Gonadectomy of male BALB/c mice increases Tim-3(+) alternatively activated M2 macrophages, Tim-3(+) T cells, Th2 cells and Treg in the heart during acute coxsackievirus-induced myocarditis

Abstract

The incidence of cardiovascular disease, including inflammatory heart diseases like myocarditis, is increased in men. Similarly, male BALB/c mice infected with coxsackievirus B3 (CVB3) develop more severe acute inflammation in the heart compared to females. To better understand the effect of male sex hormones on cardiac inflammation, we gonadectomized (Gdx) male BALB/c mice and examined acute CVB3-induced myocarditis compared to sham controls. Viral replication in the heart was not significantly altered between Gdx and sham mice. However, gonadectomy significantly reduced testosterone levels and inflammation in the heart. FACS analysis of cell populations isolated from the heart revealed that CD11b(+) cells were significantly reduced in Gdx males. However, a GR1(+)F4/80(+) subset of CD11b(+) cells was significantly increased. Because this subset also expressed the interleukin (IL)-4R and IL-10, we refer to these cells as "alternatively activated" or M2 macrophages. A greater percentage of M2 macrophages in Gdx males expressed the inhibitory receptor Tim-3, while fewer expressed IL-1beta and IL-10. Only M2 macrophages upregulated TLR4 and Tim-3, whereas GR1(-)IL-4R(lo) macrophages did not. Additionally, IL-4(+)CD4(+) Th2 cells, Foxp3(+) regulatory T (Treg) cells and Tim-3(+)CD4(+) T cells were significantly increased in the heart following Gdx. Thus, we report for the first time that the inhibitory receptor Tim-3 is expressed on M2 macrophages. Our findings show that sex hormones and/or other mediators released from the testes inhibit anti-inflammatory populations in the heart including Tim-3(+) M2, Tim-3(+)CD4(+) T cells, Th2 and Treg resulting in more severe acute cardiac inflammation in males following CVB3 infection.

Fig. 1

Gonadectomy decreases testosterone levels during acute CVB3-induced myocarditis. Male BALB/c mice were infected with 10³ PFU CVB3 ip on day 0, after recovery from gonadectomy (Gdx) or sham operation, and testosterone levels in the sera (A) or heart (B) were measured by ELISA at day 12 pi. Similar results were obtained in 3 separate experiments. Data show the mean ±SEM of 7 to 10 mice/ group. *, p < 0.05.

Fig. 2

Gonadectomy decreases acute inflammation in the heart. Male BALB/c mice were infected with 10³ PFU CVB3 ip on day 0, after recovery from gonadectomy (Gdx) or sham operation, and hearts collected on day 12 pi during acute myocarditis. Myocardial sections were stained with H&E to detect inflammation and assessed as the percentage of the heart section with inflammation compared to the overall size of the heart section (A). Representative histology sections are shown for sham (B) and Gdx (C) males (original magnification ×64). Data show the mean ±SEM of 31 mice/ group combining 4 separate experiments. Myocarditis was decreased in all 4 independent experiments, using 7 to 10 mice/ group. *** p <0.001.

Fig. 3

Viral replication is not decreased by gonadectomy. Sham or gonadectomized (Gdx) males were infected with 10³ PFU CVB3 ip on day 0 and compared for the level of viral replication in the heart at day 12 pi, during acute myocarditis. Data are presented as the mean PFU/ g heart ±SEM of 7 to 10 mice/ group. One representative example of three separate experiments is shown, p = 0.57.

