The signalling receptor for LPS, CD14, is a key marker of, and facilitator for, pro-inflammatory macrophage function. Pro-inflammatory macrophage differentiation remains a process facilitating a broad array of disease pathologies, and has recently emerged as a potential target against cytokine storm in COVID19. Here, we perform a whole-genome CRISPR screen to identify essential nodes regulating CD14 expression in myeloid cells, using the differentiation of THP-1 cells as a starting point. This strategy uncovers many known pathways required for CD14 expression and regulating macrophage differentiation while additionally providing a list of novel targets either promoting or limiting this process. To speed translation of these results, we have then taken the approach of independently validating hits from the screen using well-curated small molecules. In this manner, we identify pharmacologically tractable hits that can either increase CD14 expression on non-differentiated monocytes or prevent CD14 upregulation during macrophage differentiation. An inhibitor for one of these targets, MAP2K3, translates through to studies on primary human monocytes, where it prevents upregulation of CD14 following M-CSF induced differentiation, and pro-inflammatory cytokine production in response to LPS. Therefore, this screening cascade has rapidly identified pharmacologically tractable nodes regulating a critical disease-relevant process.

The genetic associations linking the chromosome 9 p21 (chr9p21) locus with cardiovascular diseases, such as aneurysm and coronary artery disease, are well characterised. However, the underlying molecular mechanism remains unclear. The chr9p21 locus is also associated with traits such as arterial stiffness, implicating a role in vascular cell biology. We investigated whether the risk variants affect vascular smooth muscle cell (VSMC) responses to inflammation. Methods We used recently developed and well characterised methods to differentiate VSMC from induced pluripotent stem cells (iPS) and compared the responses of risk and non-risk genotype iPS-VSMC to a range of IL-1 and TLR agonists. Results Risk and non-risk genotype iPS differentiated equally well to iPS-VSMC. However, the chr9p21 risk genotype conferred increased sensitivity to stimulation with IL-1alpha, IL-1beta and selective TLR agonists, due to increased expression of interleukin-1 receptor-associated kinase 4 (IRAK4). IRAK4-independent inflammatory responses remained unaffected by the risk genotype. IRAK4 expression was increased in risk iPS-VSMC as a consequence of altered epigenetic remodelling, including DNA methylation, of the IRAK4 promoter. Abstract 128 Figure 1 Conclusions Our study establishes a mechanistic link between the chr9p21 locus, inflammation and susceptibility to cardiovascular diseases, and provides support for the targeted use of therapies that inhibit IRAK4-dependent pathways, such as IL-1 signalling, in individuals of chr9p21 risk genotype.

Background: Abdominal aortic aneurysm (AAA) carries important morbidity and mortality and is resistant to medical therapy. Current experimental models do not accurately reproduce the major features of the human disease. There are 2 major categories of mouse models of AAA: those that induce medial dissection, which is not a major characteristic of human AAA, and those that induce aortic dilatation but are self-contained and do not progress to rupture. Methods: We hypothesized that blockade of TGFβ activity, a guardian of vascular integrity and immune homeostasis, and a major causal factor in genetically triggered thoracic aortic aneurysms in humans, would impair vascular healing in models of ‘non-dissecting’ abdominal aortic dilatation, and would lead to continuous aneurysmal growth until rupture. We tested this hypothesis in the elastase-induced abdominal aortic dilatation model in mice. We analyzed AAA development and progression using ultrasound in vivo, advanced synchrotron-based ultrahigh resolution imaging ex-vivo, and a combination of biological, histological and flow cytometry-based cellular and molecular approaches in vitro. Results: We show that systemic blockade of TGFβ activity using a neutralizing mouse monoclonal anti-TGFβ antibody induces a transition from a model of self-contained aortic dilatation to a model of sustained aneurysmal growth culminating in rupture. TGFβ blockade enhances leukocyte infiltration and extracellular matrix degradation, and leads to sustained aneurysmal aortic dilatation, associated with the formation of an intra-luminal thrombus infiltrated with neutrophils, as seen in the human disease. Persistent AAA growth throughout the duration of the experiment is associated with wall disruption without medial dissection, and culminates in fatal aortic wall rupture. Monocyte/macrophage depletion substantially limits AAA severity. Conclusions: Endogenous TGFβ activity is required for the resolution of elastase-induced aortic injury. We expect that this new model will improve our understanding of the pathophysiology of AAA, and will be useful to identify new therapeutic targets.

