Ultrastructural and cytofluorimetric analyses of blood-derived human NK cells cultured under metastatic-calcification-like conditions

Natural killer (NK) cells are innate lymphoid cells involved in the immune response against virally infected or malignant cells, playing a regulatory role in adaptive immunity in both physiological and pathological conditions. NK cells are also reported to infiltrate both atherosclerotic plaques and mineralizing aortic valve leaflets1, but whether they play a pathophysiological or compensatory role in cardiovascular calcific diseases still needs more elucidation. Here, propensity of peripheral blood human NK cells to undergo mineralization was assayed culturing cells up to 8 days with a validated pro-calcific medium simulating metastatic calcification. Cells were examined by electron microscopy, for their morphological evaluation, and multiparametric flow cytometry, for the analysis of NK cell subsets as well as major HLA class I (NKG2A, KIRs) and non-HLA class I specific (i.e. NCRs) NK receptor expression. Ultrastructurally, NK cells not exposed to the pro-calcific milieu exhibited preserved organelles. Conversely, NK cells from pro-calcific cultures showed suffering signs, but appeared free from calcification. Actually, they showed not typical features of pro-calcific degeneration, such as accumulation of membrane-derived lipid material and its layering at cell edges, as previously described for mineralizing valve interstitial cells cultured under the same pro-calcific conditions2,3. Cytofluorimetric analyses showed that treatment with the pro-calcific medium reduced NK cell counts, but without altering the ratio between cell subsets (CD56brightCD16dim/- and CD56dimCD16+) and the expression of the assayed HLA receptors, compared to NK cells cultured without the pro-calcific medium. Moreover, the pro-calcific treatment seemed to slightly upregulate the NCR NKp44, without significantly affecting the expression of the other NCRs NKp30 and NKp46. Overall, these preliminary data revealed that NK cells do not undergo mineralization as well as significant phenotypic alterations even if cultured under severe pro-calcific conditions. Further investigations are ongoing to improve knowledge on possible involvement of NK cells in calcification processes. References: 1) Passos, L.S.A.; Lupieri, A.; Becker-Greene, D.; Aikawa E. Innate and adaptive immunity in cardiovascular calcification. Atherosclerosis 2020, 306, 59-67. 2) Bonetti, A.; Della Mora, A.; Contin, M.; Tubaro, F.; Marchini, M.; Ortolani, F. Ultrastructural and spectrophotometric study on the effects of putative triggers on aortic valve interstitial cells in in vitro models simulating metastatic calcification. Anat. Rec. 2012, 295, 1117-1127. 3) Bonetti, A.; Allegri, L.; Baldan, F.; Contin, M.; Battistella, C.; Damante, G.; Marchini, M.; Ortolani, F. Critical involvement of calcium-dependent cytosolic phospholipase A2 in aortic valve interstitial cell calcification. Int. J. Mol. Sci. 2020, 21, 6398.