LDL cholesterol modulates human CD34+ HSPCs through effects on proliferation and the IL-17 G-CSF axis

Abstract

Background: Hypercholesterolemia plays a critical role in atherosclerosis. CD34+ CD45dim Lineage- hematopoietic stem/progenitor cells (HSPCs) give rise to the inflammatory cells linked to atherosclerosis. In mice, high cholesterol levels mobilize HSPCs into the bloodstream, and promote their differentiation to granulocytes and monocytes. The objective of our study was to determine how cholesterol levels affect HSPC quantity in humans.

Methods: We performed a blinded, randomized hypothesis generating study in human subjects (n=12) treated sequentially with statins of differing potencies to vary lipid levels. CD34+ HSPC levels in blood were measured by flow cytometry. Hematopoietic colony forming assays confirmed the CD34+ population studied as HSPCs with multlineage differentiation potential. Mobilizing cytokine levels were measured by ELISA.

Results: The quantity of HSPCs was 0.15 ± 0.1% of buffy coat leukocytes. We found a weak, positive correlation between CD34+ HSPCs and both total and LDL cholesterol levels (r(2)=0.096, p < 0.025). Additionally, we tested whether cholesterol modulates CD34+ HSPCs through direct effects or on the levels of mobilizing cytokines. LDL cholesterol increased cell surface expression of CXCR4, G-CSFR affecting HSPC migration, and CD47 mediating protection from phagocytosis by immune cells. LDL cholesterol also increased proliferation of CD34+ HSPCs (28 ± 5.7%, n=6, p < 0.03). Finally, the HSPC mobilizing cytokine G-CSF (r(2)=0.0683, p < 0.05), and its upstream regulator IL-17 (r(2)=0.0891, p < 0.05) both correlated positively with LDL cholesterol, while SDF-1 levels were not significantly affected.

Conclusions: Our findings support a model where LDL cholesterol levels positively correlate with CD34+ HSPC levels in humans through effects on the levels of G-CSF via IL-17 promoting mobilization of HSPCs, and by direct effects of LDL cholesterol on HSPC proliferation. The findings are provocative of further study to determine if HSPCs, like cholesterol levels, are linked to CVD events.

Figure 1. Gating Strategy and Differentiation of CD34+ Lineage- CD45dim Cells from Peripheral Blood Mononuclear Cells.

Panel A: Representative flow cytometry plots and gating strategy to quantify CD34+ Lineage- CD45dim cells in human blood. CD34 CD45 or CD34 and Lineage cocktail plots (Parent Plots) were then gated to exclude dead cells (R1 gate), and positively select CD34+ cells low in side scatter (R2 gate). Panel B: Sorted CD34+ Lineage- CD45dim cells were differentiated in methylcellulose with cytokines to granulocyte (1), erythroblast (2), and monocyte colonies(3).

Figure 2. Correlation of LDL Cholesterol Levels and CD34+ HSPCs.

LDL cholesterol and CD34+ HSPC numbers quantified across all subjects at baseline and after all statin treatments. p ≤ 0.025 determined by Pearson’s correlation.

Figure 3. Effects of LDL Cholesterol of CD34+ HSPC proliferation and Surface Receptor Expression.

Panel A: Sorted CD34+ HSPCs (n=6 individuals) were expanded in serum free medium with cytokines and Stemregenin 1, then exposed to no treatment or LDL cholesterol (100 ug/mL) for 48 hours and CD34+ Lin- low SSC cells were quantified by flow cytometry. * p < 0.03 vs. untreated cells. Panel B: Mean fluorescence intensity of CXCR4, CD47, GCSFR, VLA4 and CD34 determined from sorted CD34+ HSPCs after expansion either no treatment or LDL cholesterol (100 ug/mL); n=6 subjects, * p < 0.05.

Figure 4. Effect of LDL cholesterol on Granulocyte and Monocyte Lineage Differentiation of CD34+ HSPCs.

Sorted CD34+ HSPCs were plated in methylcellulose with cytokines with or without LDL cholesterol. Granulocyte Monocyte colonies were quantified by microscopy after 14 days in culture. n=6 subjects, * p < 0.05 vs. no LDL cholesterol, ** p < 0.05 vs. LDL cholesterol (100ng/mL).

Figure 5. Correlation Between CD34+ HSPCs and G-CSF Levels.

LDL cholesterol and G-CSF levels were determined across all subjects at baseline and after all statin treatments. p < 0.025 using Pearson’s correlation.

Figure 6. Correlation Between LDL Cholesterol and G-CSF Levels.

LDL cholesterol and G-CSF levels were determined across all subjects at baseline and after all statin treatments. p < 0.05 using Pearson’s correlation.

Figure 7. Correlation Between LDL Cholesterol and IL-17 Levels.

LDL cholesterol and IL-17 levels were determined across all subjects at baseline and after all statin treatments. p < 0.05 using Pearson’s correlation.