Abstract Background Differential leukocyte counts are usually measured based on cellular morphology or surface marker expression. It has recently been shown that leukocyte counts can also be determined by cell-type–specific DNA methylation (DNAm). Such epigenetic leukocyte counting is applicable to small blood volumes and even frozen material, but for clinical translation, the method needs to be further refined and validated. Methods We further optimized and validated targeted DNAm assays for leukocyte deconvolution using 332 venous and 122 capillary blood samples from healthy donors. In addition, we tested 36 samples from ring trials and venous blood from 266 patients diagnosed with different hematological diseases. Deconvolution of cell types was determined with various models using DNAm values obtained by pyrosequencing or digital droplet PCR (ddPCR). Results Relative leukocyte quantification correlated with conventional blood counts for granulocytes, lymphocytes, B cells, T cells (CD4 or CD8), natural killer cells, and monocytes with pyrosequencing (r = 0.84; r = 0.82; r = 0.58; r = 0.50; r = 0.70; r = 0.61; and r = 0.59, respectively) and ddPCR measurements (r = 0.65; r = 0.79; r = 0.56; r = 0.57; r = 0.75; r = 0.49; and r = 0.46, respectively). In some patients, particularly with hematopoietic malignancies, we observed outliers in epigenetic leukocyte counts, which could be discerned if relative proportions of leukocyte subsets did not sum up to 100%. Furthermore, absolute quantification was obtained by spiking blood samples with a reference plasmid of known copy number. Conclusions Targeted DNAm analysis by pyrosequencing or ddPCR is a valid alternative to quantify leukocyte subsets, but some assays require further optimization.
Einleitung: Die Osteoimmunologie befasst sich mit der Interaktion zwischen dem Immunsystem und Knochenstoffwechsel. Zytokine, wie beispielsweise Interleukin-8 (IL-8), sind wichtige Regulatoren der Osteoblasten- und Osteoklastenaktivität und spielen wahrscheinlich eine entscheidende Rolle bei Knochenstoffwechselerkrankungen wie der Osteoporose. Es ist bekannt, dass das Chemokin IL-8 einerseits die Osteoklastogenese positiv beeinflussen kann und andererseits die Knochenbildung in vivo unterstützt. In vorherigen Publikationen haben wir bereits gezeigt, dass IL-8 auch in vitro die Mineralisierung primärer humaner Osteoblasten (OB) förderte. In dieser Studie konzentrierten wir uns auf die genauere Funktion von IL-8 in der Osteogenese.
Abstract Background Cell-type specific DNA methylation (DNAm) can be employed to determine the numbers of leukocyte subsets in blood. In contrast to conventional methods for leukocyte counts, which are based on cellular morphology or surface marker protein expression, the cellular deconvolution based on DNAm levels is applicable for frozen or dried blood. Here, we further enhanced targeted DNAm assays for leukocyte counts in clinical application. Methods DNAm profiles of 40 different studies were compiled to identify CG dinucleotides (CpGs) with cell-type specific DNAm using a computational framework, CimpleG. DNAm levels at these CpGs were then measured with digital droplet PCR in venous blood from 160 healthy donors and 150 patients with various hematological disorders. Deconvolution was further validated with venous blood (n = 75) and capillary blood (n = 31) that was dried on Whatman paper or on Mitra microsampling devices. Results In venous blood, automated cell counting or flow cytometry correlated well with epigenetic estimates of relative leukocyte counts for granulocytes (r = 0.95), lymphocytes (r = 0.97), monocytes (r = 0.82), CD4 T cells (r = 0.84), CD8 T cells (r = 0.94), B cells (r = 0.96), and NK cells (r = 0.72). Similar correlations and precisions were achieved for dried blood samples. Spike-in with a reference plasmid enabled accurate epigenetic estimation of absolute leukocyte counts from dried blood samples, correlating with conventional venous (r = 0.86) and capillary (r = 0.80) blood measurements. Conclusions The advanced selection of cell-type specific CpGs and utilization of digital droplet PCR analysis provided accurate epigenetic blood counts. Analysis of dried blood facilitates self-sampling with a finger prick, thereby enabling easier accessibility to testing.
Abstract Myeloproliferative neoplasms (MPNs) are caused by somatic driver mutations, such as JAK2 V617F , which might also affect cellular aging and senescence. Here, we analyzed the heterogeneity of aging in MPN patients and if this can be used to specifically target malignant cells. The mean epigenetic age was significantly accelerated in 129 MPN patients across all disease-entities, whereas premature telomere attrition was particularly observed in primary myelofibrosis. Overall, accelerated cellular aging correlated with JAK2 V617F allele frequency and was more pronounced in colony forming cells with JAK2 V617F as compared to JAK2 wild- type colonies. JAK2 V617F mutation did not evoke clear acceleration of aging in syngeneic iPSC models upon short-term hematopoietic differentiation. On the other hand, a murine Jak2 V617F model revealed epigenetic age-acceleration that therefore appears as sequel of disease progression. To investigate if the malignant clone might be targeted, we tested eight senolytic compounds, of which JQ1 and piperlongumine showed a reduction in allele burden and an increase in telomere length. Notably, treatment with the telomerase inhibitor BIBR-1532 reduced mutated colonies, particularly in patients with preexisting short telomeres. Our results indicate that cellular aging is accelerated in malignant MPN clones and this can provide a target for treatment with senolytic drugs or telomerase inhibitors.