The inflammasome is a cytoplasmic protein complex that processes interleukins (IL)-1β and IL-18, and drives a form of cell death known as pyroptosis. Oligomerization of this complex is actually the second step of activation, and a priming step must occur first. This involves transcriptional upregulation of pro-IL-1β, inflammasome sensor NLRP3, or the non-canonical inflammasome sensor caspase-11. An additional aspect of priming is the post-translational modification of particular inflammasome constituents. Priming is typically accomplished in vitro using a microbial Toll-like receptor (TLR) ligand. However, it is now clear that inflammasomes are activated during the progression of sterile inflammatory diseases such as atherosclerosis, metabolic disease, and neuroinflammatory disorders. Therefore, it is time to consider the endogenous factors and mechanisms that may prime the inflammasome in these conditions.

Objective— Current experimental models of abdominal aortic aneurysm (AAA) do not accurately reproduce the major features of human AAA. We hypothesized that blockade of TGFβ (transforming growth factor-β) activity—a guardian of vascular integrity and immune homeostasis—would impair vascular healing in models of nondissecting AAA and would lead to sustained aneurysmal growth until rupture. Approach and Results— Here, we test this hypothesis in the elastase-induced AAA model in mice. We analyze AAA development and progression using ultrasound in vivo, synchrotron-based ultrahigh resolution imaging ex vivo, and a combination of biological, histological, and flow cytometry-based cellular and molecular approaches in vitro. Systemic blockade of TGFβ using a monoclonal antibody induces a transition from a self-contained aortic dilatation to a model of sustained aneurysmal growth, associated with the formation of an intraluminal thrombus. AAA growth is associated with wall disruption but no medial dissection and culminates in fatal transmural aortic wall rupture. TGFβ blockade enhances leukocyte infiltration both in the aortic wall and the intraluminal thrombus and aggravates extracellular matrix degradation. Early blockade of IL-1β or monocyte-dependent responses substantially limits AAA severity. However, blockade of IL-1β after disease initiation has no effect on AAA progression to rupture. Conclusions— Endogenous TGFβ activity is required for the healing of AAA. TGFβ blockade may be harnessed to generate new models of AAA with better relevance to the human disease. We expect that the new models will improve our understanding of the pathophysiology of AAA and will be useful in the identification of new therapeutic targets.

Studies made on transgenic Marfan syndrome models of mouse showed the pathogenic role of a superactivation of the TGFβ (Transforming Growth Factor β) pathway. The role of this cytokine is not yet well elucidated in the pathophysiology of abdominal aortic aneurysms (AAA). The objective of our study was to evaluate the effect of the neutralization of TGFβ in a murine model of AAA induced by elastase.

The T2 ribonuclease omega-1 is a powerful Th2-inducing factor secreted by the eggs of the blood fluke Schistosoma mansoni. Omega-1 can modulate pattern recognition receptor-induced inflammatory signatures and alter antigen presentation by dendritic cells. Recent findings have suggested that component(s) contained in or secreted by S. mansoni eggs (soluble egg antigen) can also enhance IL-1β secretion by dendritic cells stimulated with pattern recognition receptor ligands. Here we show that omega-1 enhances IL-1β secretion in macrophages stimulated with Toll-like receptor 2 ligand, and propose omega-1 as the factor in soluble egg antigen capable of regulating inflammasome activity. This effect is dependent on the C-type lectin receptor Dectin-1, caspase-8 and the ASC inflammasome adaptor protein, highlighting the ability of omega-1 to regulate multiple pattern recognition receptor signalling pathways. These mechanistic insights into manipulation of host immunity by a parasite product have implications for the design of anti-inflammatory therapeutic drugs.

Obesity increases the risk of developing life-threatening metabolic diseases including cardiovascular disease, fatty liver disease, diabetes, and cancer. Efforts to curb the global obesity epidemic and its impact have proven unsuccessful in part by a limited understanding of these chronic progressive diseases. It is clear that low-grade chronic inflammation, or metaflammation, underlies the pathogenesis of obesity-associated type 2 diabetes and atherosclerosis. However, the mechanisms that maintain chronicity and prevent inflammatory resolution are poorly understood. Here, we show that inhibitor of κB kinase epsilon (IKBKE) is a novel regulator that limits chronic inflammation during metabolic disease and atherosclerosis. The pathogenic relevance of IKBKE was indicated by the colocalization with macrophages in human and murine tissues and in atherosclerotic plaques. Genetic ablation of IKBKE resulted in enhanced and prolonged priming of the NLRP3 inflammasome in cultured macrophages, in hypertrophic adipose tissue, and in livers of hypercholesterolemic mice. This altered profile associated with enhanced acute phase response, deregulated cholesterol metabolism, and steatoheptatitis. Restoring IKBKE only in hematopoietic cells was sufficient to reverse elevated inflammasome priming and these metabolic features. In advanced atherosclerotic plaques, loss of IKBKE and hematopoietic cell restoration altered plaque composition. These studies reveal a new role for hematopoietic IKBKE: to limit inflammasome priming and metaflammation.