test. GAS, group A streptococcus; Ig, immunoglobulin; PBS, phosphate‐buffered saline; SDSE, Streptococcus dysgalactiae subspecies equisimilis. [Colour figure can be viewed at…

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  Figure 6. Representative western blot images illustrate the binding pattern of the IgG from pooled sera of rats injected with GAS rM5 (a) and SDSE Stg480 (b) with purified DR1 and DR2, tubulin and lysogangliosideGM1 and bands on these blots were observed at ~48, ~50 and ~52 kDa. Antibodies in pooled sera from rats injected with GAS rM5 (c) and SDSE Stg480 (d) to lysates of the striatum and cerebellum from naïve rats. Bands on these blots were observed at ~55 and ~70 kDa, which is consistent with the known molecular weights of DR1, DR2, lysogangliosideGM1 and tubulin, respectively. Moreover, these sera did not recognize proteins in kidney lysates used as non‐neuronal control. Data are from one of two experiments with age‐matched rats injected with GAS rM5 (n = 5) and SDSE Stg480 (n = 6). DR, dopamine receptor; GAS, group A streptococcus; Ig, immunoglobulin; SDSE, Streptococcus dysgalactiae subspecies equisimilis; GAPDH, Glyceraldehyde‐3‐phosphate dehydrogenase.