Final Report Summary - MYCMACS (Role of c-Myc in atherosclerotic macrophages)
In the MycMac CIG – 303850 project I proposed to elucidate the role of c-Myc in macrophages involved in atherosclerosis in vitro and in vivo.
We first characterized the behaviour of wild-type macrophages in vitro when c-Myc activity is blocked using 1) a c-Myc chemical inhibitor or 2) sh-RNA against c-Myc to abrogated c-Myc expression. Macrophage proliferation was significantly reduced in both conditions, without affecting cell survival. In addition, uptake of acetylated-LDLs was slightly but significantly reduced in presence of a c-Myc chemical inhibitor.
We next wanted to study the role of c-Myc macrophages in vivo during atherosclerosis development. We characterized the c-Mycfl/fl LysMcre/+ C57BL/6 mice (Mac-cMyc-KO) carrying a myeloid cell-specific c-Myc inactivation. This new mouse model was useful to study the atherosclerotic behavior of c-Myc-lacking macrophages in vivo and in vitro.Compared with control counterparts with intact c-Myc (c-Mycfl/fl), macrophages from Mac-cMyc-KO mice showed lower c-Myc levels (70-100% lower across all analyzed mice), while expression of c-Myc was unaffected in other tissues such as liver, kidney and testis. In vitro, macrophages from Mac-cMyc-KO mice showed reduced proliferative activity.
Under steady-state conditions, Mac-cMyc-KO mice exhibited normal immune system parameters, including numbers of total bone marrow cells and bone marrow hematopoietic precursors. Moreover, Mac-cMyc-KO and control c-Mycfl/fl mice showed similar circulating cell counts, including total white blood cells, lymphocytes, neutrophils, classical- and non-classical monocytes, granulocytes, erythrocytes and platelets, as well as comparable numbers of similarly distributed infiltrated CD68+-macrophages in spleen, thymus and lymph nodes.
In a model of melanoma, however, tumor associated macrophages lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in Mac-cMyc-KO mice. Macrophage-specific c-Myc deletion also diminished fibrosarcoma growth.
Given that c-Myc is expressed in M2/anti-inflammatory macrophages, we hypothesized that genetic deletion of c-Myc in macrophages will block the acquisition of an anti-inflammatory/anti-atherosclerotic phenotype, leading to an increase in lesion size.
To investigate the role of c-Myc expression in the development of an anti-atherosclerotic macrophage response, we generated mice with macrophage-specific c-Myc deficiency in an apoE-KO atherosclerotic background by crossing c-Mycfl/fl LysMcre/+ mice with apoE-KO mice, yielding c-Mycfl/fl LysMcre/+ apoE-KO mice doubly deficient for c-Myc and apoE (Mac-cMyc apoE-DKO mice) and control c-Mycfl/fl apoE-KO littermates with intact c-Myc.
When animals were 2 months old, they were subsequently fed a high-cholesterol diet. Our results show that, compared with similarly treated control littermates, fat-fed Mac-c-Myc apoE-DKO mice mount a faster and stronger inflammatory response characterized by higher levels of IL-6-producing Ly6Chigh monocytes, neutrophilia and fibrin deposition.
Only after 3 weeks high cholesterol diet, both genotypes showed peripheral blood monocytosis. However, in Mac-c-Myc apoE-DKO mice we observed an unbalanced increase of different monocyte subsets, being mainly dominated by Ly6Chigh monocytes, which are higher producers of inflammatory cytokines (such IL-6) and chemokines (such Cxcl1).
Activation of the IL-6/Cxcl1 pathway has been related with higher inflammatory neutrophilia. We could observe higher numbers of neutrophils in Mac-c-Myc apoE-DKO mice peripheral blood after 3 weeks of high-cholesterol diet, compared with control littermates. In addition, higher neutrophil-associated fibrin deposits were identified in the hearts of these mice.
Fibrin/fibrinogen deposits are considered inflammatory markers of early atherosclerosis and could affect heart activity provoking even death. Consequently, high-cholesterol fed Mac-c-Myc apoE-DKO mice show compromised survival during 8-weeks high-cholesterol diet treatment and survivors showed a greater atherosclerotic burden in both aortic arch and thoracic aorta, compared with control littermates.
Overall, our studies demonstrate that c-Myc absence in macrophages leads to an inflammatory macrophage state that contributes to atherosclerosis development and suggest that increasing macrophage c-Myc expression has the potential to reduce atherosclerosis progression.
