Molecular mechanisms underlying NLRP3 inflammasome activation and IL-1ß production in air pollution fine particulate matter (PM2.5)-primed macrophages.

Exposure to air pollution fine particulate matter (PM2.5) aggravates respiratory and cardiovascular diseases. It has been proposed that PM2.5 uptake by alveolar macrophages promotes local inflammation that ignites a systemic response, but precise underlying mechanisms remain unclear. Here, we demonstrate that PM2.5 phagocytosis leads to NLRP3 inflammasome activation and subsequent release of the pro-inflammatory master cytokine IL-1ß. Inflammasome priming and assembly was time- and dose-dependent in inflammasome-reporter THP-1-ASC-GFP cells, and consistent across PM2.5 samples of variable chemical composition. While inflammasome activation was promoted by different PM2.5 surrogates, significant IL-1ß release could only be observed after stimulation with transition-metal rich Residual Oil Fly Ash (ROFA) particles. This effect was confirmed in primary human monocyte-derived macrophages and murine bone marrow-derived macrophages (BMDMs), and by confocal imaging of inflammasome-reporter ASC-Citrine BMDMs. IL-1ß release by ROFA was dependent on the NLRP3 inflammasome, as indicated by lack of IL-1ß production in ROFA-exposed NLRP3-deficient (Nlrp3-/-) BMDMs, and by specific NLRP3 inhibition with the pharmacological compound MCC950. In addition, while ROFA promoted the upregulation of pro-inflammatory gene expression and cytokines release, MCC950 reduced TNF-α, IL-6, and CCL2 production. Furthermore, inhibition of TNF-α with a neutralizing antibody decreased IL-1ß release in ROFA-exposed BMDMs. Using electron tomography, ROFA particles were observed inside intracellular vesicles and mitochondria, which showed signs of ultrastructural damage. Mechanistically, we identified lysosomal rupture, K+ efflux, and impaired mitochondrial function as important prerequisites for ROFA-mediated IL-1ß release. Interestingly, specific inhibition of superoxide anion production (O2•-) from mitochondrial respiratory Complex I, but not III, blunted IL-1ß release in ROFA-exposed BMDMs. Our findings unravel the mechanism by which PM2.5 promotes IL-1ß release in macrophages and provide a novel link between innate immune response and exposure to air pollution PM2.5.

ADP as a novel stimulus for NLRP3-inflammasome activation in mice fails to translate to humans.

The NLRP3-inflammasome is a cytosolic multiprotein complex that triggers an inflammatory response to certain danger signals. Recently adenosine diphosphate (ADP) was found to activate the NLRP3-inflammasome in murine macrophages via the P2Y1 receptor. Blockade of this signaling pathway reduced disease severity in a murine colitis-model. However, the role of the ADP/P2Y1-axis has not yet been studied in humans. This present study confirmed ADP-dependent NLRP3-inflammasome activation in murine macrophages, but found no evidence for a role of ADP in inflammasome activation in humans. We investigated the THP1 cell line as well as primary monocytes and further looked at macrophages. Although all cells express the three human ADP-receptors P2Y1, P2Y12 and P2Y13, independent of priming, neither increased ASC-speck formation could be detected with flow cytometry nor additional IL-1ß release be found in the culture supernatant of ADP stimulated cells. We now show for the first time that the responsiveness of monocytes and macrophages to ADP as well as the regulation of its purinergic receptors is very much dependent on the species. Therefore the signaling pathway found to contribute to colitis in mice is likely not applicable to humans.

Elevated platelet-leukocyte complexes are associated with, but dispensable for myocardial ischemia-reperfusion injury.

AIMS: P-selectin is an activatable adhesion molecule on platelets promoting platelet aggregation, and platelet-leukocyte complex (PLC) formation. Increased numbers of PLC are circulating in the blood of patients shortly after acute myocardial infarction and predict adverse outcomes. These correlations led to speculations about whether PLC may represent novel therapeutic targets. We therefore set out to elucidate the pathomechanistic relevance of PLC in myocardial ischemia and reperfusion injury. METHODS AND RESULTS: By generating P-selectin deficient bone marrow chimeric mice, the post-myocardial infarction surge in PLC numbers in blood was prevented. Yet, intravital microscopy, flow cytometry and immunohistochemical staining, echocardiography, and gene expression profiling showed unequivocally that leukocyte adhesion to the vessel wall, leukocyte infiltration, and myocardial damage post-infarction were not altered in response to the lack in PLC. CONCLUSION: We conclude that myocardial infarction associated sterile inflammation triggers PLC formation, reminiscent of conserved immunothrombotic responses, but without PLC influencing myocardial ischemia and reperfusion injury in return. Our experimental data do not support a therapeutic concept of selectively targeting PLC formation in myocardial infarction.

P2X4 deficiency reduces atherosclerosis and plaque inflammation in mice.

