Muscle Tissue Damage and Recovery After EV71 Infection Correspond to Dynamic Macrophage Phenotypes

Figures

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  Figure 1. Mouse pups infected with EV71 show rapid induction of inflammatory cytokines and anti-viral molecules in skeletal muscle. (A) Mouse pups (P11-P12, n = 23) were injected i.p. with EV71 virus (8 × 105 PFU) at day 0 and monitored daily for limb paralysis. Clinical scores: 0, no sign of disease; 1, weakness in one or two hindlimbs (wobbling); 2, paralysis in one hindlimb; 3, paralysis in both hindlimbs; 4, paralysis in both forelimbs plus both hindlimbs; 5, death. (B) Gastrocnemius and soleus muscles were collected from sham-infected or EV71-infected mice at indicated times, and real-time quantitative PCR was performed to determine gene expression levels of EV71 viral gene vp1 (left panel). Lysates from gastrocnemius and soleus muscles of EV71-infected mice at day 3 and day 5 post-infection, and at recovery phase (R) were subjected to plaque assay (right panel). R represents mice that recovered from limb paralysis and wobbly status (score = 1). Each symbol represents one mouse (n = 4-8). B.D. represents below determination. (C) Cryosections of gastrocnemius muscle from sham-infected or EV71-infected mice at day 5 post-infection were subjected to immunofluorescence staining with the…

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  Figure 2. Histopathological staining of the gastrocnemius muscle from EV71-infected mice. Gastrocnemius muscle sections from sham-infected or EV71-infected mice at day 3 (score = 1), day 6 (score = 3.5), day 14 (score = 1), day 24 (score = 0.5), day 34 (score = 0), and day 43 (score = 0) were stained with H&E staining (A-G) or Alizarin Red S staining (I-O). (H) Photos of representative sham-infected and EV71-infected mice at day 8 post-infection (score = 3) are shown. The EV71-infected mouse (d8) showed a white appearance in muscles of body trunk and limbs. The magnified image highlights the aberrant structure of muscles with white appearance. (P) The percentage of calcification areas of muscles were measured and calculated as mentioned in Supplementary Figure 1 . The number of mice used for the quantification of image and their paralysis scores were indicated below: sham-infected (n = 2) or EV71-infected mice at day 3 (n = 3, score = 0, 0.5-1), day 6 (n = 2, score = 3.5), day 14 (n = 4, score = 0.5~1), day 24 (n = 3. score = 0.5~1.5), day 34 (n = 2, score = 0) and day 43 (n = 2, score = 0) post-infection. P value was calculated using one-way ANOVA. P < 0.0001.

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  Figure 3. Macrophage phenotypes change in accordance with EV71-induced myositis and muscle regeneration. (A-C) FACS analysis of myeloid populations in muscle tissues. EV71-infected mice were sacrificed on the indicated day post-infection. Cells isolated from hindlimb muscles (gastrocnemius and soleus) were subjected to surface staining for CD45, CD11b, Ly6C, Ly6G and intracellular staining for CD206. Live single muscle cells were first gated on myeloid cells (CD45+CD11b+) followed by further gating on neutrophils (Ly6G+Ly6Cint), monocytes/inflammatory macrophages (Ly6C+Ly6G-) and anti-inflammatory/restorative macrophages (Ly6Clow/-Ly6G-). Representative pseudo-color plots are shown (A). The frequencies of myeloid populations in muscles of EV71-infected mice are shown (B). The ratio of inflammatory macrophages to anti-inflammatory/restorative macrophages is shown (C). Each symbol represents one mouse. (D, E) FACS analysis of CD206 expression in anti-inflammatory/restorative macrophages (CD45+CD11b+Ly6Clow/-Ly6G-). Representative pseudo-color plots (D) and the frequencies (E) are shown. Each symbol represents one mouse (n = 3-5). (F-H) Cryosections of gastrocnemius muscle in representative…

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  Figure 4. Defects in postsynaptic NMJ structure in the gastrocnemius muscle of EV71-infected mice. (A, B) Cryosections of gastrocnemius muscle from sham-infected young mice (P15), sham-infected adult (P34) (A), or EV71-infected mice at 16 h (score = 0), day 3 (score = 2), day 14 (score = 1), day 21 (score = 0.5), day 34 (score = 0.5), and day 51 (score = 0) post-infection (B) were stained with tetramethylrhodamine α-BTX and observed under LSM700 confocal microscopy.

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  Figure 5. The levels of gene expression of muscle-related transcription factors are increased in damaged muscles after EV71 infection. The gastrocnemius and soleus muscles of sham-infected (P14, n = 6; P29, n = 6) or EV71-infected mice at day 3 and day 6 post-infection, and at the recovery phase were collected (day 15-23 post-infection) and subjected to real-time quantitative PCR analysis to determine gene expression levels of Pax7, MyoD, and Myogenin. The relative gene expression levels were normalized to GAPDH. Each symbol represents one mouse (n = 6-12). P values were calculated by Kruskal-Wallis test with Dunn’s multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.