Contributions
Abstract: S300
Type: Oral Presentation
Session title: Dilemmas in coagulation and thrombosis
Background
Recent epidemiological studies showed the significant association between cardiovascular diseases (CVD) and clonal hematopoiesis of indeterminate potential (CHIP). CHIP is defined as the clonal expansion of blood cells driven by somatic mutations in hematopoietic genes, such as DNMT3A, ASXL1, TET2, or JAK2. Previous studies have shown that TET2-, DNMT3A- and JAK2-driven CHIP in fact promote atherosclerosis or worsen heart failure using mice models. In contrast, whether and how ASXL1 mutations contribute to atherosclerosis remains unknown despite the robust clinical association between mutant ASXL1-driven CHIP and CVD.
Aims
We aim to investigate the crosstalk between hematopoiesis with mutant ASXL1 and CVDs.
Methods
We previously established knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT). Using these mice, we assessed if the high-fat diet (HFD) affects the reconstitution and differentiation of hematopoietic cells with ASXL1-MT. We also assessed the role of hematopoietic cells derived from ASXL1-MT KI mice in the development of atherosclerotic CVDs using the atherosclerosis-prone Ldlr-/- mice. To study the mechanisms underlying the promotive effect of ASXL1 mutation on atherosclerosis, we used mouse and human cell lines (RAW264.7 cells, 293T cells), BaF/3 cells expressing Toll-like receptor (TLR), and bone marrow-derived macrophages derived from the ASXL1-MT KI mice.
Results
The Ldlr-/- mice transplanted with bone marrow from ASXL1-MT KI mice showed significantly larger atherosclerotic lesions in the aorta and aortic valves than the control mice. We also found that HFD conferred growth advantage to ASXL1-MT cells over wild-type cells, particularly in the population of the inflammatory monocytes. Consistent with these findings, RNA-seq of the plaque-macrophages derived from ASXL1-MT KI mice showed more inflammatory signatures than those from control mice. Thus, these data suggest that mutant ASXL1 promotes HFD-induced expansion of inflammatory monocytes and macrophages, thereby accelerates atherosclerosis.
Mechanistically, we found that wild-type and mutant ASXL1 have different effects on the innate immune pathway. NF-κB reporter assay revealed that wild-type ASXL1 suppressed, while mutant ASXL1 promoted, the reporter activation induced by the TLR stimulation. Furthermore, we found that wild-type ASXL1, but not mutant ASXL1, suppresses the activation of TAK1 in TLR-NF-κB signaling pathway through direct interaction with IRAK1 and TRAF6. These results suggest that ASXL1 inhibits the innate immune pathway through interactions with IRAK1/TRAF6/TAK1 in myeloid cells, while mutant ASXL1 loses this regulatory function. Finally, we assessed the effect of IRAK1/4 inhibitor on atherosclerosis in HFD-fed ASXL1-MT mice. As expected, IRAK1/4 inhibition decreased the number of inflammatory monocytes and attenuated the development of atherosclerosis driven by ASXL1-MT.
Conclusion
Our study demonstrated the causative role of the CHIP-associated ASXL1 mutation to promote atherosclerosis. Inhibition of the innate immune pathway may prevent the development of CVD associated with CHIP harboring ASXL1 mutations.
Keyword(s): Atherosclerosis, Hematopoiesis, Inflammation, Macrophage
Abstract: S300
Type: Oral Presentation
Session title: Dilemmas in coagulation and thrombosis
Background
Recent epidemiological studies showed the significant association between cardiovascular diseases (CVD) and clonal hematopoiesis of indeterminate potential (CHIP). CHIP is defined as the clonal expansion of blood cells driven by somatic mutations in hematopoietic genes, such as DNMT3A, ASXL1, TET2, or JAK2. Previous studies have shown that TET2-, DNMT3A- and JAK2-driven CHIP in fact promote atherosclerosis or worsen heart failure using mice models. In contrast, whether and how ASXL1 mutations contribute to atherosclerosis remains unknown despite the robust clinical association between mutant ASXL1-driven CHIP and CVD.
Aims
We aim to investigate the crosstalk between hematopoiesis with mutant ASXL1 and CVDs.
Methods
We previously established knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT). Using these mice, we assessed if the high-fat diet (HFD) affects the reconstitution and differentiation of hematopoietic cells with ASXL1-MT. We also assessed the role of hematopoietic cells derived from ASXL1-MT KI mice in the development of atherosclerotic CVDs using the atherosclerosis-prone Ldlr-/- mice. To study the mechanisms underlying the promotive effect of ASXL1 mutation on atherosclerosis, we used mouse and human cell lines (RAW264.7 cells, 293T cells), BaF/3 cells expressing Toll-like receptor (TLR), and bone marrow-derived macrophages derived from the ASXL1-MT KI mice.
Results
The Ldlr-/- mice transplanted with bone marrow from ASXL1-MT KI mice showed significantly larger atherosclerotic lesions in the aorta and aortic valves than the control mice. We also found that HFD conferred growth advantage to ASXL1-MT cells over wild-type cells, particularly in the population of the inflammatory monocytes. Consistent with these findings, RNA-seq of the plaque-macrophages derived from ASXL1-MT KI mice showed more inflammatory signatures than those from control mice. Thus, these data suggest that mutant ASXL1 promotes HFD-induced expansion of inflammatory monocytes and macrophages, thereby accelerates atherosclerosis.
Mechanistically, we found that wild-type and mutant ASXL1 have different effects on the innate immune pathway. NF-κB reporter assay revealed that wild-type ASXL1 suppressed, while mutant ASXL1 promoted, the reporter activation induced by the TLR stimulation. Furthermore, we found that wild-type ASXL1, but not mutant ASXL1, suppresses the activation of TAK1 in TLR-NF-κB signaling pathway through direct interaction with IRAK1 and TRAF6. These results suggest that ASXL1 inhibits the innate immune pathway through interactions with IRAK1/TRAF6/TAK1 in myeloid cells, while mutant ASXL1 loses this regulatory function. Finally, we assessed the effect of IRAK1/4 inhibitor on atherosclerosis in HFD-fed ASXL1-MT mice. As expected, IRAK1/4 inhibition decreased the number of inflammatory monocytes and attenuated the development of atherosclerosis driven by ASXL1-MT.
Conclusion
Our study demonstrated the causative role of the CHIP-associated ASXL1 mutation to promote atherosclerosis. Inhibition of the innate immune pathway may prevent the development of CVD associated with CHIP harboring ASXL1 mutations.
Keyword(s): Atherosclerosis, Hematopoiesis, Inflammation, Macrophage