Clonal haematopoiesis, which is highly prevalent in older individuals, arises from somatic mutations that endow a proliferative advantage to haematopoietic cells. Clonal haematopoiesis increases the risk of myocardial infarction and stroke independently of traditional risk factors 1 . Among the common genetic variants that give rise to clonal haematopoiesis, the JAK2 V617F (JAK2 VF ) mutation, which increases JAK-STAT signalling, occurs at a younger age and imparts the strongest risk of premature coronary heart disease 1,2 . Here we show increased proliferation of macrophages and prominent formation of necrotic cores in atherosclerotic lesions in mice that express Jak2 VF selectively in macrophages, and in chimeric mice that model clonal haematopoiesis. Deletion of the essential inflammasome components caspase 1 and 11, or of the pyroptosis executioner gasdermin D, reversed these adverse changes. Jak2 VF lesions showed increased expression of AIM2, oxidative DNA damage and DNA replication stress, and Aim2 deficiency reduced atherosclerosis. Single-cell RNA sequencing analysis of Jak2 VF lesions revealed a landscape that was enriched for inflammatory myeloid cells, which were suppressed by deletion of Gsdmd. Inhibition of the inflammasome product interleukin-1β reduced macrophage proliferation and necrotic formation while increasing the thickness of fibrous caps, indicating that it stabilized plaques. Our findings suggest that increased proliferation and glycolytic metabolism in Jak2 VF macrophages lead to DNA replication stress and activation of the AIM2 inflammasome, thereby aggravating atherosclerosis. Precise application of therapies that target interleukin-1β or specific inflammasomes according to clonal haematopoiesis status could substantially reduce cardiovascular risk.Atherosclerotic cardiovascular disease (ACVD) is the major cause of death and disability in the developed world 3 . A large burden of residual ACVD risk remains despite current therapies, including intensive lowering of low-density lipoprotein levels 3 , which highlights the need for new treatments. In the Canakinumab Antiinflammatory Thrombosis Outcomes Study (CANTOS), inhibition of IL-1β reduced cardiovascular events, thereby validating the contribution of inflammation to ACVD 4 . However, canakinumab therapy was associated with a small risk of infections and has not been approved for cardiovascular conditions. Thus, a more precise way to identify patients who may benefit most from anti-inflammatory therapy is required. Clonal haematopoiesis usually arises from somatic mutations in haematopoietic stem and progenitor cells (HSPCs) in one of four genes (TET2, ASXL1, DNMT3A or JAK2), which lead to clonal expansion of haematopoietic cells. The prevalence of clonal haematopoiesis increases with age, and it affects more than 10% of people who are over 70 years old 1 . Although clonal haematopoiesis conferred an increased risk of haematological malignancies of 0.5-1% per year, this modest increase was not nearly enough to account for the 40% incr...

SUMMARYAdipocytes undergo considerable volumetric expansion in the setting of obesity. It has been proposed that such marked increases in adipocyte size may be sensed via adipocyte-autonomous mechanisms to mediate size-dependent intracellular signaling. Here, we show that SWELL1 (LRRC8a), a member of the Leucine Rich Repeat Containing protein family, is an essential component of a volume-sensitive ion channel (VRAC) in adipocytes. We find that SWELL1-mediated VRAC is augmented in hypertrophic murine and human adipocytes in the setting of obesity. SWELL1 regulates adipocyte insulin-PI3K-AKT2-GLUT4 signaling, glucose uptake and lipid content via SWELL1 C-terminal leucine-rich repeat domain interactions with GRB2/Cav1. Silencing GRB2 in SWELL1 KO adipocytes rescues insulin-pAKT2 signaling. In vivo, shRNA-mediated SWELL1 knock-down and adipose-targeted SWELL1 knock-out reduce adiposity and adipocyte size in obese mice while impairing systemic glycaemia and insulin-sensitivity. These studies identify SWELL1 as a cell-autonomous sensor of adipocyte size that regulates adipocyte growth, insulin sensitivity and glucose tolerance.

