Figure 1.
Mice have increased myocardial fibrosis after CVB3 infection.
(A) Groups of 8 mice were infected with 10 TCID50 dose of CVB3, the myocardial inflammation was shown by H&E staining 4 wks after infection. Representative heart sections were shown for each week (magnification: 200×). Arrows indicate inflammatory cell infiltrate. (B) At 1,2,3,4 wks after CVB3 infection, the mRNA expression levels of matrix proteins (collagen I, MMP-1, MMP-3, TIMP-1) were determined by quantitative real time-PCR. Data are from one representative experiment of three performed ones and represent as the mean±SEM. (C) Picrosirius-red stained heart sections of CVB3-infected mice revealed increased fibrosis (red) (magnification: 200×). Arrows indicate myocardial interstitial and perivascular fibrosis. The severity of fibrosis was scored by collagen volume fractions and represent as mean ± SEM of three separate experiments. *, P<0.05 compared to the non-infected mice (wk0).
Figure 2.
Treg frequency has negative correlation with severity of cardiac fibrosis.
Groups of 20 mice were infected with 10 TCID50 dose of CVB3, 1,2,3,4 wks after infection, spleen and myocardial infiltrated cells were subjected to, flow cytometry protocol and stained for PerCP-CD4 and PE-Foxp3. (A) Frequencies of peripheral and myocardial Tregs were shown, graph representative of three independent experiments. (B) Relative numbers of Treg frequency. Data. represent as mean ±SEM. *, P<0.05 compared to non-infected mice (wk0). N=5 mice per group (C) Myocardial Treg frequency and the relative collagen volume fraction (CVF) of heart tissue were parallelly shown in one graph. (D and E) 4 weeks after CVB3 infection, peripheral Treg frequency and CVF or collagen I mRNA in each mouse were analyzed and correlated.
Figure 3.
Mice Depleted of Tregs had aggravated cardiac fibrosis.
(A) Groups of 5 mice were treated with 8 doses of 0.1 mg anti-CD25 or isotype mAb via intraperitoneal injection during the course of CVB3 infection to deplete Tregs. The peripheral Treg frequency at wk 1 was determined to confirm the depletion effect. Graph representative of three independent experiments. Relative numbers of Treg frequency are represent as the mean ±SEM. (B) 4 wks after CVB3 infection, mRNA levels of collagen I, TIMP-1 and relative expression of TIMP-1/MMP-1 as well as TIMP-1/MMP-3 in the heart tissues were detected. Data are from one representative experiment of three performed ones and represent as the mean ±SEM. (C) Representative Picrosirius-red stained heart sections (magnification: 200×) and the collagen volume fraction (CVF) at week 4. Data represent the mean ± SEM. Arrows indicate myocardial interstitial and perivascular fibrosis. Individual experiments were performed three times with similar results. *, P<0.05 compared to mice receiving isotype Ab.
Figure 4.
Adoptive transfer of Tregs ameliorated cardiac fibrosis in CVB3-infected mice.
(A) Groups of 5 mice were transferred with 1×106 Tregs per mouse one day before CVB3 challenge via tail vein. The peripheral Treg frequency at wk 1 was determined. Graph representative of three independent experiments. Relative numbers of Treg frequency are represent as the mean ±SEM. (B) RT-PCR measurement of cardiac gene expression levels of collagen I and TIMP-1, TIMP-1/MMP-1 and TIMP-1/MMP-3 4 wks after CVB3 infection. Data are from one representative experiment of three performed ones and represent the mean ±SEM. (C) Picrosirius-red stained heart sections at week 4 (magnification: 200×) were shown and the collagen volume fraction (CVF) was evaluated. Arrows indicate myocardial interstitial and perivascular fibrosis. Individual experiments were performed three times with similar results. *, P<0.05 compared to non-transferred mice.
Figure 5.
IL-10 played an important role in Tregs-mediated inhibition of cardiac fibrosis.
(A) Groups of 10 mice were adoptively transferred with 1×106 Tregs one day before CVB3 infection. 1 hr after CVB3 infection, mice were injected with 100 μg of anti-IL-10 neutralizing mAb per mouse twice a week for 4 weeks. Representative Picrosirius-red stained heart sections 4 weeks after CVB3 infection (magnification: 200×) and the collagen volume fraction (CVF) was shown. Arrows indicate myocardial interstitial and perivascular fibrosis. Data represent the mean ± SEM. *, Individual experiments were performed three times. P<0.05 compared to mice given isotype Ab after Treg transfer. (B) RT-PCR measurement of cardiac fibroblast gene expression levels of collagen I and TIMP-1 24 hours after isolated Tregs were co-cultured with cardiac fibroblasts (CFs) at different cell ratios (1:1, 1:2.5, 1:5). Data are from one representative experiment of three performed ones and represent as the mean ±SEM. *, P<0.05 compared to cardiac fibroblasts culture alone. (C) The relative expression of collagen I and TIMP-1 after anti-IL-10 mAb was added in the co-culture system. Data represent the mean ± SEM, representative of three independent experiments. *, P<0.05, compared to co-culture system with no IL-10.