Taking AIM at Clonal Hematopoiesis-associated Atherosclerosis

Clonal hematopoiesis (CH) is defined as the presence of an expanded somatic blood-cell clone in persons without other hematologic abnormalities. The presence of CH increases with age and is usually present in 10 to 20 percent of people older than 70 years.¹  The most common myeloid mutations associated with CH are TET2, ASXL1, DNMT3A, and JAK2. Carriers of CH have an approximately twofold increased risk of atherosclerotic cardiovascular disease (CVD).^1,2  Mouse models of TET2 CH have shown increased macrophage inflammation and atherosclerosis, heightened IL-1β/IL-6 signaling, and also that inhibition of the NLRP3 inflammasome (that activates IL-1β, a key inducer of IL-6) ameliorates atherosclerosis.^1,3  These observations suggest that inhibiting the IL-6/IL-1β pathway may be effective in reducing CVD risk in humans with CH.^1,3  This is of particular interest as a trial of anti-IL-1ß (canakinumab) found up to 15 percent reduction in cardiovascular events; however, whether response was dependent on the presence of an underlying CH mutation is unknown. Furthermore, whether different myeloid mutations increase the risk of thrombosis through different mechanisms is also unknown.

JAK2 V617F mutation (JAK2^VF) –positive CH is associated with a particularly high risk of premature coronary artery disease. This is of particular relevance as JAK2^VF-driven myeloproliferative neoplasms are also associated with a significantly increased risk of thrombosis and vascular disease, which remains the major cause of morbidity and early mortality in these conditions (thrombosis risk, 2.5-10.9% per annum).^4,5  Although thrombosis risk is highest around the time of initial diagnosis, over time the risk of arterial thrombosis remains about twofold higher, and this risk is relatively higher for younger people with myeloproliferative neoplasms.⁶  The JAK2^VF driver mutation results in constitutive activation of JAK2 and downstream activation of the signal transducer and activator of transcription (STAT) 1, 3, and 5; mitogen-activated protein kinase; and phosphoinositide 3-kinase pathways. Exactly how this may result in increased atherosclerosis and thrombosis is not yet well understood. However, it is noteworthy that although individual randomized controlled trials of ruxolitinib, a tyrosine kinase inhibitor that inhibits JAK1 and JAK2, have not been powered to study thromboembolic events, preliminary evidence suggests that ruxolitinib treatment may reduce thromboembolic events.^7,8 

To understand the mechanism by which the JAK2^VF mutation might lead to an increased risk of vascular disease, Dr. Trevor Fidler and colleagues first studied gene expression changes associated with JAK2^VF expression in splenic myeloid cells from wildtype or hyperlipidemic (Ldlr^-/-) mice. This analysis revealed enrichment of genes associated with cellular proliferation, DNA damage repair, and metabolic pathways associated with presence of JAK2^VF mutation in myeloid cells. To specifically model the impact of myeloid cell–specific CH, chimeric mice were generated where JAK2^VF was expressed in only a proportion of blood cells, including a series of elegant experiments using an array of different mouse models, which allow induction of the JAK2^VF mutation in a myeloid cell–specific manner (i.e., only in neutrophils or monocytes). Mice in which JAK2^VF was expressed specifically in neutrophils showed no changes in atherosclerotic plaque area or morphology in hyperlipidemic mice. In contrast, both the chimeric mice modelling CH and mice that expressed JAK2^VF selectively in macrophages showed increased atherosclerotic plaque area with proliferation of macrophages and increased necrotic cores. These atherosclerotic lesions showed increased expression of the inflammasome gene Aim2 as well as oxidative DNA damage and DNA replication stress. Single-cell RNA sequencing analysis of JAK2^VF lesions demonstrated anenriched inflammatory myeloid cell landscape. Even though both Nlrp3 and AIM2 inflammasomes could be activated in cultured JAK2^VF macrophages, Nlrp3 deficiency had no significant effect on the lesions or necrotic cores, whereas Aim2 deficiency markedly reduced both (in contrast to the observations made in Tet2 deficient-mice mentioned earlier). Thus, the AIM2 inflammasome was the key pathway promoting atherosclerotic lesion formation in these JAK2^VF mice, possibly reflecting increased expression of AIM2 downstream of JAK/STAT signaling. Deletion of the downstream inflammasome components caspase 1 and 11 or pyroptosis executioner gasdermin D also partially reversed the changes and increased the stability of the plaques. These findings suggest that JAK2^VF mutations drive increased atherosclerosis primarily through the impact on macrophages, by increased proliferation, glycolytic metabolism, DNA replication stress, and activation of the AIM2 inflammasome, and increased IL-1ß. The group went on to study potential therapeutic interventions. Ruxolitinib treatment was associated with slightly smaller atherosclerotic lesions, but concerningly, they had increased necrotic cores and thinner caps, suggesting less-stable plaques and indicating that ruxolitinib promoted lesional necrosis. In contrast, inhibition of IL-1 or the inflammasome product IL-1ß reduced macrophage proliferation and necrotic core formation while increasing the thickness of fibrous caps, indicating plaque stabilization.