Rationale: The mechanisms driving athero-thrombotic risk in individuals with JAK2V617F (Jak2VF) positive clonal hematopoiesis (CH) or myeloproliferative neoplasms (MPN) are poorly understood. Objective: The goal of this study was to assess atherosclerosis and underlying mechanisms in hypercholesterolemic mice with hematopoietic Jak2VF expression. Methods and Results: Irradiated low-density lipoprotein receptor knockout (Ldlr−/−) mice were transplanted with bone marrow from WT or Jak2VF mice and fed a high fat high cholesterol Western diet (WD). Hematopoietic functions and atherosclerosis were characterized. After 7 weeks of WD Jak2VF mice showed increased atherosclerosis. Early atherosclerotic lesions showed increased neutrophil adhesion and content, correlating with lesion size. After 12 weeks of WD Jak2VF lesions showed increased complexity, with larger necrotic cores, defective efferocytosis, prominent iron deposition and co-staining of erythrocytes and macrophages suggesting erythrophagocytosis. Jak2VF erythrocytes were more susceptible to phagocytosis by WT macrophages and showed decreased surface expression of CD47, a “don’t eat me” signal. Human JAK2VF erythrocytes were also more susceptible to erythrophagocytosis. Jak2VF macrophages displayed increased expression and production of pro-inflammatory cytokines and chemokines, prominent inflammasome activation, increased p38 MAP kinase signaling and reduced levels of MerTK, a key molecule mediating efferocytosis. Increased erythrophagocytosis also suppressed efferocytosis. Conclusions: Hematopoietic Jak2VF expression promotes early lesion formation and increased complexity in advanced atherosclerosis. In addition to increasing hematopoiesis and neutrophil infiltration in early lesions, Jak2VF caused cellular defects in erythrocytes and macrophages, leading to increased erythrophagocytosis but defective efferocytosis. These changes promote accumulation of iron in plaques and increased necrotic core formation which, together with exacerbated pro-inflammatory responses, likely contribute to plaque instability.

The oncoprotein Bcr-Abl drives aberrant downstream activity through trans-autophosphorylation of homo-oligomers in chronic myelogenous leukemia (CML).1,2 The formation of Bcr-Abl oligomers is achieved through the coiled-coil domain at the N-terminus of Bcr.3, 4 We have previously reported a modified version of this coiled-coil domain, CCmut2, which exhibits disruption of Bcr-Abl oligomeric complexes and results in decreased proliferation of CML cells and induction of apoptosis.5 A major contributing factor to these enhanced capabilities is the destabilization of the CCmut2 homo-dimers, increasing the availability to interact with and inhibit Bcr-Abl. Here, we included an additional mutation (K39E) that could in turn further destabilize the mutant homo-dimer. Incorporation of this modification into CCmut2 (C38A, S41R, L45D, E48R, Q60E) generated what we termed CCmut3, and resulted in further improvements in the binding properties with the wild-type coiled-coil domain representative of Bcr-Abl. A separate construct containing one revert mutation, CCmut4, did not demonstrate improved oligomeric properties and indicated the importance of the L45D mutation. CCmut3 demonstrated improved oligomerization via a two-hybrid assay as well as through colocalization studies, in addition to showing similar biologic activity as CCmut2. The improved binding between CCmut3 and the Bcr-Abl coiled-coil may be used to redirect Bcr-Abl to alternative subcellular locations with interesting therapeutic implications.

Despite the initial success of some drugs and vaccines targeting COVID-19, understanding the mechanism underlying SARS-CoV-2 disease pathogenesis remains crucial for the development of further approaches to treatment. Some patients with severe Covid-19 experience a cytokine storm and display evidence of inflammasome activation leading to increased levels of IL-1β and IL-18; however, other reports have suggested reduced inflammatory responses to Sars-Cov-2. In this study we have examined the effects of the Sars-Cov-2 envelope (E) protein, a virulence factor in coronaviruses, on inflammasome activation and pulmonary inflammation. In cultured macrophages the E protein suppressed inflammasome priming and NLRP3 inflammasome activation. Similarly, in mice transfected with E protein and treated with poly(I:C) to simulate the effects of viral RNA, the E protein, in an NLRP3-dependent fashion, reduced expression of pro-IL-1β, levels of IL-1β and IL-18 in broncho-alveolar lavage fluid, and macrophage infiltration in the lung. To simulate the effects of more advanced infection, macrophages were treated with both LPS and poly(I:C). In this setting the E protein increased NLRP3 inflammasome activation in both murine and human macrophages. Thus, the Sars-Cov-2 E protein may initially suppress the host NLRP3 inflammasome response to viral RNA while potentially increasing NLRP3 inflammasome responses in the later stages of infection. Targeting the Sars-Cov-2 E protein especially in the early stages of infection may represent a novel approach to Covid-19 therapy.