Fig. 4

Males develop significantly reduced CD45⁺ and CD11b⁺ inflammation in the heart following gonadectomy. Sham or gonadectomized (Gdx) males were infected with 10³ PFU CVB3 ip on day 0 and compared by FACS analysis on day 10 pi during acute myocarditis for the percentage of CD45⁺ leukocytes compared to total heart cells (A and B) or the relative proportion of individual immune cells (C and D). The following cell types were evaluated by FACS (D): macrophages/ neutrophils/ MCs/ DCs (CD11b), B cells (B220), CD4⁺ T cells (CD4), CD8⁺ T cells (CD8), and MCs (CD117). One representative sham (light line/ red) and Gdx (heavy line/ blue) are compared by FACS for CD45 (B) or CD11b (C) expression within the gated region. Isotype controls are depicted as shaded grey (B and C). Similar results were obtained in 2 separate experiments. Data show the mean ±SEM of 10 pooled samples/ group. Each pooled sample consists of cells from 3 mice for a total of 30 mice/ group. **, p < 0.01.

Fig. 5

Gonadectomy increases Th2, Tim-3⁺ and CTLA-4⁺ T cells in the heart of males. Mice received 10³ PFU CVB3 ip on day 0 after recovery from operations and CD45⁺ immune cells were isolated from the heart on day 10 pi, during acute myocarditis. FACS analysis depicts expression of Tim-3, CTLA-4 (intracellular) or Foxp3 (intracellular) on/ in CD4⁺ T cells (A). Intracellular levels of IL-4 (Th2), IFN-γ (Th1) or IL-17 (Th17) were assessed in CD4⁺ T cells after stimulation for 4 hours with a leukocyte activation cocktail of PMA, Ionomycin and Brefeldin A (B). Similar results were obtained in 2 separate experiments. Data show the mean ±SEM of 5 pooled samples/ group. Each pooled sample consists of cells from 3 mice for a total of 15 mice/ group. *, p < 0.05; **, p ≤ 0.01.

Fig. 6

Gonadectomy reduces IL-1β and IL-10 levels in the heart while increasing IL-4 during acute myocarditis. Sham or gonadectomized (Gdx) males were infected with 10³ PFU CVB3 ip on day 0 and compared for cytokines levels in the heart at day 12 pi by ELISA. Data are presented as the mean pg cytokines/ g heart ±SEM of 7 to 10 mice/ group. One representative example of four separate experiments is shown. *, p < 0.05; **, p < 0.01.

Fig. 7

Gonadectomy decreases GR1⁻F4/80⁺ M1 macrophages (A) and increases GR1⁺F4/80⁺ M2 macrophages (B and C) in the heart of males. Mice received 10³ PFU CVB3 ip on day 0 and CD45⁺ immune cells were isolated from the heart on day 10 pi, during acute myocarditis. An example of the shift in M2 population in sham (middle) vs. Gdx (right) and isotype control (left) is shown in (C). Similar results were obtained in 2 separate experiments. Data show the mean ±SEM of 5 pooled samples/ group. Each pooled sample consists of cells from 3 mice for a total of 15 mice/ group. *, p < 0.05.

Fig. 8

Gonadectomy increases Tim-3 expression and decreases IL-1β/ IL-10 expression on/ in GR1⁺F4/80⁺ M2 macrophages during acute myocarditis. Mice received 10³ PFU CVB3 ip on day 0 and CD45⁺ immune cells were isolated from the heart on day 10 pi, during acute myocarditis. Expression levels of TLR4, Tim-3, IL-4R, IL-1β and IL-10 are shown for GR1⁻F4/80⁺ M1 (A) and GR1⁺F4/80⁺ M2 (B) macrophages. Similar results were obtained in 2 separate experiments. Data show the mean ±SEM of 5 pooled samples/ group. Each pooled sample consists of cells from 3 mice for a total of 15 mice/group. *, p < 0.05.

Fig. 9

Sex hormones or other mediators released from the testes (male gonads) increase inflammation in the heart following CVB3 infection by increasing proinflammatory GR1⁻F4/80⁺ M1 and IL-1β⁺IL-10⁺ M2 macrophage populations, and by inhibiting regulatory populations like Tim-3⁺ M2 macrophages, Tim-3⁺ T cells, Th2 cells and Treg during acute CVB3-induced myocarditis.