Coxsackievirus B3 induction of NFAT: requirement for myocarditis susceptibility

Abstract

Ultraviolet (u.v.) inactivated coxsackievirus B3 (CVB3) induces rapid calcium flux in naïve BALB/c CD4+ T cells. CD4+ cells lacking decay accelerating factor (DAF-/-) show little calcium flux indicating that virus cross-linking of this virus receptor protein is necessary for calcium signaling in CVB3 infection. Interaction of CVB3 with CD4+ cells also activates NFAT DNA binding. To show that NFAT activation is crucial to CVB3 induced disease, wild-type mice and transgenic mice expressing dominant-negative NFAT (dnNFAT) mutant in T cells were infected and evaluated for myocarditis and pancreatitis 7 days later. Inhibition of NFAT in T cells prevented myocarditis but had no effect on pancreatitis. Virus titers in pancreas were equivalent in wild-type and dnNFAT animals but cardiac virus titers were increased in dnNFAT mice. Interferon-gamma (IFN gamma) expression was reduced in both CD4+ and V gamma 4+ T cells from dnNFAT mice compared to controls. FasL expression by V gamma 4+ cells was also suppressed. Inhibition of FasL expression by V gamma 4+ cells is consistent with myocarditis protection in dnNFAT mice.

Figure 1

Calcium flux on mesenteric lymph node cells. Enriched CD4+ lymphocytes were isolated from naïve BALB/c and BALB/c DAF-/- mice and loaded with Indo-1. Basal levels of calcium flux were determined on unstimulated cells, then 4 × 10⁸ PFU equivalent of u.v. inactivated H3 virus was added and calcium flux determination was continued for the time indicated. As a positive control, ionomycin at a concentration of 250 ng/ml was added to the cells to demonstrate maximum flux.

Figure 2

H3 virus induces NFAT activation. (A) CD4+ T cells were treated with medium (M), inactivated H3 (H3) or PMA (5 ng/ml) and ionomycin (250 ng/ml) (PI) for 6 or 12 h. Nuclear extracts were generated and examined by EMSA using an oligo containing consensus NFAT site. (B) CD4+ T cells were activated for 6 and 12 h with H3 virus. Nuclear extracts were generated and examined by EMSA for NFAT DNA binding. Binding reactions were performed in the absence (-) or presence or an anti-NFATc1 (c1) or anti-NFATc2 (c2) antibodies. (C) CD4+ T cells from BALB/c and BALB/c DAF-/- mice were activated for 12 h with u.v. inactivated H3 virus, 250 ng/ml ionomycin (Ion) or 0.1 ml Hela cell extract then nuclear extracts were examined by EMSA for NFAT binding.

Figure 3

Hearts and pancreases from B10.BR and dnNFAT B10.BR male mice infected 7 days earlier with 10⁵ PFU H3 virus were formalin fixed, sectioned and stained with hematoxylin and eosin. Arrow indicates inflammation.

Figure 4

B10.BR and dnB10.BR mice were infected 7 days earlier with 10⁵ PFU H3 virus. (A) and (B) Heart and pancreas were titered for virus. Data is presented as log₁₀ PFU/mg tissue. (C) and (D) Histology score for inflammation.

Figure 5

Splenocytes were isolated and labeled with antibodies to Vγ4, FasL and intracellularly for IFNγ. Cells were evaluated by flow cytometry for percent of splenic lymphocytes positive for these markers. Data represents the mean ± SEM for 5-8 individual mice/group. * dnNFAT mice significantly differ from B10.BR animals at p≤0.05.

Figure 6

Flow cytometry on splenic lymphocytes from infected mice. Cells were labeled with antibodies to Vγ4, FasL and CD4. Some cells were intracellularly labled with antibody to IFNγ. Numbers in upper right indicate percent of cells in each quadrant.