We first characterized the behaviour of wild-type macrophages in vitro when c-Myc activity is blocked using 1) a c-Myc chemical inhibitor or 2) sh-RNA against c-Myc to abrogated c-Myc expression. Macrophage proliferation was significantly reduced in both conditions, without affecting cell survival. In addition, uptake of acetylated-LDLs was slightly but significantly reduced in presence of a c-Myc chemical inhibitor.
We next wanted to study the role of c-Myc macrophages in vivo during atherosclerosis development. We characterized the c-Mycfl/fl LysMcre/+ C57BL/6 mice (Mac-cMyc-KO) carrying a myeloid cell-specific c-Myc inactivation. This new mouse model was useful to study the atherosclerotic behavior of c-Myc-lacking macrophages in vivo and in vitro.Compared with control counterparts with intact c-Myc (c-Mycfl/fl), macrophages from Mac-cMyc-KO mice showed lower c-Myc levels (70-100% lower across all analyzed mice), while expression of c-Myc was unaffected in other tissues such as liver, kidney and testis. In vitro, macrophages from Mac-cMyc-KO mice showed reduced proliferative activity.
Under steady-state conditions, Mac-cMyc-KO mice exhibited normal immune system parameters, including numbers of total bone marrow cells and bone marrow hematopoietic precursors. Moreover, Mac-cMyc-KO and control c-Mycfl/fl mice showed similar circulating cell counts, including total white blood cells, lymphocytes, neutrophils, classical- and non-classical monocytes, granulocytes, erythrocytes and platelets, as well as comparable numbers of similarly distributed infiltrated CD68+-macrophages in spleen, thymus and lymph nodes.
In a model of melanoma, however, tumor associated macrophages lacking c-Myc displayed a delay in maturation and showed an attenuation of pro-tumoral functions that was associated with impaired tissue remodeling and angiogenesis and limited tumor growth in Mac-cMyc-KO mice. Macrophage-specific c-Myc deletion also diminished fibrosarcoma growth.
Given that c-Myc is expressed in M2/anti-inflammatory macrophages, we hypothesized that genetic deletion of c-Myc in macrophages will block the acquisition of an anti-inflammatory/anti-atherosclerotic phenotype, leading to an increase in lesion size.
To investigate the role of c-Myc expression in the development of an anti-atherosclerotic macrophage response, we generated mice with macrophage-specific c-Myc deficiency in an apoE-KO atherosclerotic background by crossing c-Mycfl/fl LysMcre/+ mice with apoE-KO mice, yielding c-Mycfl/fl LysMcre/+ apoE-KO mice doubly deficient for c-Myc and apoE (Mac-cMyc apoE-DKO mice) and control c-Mycfl/fl apoE-KO littermates with intact c-Myc.
When animals were 2 months old, they were subsequently fed a high-cholesterol diet. Our results show that, compared with similarly treated control littermates, fat-fed Mac-c-Myc apoE-DKO mice mount a faster and stronger inflammatory response characterized by higher levels of IL-6-producing Ly6Chigh monocytes, neutrophilia and fibrin deposition.
Only after 3 weeks high cholesterol diet, both genotypes showed peripheral blood monocytosis. However, in Mac-c-Myc apoE-DKO mice we observed an unbalanced increase of different monocyte subsets, being mainly dominated by Ly6Chigh monocytes, which are higher producers of inflammatory cytokines (such IL-6) and chemokines (such Cxcl1).
Activation of the IL-6/Cxcl1 pathway has been related with higher inflammatory neutrophilia. We could observe higher numbers of neutrophils in Mac-c-Myc apoE-DKO mice peripheral blood after 3 weeks of high-cholesterol diet, compared with control littermates. In addition, higher neutrophil-associated fibrin deposits were identified in the hearts of these mice.
Fibrin/fibrinogen deposits are considered inflammatory markers of early atherosclerosis and could affect heart activity provoking even death. Consequently, high-cholesterol fed Mac-c-Myc apoE-DKO mice show compromised survival during 8-weeks high-cholesterol diet treatment and survivors showed a greater atherosclerotic burden in both aortic arch and thoracic aorta, compared with control littermates.
Overall, our studies demonstrate that c-Myc absence in macrophages leads to an inflammatory macrophage state that contributes to atherosclerosis development and suggest that increasing macrophage c-Myc expression has the potential to reduce atherosclerosis progression.