Gene profile of myeloid-derived suppressive cells from the bone marrow of lysosomal acid lipase knock-out mice

Abstract

Background: Lysosomal acid lipase (LAL) controls development and homeostasis of myeloid lineage cells. Loss of the lysosomal acid lipase (LAL) function leads to expansion of myeloid-derived suppressive cells (MDSCs) that cause myeloproliferative neoplasm.

Methodology/principal findings: Affymetrix GeneChip microarray analysis identified detailed intrinsic defects in Ly6G(+) myeloid lineage cells of LAL knock-out (lal-/-) mice. Ingenuity Pathway Analysis revealed activation of the mammalian target of rapamycin (mTOR) signaling, which functions as a nutrient/energy/redox sensor, and controls cell growth, cell cycle entry, cell survival, and cell motility. Loss of the LAL function led to major alteration of large GTPase and small GTPase signal transduction pathways. lal-/- Ly6G(+) myeloid cells in the bone marrow showed substantial increase of cell proliferation in association with up-regulation of cyclin and cyclin-dependent kinase (cdk) genes. The epigenetic microenvironment was significantly changed due to the increased expression of multiple histone cluster genes, centromere protein genes and chromosome modification genes. Gene expression of bioenergetic pathways, including glycolysis, aerobic glycolysis, mitochondrial oxidative phosphorylation, and respiratory chain proteins, was also increased, while the mitochondrial function was impaired in lal-/- Ly6G(+) myeloid cells. The concentration of reactive oxygen species (ROS) was significantly increased accompanied by up-regulation of nitric oxide/ROS production genes in these cells.

Conclusions/significance: This comprehensive gene profile study for the first time identifies and defines important gene pathways involved in the myeloid lineage cells towards MDSCs using lal-/- mouse model.

Figure 1. Affymetrix GeneChip microarray and Ingenuity Pathway analyses of lal−/− MDSCs.

A). Affymetrix GeneChip microarray analysis of Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice. Numbers represent percentages of changed genes in each category vs total changed genes between lal−/− mice and lal+/+ mice; B). Differential gene expression of Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice was analyzed by Ingenuity Pathway Analysis. Changed genes in the PPARγ pathway, cholesterol biosynthesis and mTOR signaling pathway are presented; C) Flow cytometry of mTOR downstream effectors S6 and 4E-BP1 in Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice. n = 4.

Figure 2. ROS Pathway Analysis of lal−/− bone marrow MDSCs.

A). Differential gene expression of CD11b⁺Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice was analyzed by Ingenuity Pathway Analysis. Changed genes in the nitric oxide and reactive oxygen species production genes are presented; B). ROS production in CD11b⁺Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice. The ROS signal was statistically analyzed by mean fluorescent intensity (MFI). Results are means of 4 independent FACS experiments. n = 4, **, p<0.01.

Figure 3. Cell cycle analysis of lal−/− bone marrow MDSCs.

A). Cell cycle analysis of CD11b⁺ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice; B). The G1/G0 phase, S phase and G2/M phase of CD11b⁺Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice were statistically analyzed. Results are means of 4 independent FACS experiments. n = 4, **, p<0.01, *, p<0.5.

Figure 4. ATP production and mitochondrial potential analyses of lal−/− bone marrow MDSCs.

A). ATP concentrations in Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice. Results are means of 4 independent experiments. n = 3, **, p<0.01, *, p<0.5; B). Mitochondrial membrane potential of CD11b⁺Ly6G⁺ MDSCs from the bone marrow of lal+/+ mice and lal−/− mice was analyzed by JC-1 staining. Positive staining of JC-1 red represents healthy mitochondria. Transition from negative staining of JC-1 red to positive staining of JC-1 green represents impaired mitochondrial potential.