Blood-derived NK cells modulate the calcification process in primary cultured human fibroblasts

Natural killer (NK) cells are lymphoid cells that exert cytotoxic activity against virally infected or malignant cells. Although NK cells have been identified within mineralized tissues,1,2 their potential pathophysiological or compensatory role in calcification remains largely unclear. Using validated pro-calcific models,3 peripheral blood-derived human NK cells were previously found neither to undergo calcification, as demonstrated by ultrastructural analyses, nor to exhibit significant phenotypic changes, as revealed by flow cytometric assays, even when exposed to high, metastatic-like concentrations of inorganic phosphate. To assess whether NK cells might influence cell calcification, primary co-cultures of blood-derived human NK cells and human fibroblasts isolated from abdominal connective tissue were established and treated for up to 8 days with a pro-calcific medium simulating metastatic calcification. Two different co-culture conditions were tested: one in which NK cells were added only at the beginning of stimulation, and another in which NK cells were added every three days, coinciding with medium renewal. Additional pro-calcific cultures of fibroblasts alone, as well as untreated cultures of both fibroblasts and NK cells alone, were also prepared. Cultures were analysed by transmission electron microscopy to evaluate the extent of calcification, alongside flow cytometric analyses to assess the phenotypic features of NK cells. Compared to fibroblasts cultured alone in pro-calcific conditions, those co-cultured with NK cells exhibited fewer degenerative changes, with some cells showing features similar to untreated fibroblasts. The protective effects of co-culturing were more pronounced when NK cells were added repeatedly throughout the treatment period. Flow cytometric analyses revealed that, in pro-calcific co-cultures, a higher proportion of NK cells retained elevated expression of the activating receptor CD16 and upregulated NKp46, one of the main receptors triggering NK cell-mediated cytotoxicity, compared to control conditions. These preliminary data suggest that, in vivo, blood NK cells might exert a protective effect against cell calcification, as previously observed for mineralized vascular smooth muscle cells in a mouse model of hypoxic pulmonary hypertension.4 1) Morishima T, et al. Eur J Neurol 2002; 9:521-5. 2) Bobryshev YV and Lord RSA. Atherosclerosis 2005; 180:423-7. 3) Bonetti A, et al. Anat. Rec 2012; 295:1117-27. 4) Mao M, et al. Cell Death Dis 2018; 9:221.