Regulatory T-cells modulate ectopic calcification in nephrocalcinosis and dystrophic cardiac calcinosis

Abstract

Purpose: Ectopic calcification is often associated with soft tissue inflammation and is seen in a multitude of clinical settings such as chronic renal failure and primary hyperparathyroidism. To further evaluate the specific pathophysiological role of T cells in nephrocalcinosis and dystrophic cardiac calcinosis, we used DBA/2 mice that have a natural splice variant in the ABCC6 gene and are prone to develop ectopic calcifications under high-phosphate diet. Methods: Female DBA/2 mice were depleted of T cells (n=10) or regulatory T-cells (Tregs) (n=15) using either an anti-CD3ɛ or an anti-CD25 monoclonal antibody, and were compared to isotype-treated controls, respectively. After this immunomodulation, the DBA/2 mice were set on a high-phosphate diet for 9 days and the degree of calcification was assessed by micro-computed tomography. Successful depletion was confirmed by flow cytometry of splenocytes. Moreover, we registered continuous telemetric ECG readings (n=5) to monitor the electrophysiological impact of ectopic calcification on heart rate and mortality. Results: In DBA/2 mice, high-phosphate diet induced a phenotype of and nephrocalcinosis and dystrophic cardiac calcinosis. T-cell depletion significantly increased renal calcification in micro-computed tomography (p=0.022). Concordantly, Treg depletion significantly deteriorated nephrocalcinosis (p=0.039) and was associated with significantly increased serum FGF23 levels (p=0.005). Semi-quantitative histopathological evaluations with Alizarin Red stainings independently confirmed the respective radiological measurements. Most importantly, Treg depletion was also associated with a significantly increased mortality rate (p=0.02). Immunomodulation had no impact on the absolute amount of cardiac calcification. However, continuous telemetric ECG readings revealed negative chronotropic and negative dromotropic effects in mice suffering from dystrophic cardiac calcinosis that ultimately led to atrioventricular conduction block, cardiac arrest and death. Conclusion: In summary, our data suggest a pivotal role of T-cells and particularly regulatory T cells in the progression of ectopic calcification, and emphasize the fact that soft-tissue inflammation deteriorates the outcome in nephrocalcinosis and dystrophic cardiac calcinosis.