Extracellular adenosine-5'-triphosphate (ATP) acts as an import signaling molecule mediating inflammation via purinergic P2 receptors. ATP binds to the purinergic receptor P2X4 and promotes inflammation via increased expression of pro-inflammatory cytokines. Because of the central role of inflammation, we assumed a functional contribution of the ATP-P2X4-axis in atherosclerosis. Expression of P2X4 was increased in atherosclerotic aortic arches from low-density lipoprotein receptor-deficient mice being fed a high cholesterol diet as assessed by real-time polymerase chain reaction and immunohistochemistry. To investigate the functional role of P2X4 in atherosclerosis, P2X4-deficient mice were crossed with low-density lipoprotein receptor-deficient mice and fed high cholesterol diet. After 16 weeks, P2X4-deficient mice developed smaller atherosclerotic lesions compared to P2X4-competent mice. Furthermore, intravital microscopy showed reduced ATP-induced leukocyte rolling at the vessel wall in P2X4-deficient mice. Mechanistically, we found a reduced RNA expression of CC chemokine ligand 2 (CCL-2), C-X-C motif chemokine-1 (CXCL-1), C-X-C motif chemokine-2 (CXCL-2), Interleukin-6 (IL-6) and tumor necrosis factor α (TNFα) as well as a decreased nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)-inflammasome priming in atherosclerotic plaques from P2X4-deficient mice. Moreover, bone marrow derived macrophages isolated from P2X4-deficient mice revealed a reduced ATP-mediated release of CCL-2, CC chemokine ligand 5 (CCL-5), Interleukin-1ß (IL-1ß) and IL-6. Additionally, P2X4-deficient mice shared a lower proportion of pro-inflammatory Ly6Chigh monocytes and a higher proportion of anti-inflammatory Ly6Clow monocytes, and expressend less endothelial VCAM-1. Finally, increased P2X4 expression in human atherosclerotic lesions from carotid endarterectomy was found, indicating the importance of potential implementations of this study's findings for human atherosclerosis. Collectively, P2X4 deficiency reduced experimental atherosclerosis, plaque inflammation and inflammasome priming, pointing to P2X4 as a potential therapeutic target in the fight against atherosclerosis.

Myeloid cell-specific Irf5 deficiency stabilizes atherosclerotic plaques in Apoe-/- mice.

OBJECTIVE: Interferon regulatory factor (IRF) 5 is a transcription factor known for promoting M1 type macrophage polarization in vitro. Given the central role of inflammatory macrophages in promoting atherosclerotic plaque progression, we hypothesize that myeloid cell-specific deletion of IRF5 is protective against atherosclerosis. METHODS: Female Apoe-/-LysmCre/+Irf5fl/fl and Apoe-/-Irf5fl/fl mice were fed a high-cholesterol diet for three months. Atherosclerotic plaque size and compositions as well as inflammatory gene expression were analyzed. Mechanistically, IRF5-dependent bone marrow-derived macrophage cytokine profiles were tested under M1 and M2 polarizing conditions. Mixed bone marrow chimeras were generated to determine intrinsic IRF5-dependent effects on macrophage accumulation in atherosclerotic plaques. RESULTS: Myeloid cell-specific Irf5 deficiency blunted LPS/IFNγ-induced inflammatory gene expression in vitro and in the atherosclerotic aorta in vivo. While atherosclerotic lesion size was not reduced in myeloid cell-specific Irf5-deficient Apoe-/- mice, plaque composition was favorably altered, resembling a stable plaque phenotype with reduced macrophage and lipid contents, reduced inflammatory gene expression and increased collagen deposition alongside elevated Mertk and Tgfß expression. Irf5-deficient macrophages, when directly competing with wild type macrophages in the same mouse, were less prone to accumulate in atherosclerotic lesion, independent of monocyte recruitment. Irf5-deficient monocytes, when exposed to oxidized low density lipoprotein, were less likely to differentiate into macrophage foam cells, and Irf5-deficient macrophages proliferated less in the plaque. CONCLUSION: Our study provides genetic evidence that selectively altering macrophage polarization induces a stable plaque phenotype in mice.

Residual inflammatory risk in coronary heart disease: incidence of elevated high-sensitive CRP in a real-world cohort.

BACKGROUND: Inflammation drives atherosclerosis and its complications. Anti-inflammatory therapy with interleukin 1 beta (IL-1ß) antibody reduces cardiovascular events in patients with elevated high-sensitive C-reactive protein (hsCRP). This study aims to identify the share of patients with coronary heart disease (CHD) and residual inflammation who may benefit from anti-inflammatory therapy. METHODS: hsCRP and low-density lipoprotein (LDL) levels were determined in 2741 all-comers admitted to the cardiological ward of our tertiary referral hospital between June 2016 and June 2018. Patients without CHD, with acute coronary syndrome, chronic or recurrent systemic infection, use of immunosuppressant or anti-inflammatory agents, chronic inflammatory diseases, chemotherapy, terminal organ failure, traumatic injury and pregnancy were excluded. RESULTS: 856 patients with stable CHD were included. 42.7% of those had elevated hsCRP ≥ 2 mg/l. Within the group of patients with LDL-cholesterol < 70 mg/dl, 30.9% shared increased hsCRP indicating residual inflammation. After multivariate adjusted backward selection elevated Lipoprotein (a) (OR 1.61, p = 0.048), elevated proBNP (OR 2.57, p < 0.0001), smoking (OR 1.70, p = 0.022), and obesity (OR 2.28, p = 0.007) were associated with elevated hsCRP. In contrast, the use of ezetimibe was associated with normal hsCRP (OR 0.51, p = 0.014). In the subgroup of patients with on-target LDL-cholesterol < 70 mg/dl, backward selection identified elevated proBNP (OR 3.49, p = 0.007) as independent predictor of elevated hsCRP in patients with LDL-cholesterol < 70 mg/dl. CONCLUSION: One-third of all-comers patients with CHD showed increased levels of hsCRP despite a LDL-cholesterol < 70 mg/dl potentially qualifying for an anti-inflammatory therapy. Elevated proBNP is an independent risk factor for hsCRP elevation.

Purinergic receptor Y2 (P2Y2)- dependent VCAM-1 expression promotes immune cell infiltration in metabolic syndrome.

Sterile inflammation of visceral fat, provoked by dying adipocytes, links the metabolic syndrome to cardiovascular disease. Danger-associated molecular patterns, such as adenosine triphosphate (ATP), are released by activated or dying cells and orchestrate leukocyte infiltration and inflammation via the purinergic receptor P2Y2. The gene expression of ATP receptor P2Y2 did not change in several tissues in the course of obesity, but was increased within epididymal fat. Adipose tissue from P2Y 2-/- mice consuming high-fat diet (HFD) contained less crown-like structures with a reduced frequency of adipose tissue macrophages (ATMs). This was likely due to decreased leukocyte migration because of missing VCAM-1 exposition on P2Y2 deficient hypertrophic adipose tissue endothelial cells. Accordingly, P2Y 2-/- mice showed blunted traits of the metabolic syndrome: they gained less weight compared to P2Y 2+/+ controls, while intake of food and movement behaviour remained unchanged. Liver and adipose tissue were smaller in P2Y 2-/- animals. Insulin tolerance testing (ITT) performed in obese P2Y 2-/- mice revealed a better insulin sensitivity as well as lower plasma C-peptide and cholesterol levels. We demonstrate that interfering with somatic P2Y2 signalling prevents excessive immune cell deposition in diet-induced obesity (DIO), both attenuating adipose tissue inflammation and ameliorating the metabolic phenotype. Thus, blocking the P2Y2 cascade may be a promising strategy to limit metabolic disease and its sequelae.

P2X7 Deficiency Blocks Lesional Inflammasome Activity and Ameliorates Atherosclerosis in Mice.

BACKGROUND: Extracellular adenosine triphosphate (ATP) binds as a danger signal to purinergic receptor P2X7 and promotes inflammasome assembly and interleukin-1ß expression. We hypothesized a functional role of the signal axis ATP-P2X7 in inflammasome activation and the chronic inflammation driving atherosclerosis. METHODS: P2X7-competent and P2X7-deficient macrophages were isolated and stimulated with lipopolysaccharide, ATP, or both. To assess whether P2X7 may have a role in atherosclerosis, P2X7 expression was analyzed in aortic arches from low density lipoprotein receptor-/- mice consuming a high-cholesterol or chow diet. P2X7+/+ and P2X7-/- low density lipoprotein receptor-/- mice were fed a high-cholesterol diet to investigate the functional role of P2X7 knockout in atherosclerosis. Human plaques were derived from carotid endarterectomy and stained against P2X7. RESULTS: Lipopolysaccharide or ATP stimulation alone did not activate caspase 1 in isolated macrophages. However, priming with lipopolysaccharide, followed by stimulation with ATP, led to an activation of caspase 1 and interleukin-1ß in P2X7-competent macrophages. In contrast, P2X7-deficient macrophages showed no activation of caspase 1 after sequential stimulation while still expressing a basal amount of interleukin-1ß. P2X7 receptor was higher expressed in murine atherosclerotic lesions, particularly by lesional macrophages. After 16 weeks of a high-cholesterol diet, P2X7-deficient mice showed smaller atherosclerotic lesions than P2X7-competent mice (0.162 cm2±0.023 [n=9], P2X7-/- low density lipoprotein receptor-/- : 0.084 cm2±0.01 [n=11], P=0.004) with a reduced amount of lesional macrophages. In accord with our in vitro findings, lesional caspase 1 activity was abolished in P2X7-/- mice. In addition, intravital microscopy revealed reduced leukocyte rolling and adhesion in P2X7-deficient mice. Last, we observe increased P2X7 expression in human atherosclerotic lesions, suggesting that our findings in mice are relevant for human disease. CONCLUSIONS: P2X7 deficiency resolved plaque inflammation by inhibition of lesional inflammasome activation and reduced experimental atherosclerosis. Therefore, P2X7 represents an interesting potential new target to combat atherosclerosis.