Background: Cardiovascular risk in diabetes remains elevated despite glucose lowering therapies. We hypothesised that hyperglycaemia induces trained immunity in macrophages, promoting persistent pro-atherogenic characteristics. Methods: Bone marrow derived macrophages from control and mice with diabetes were grown in physiological glucose (5 mM) and subject to RNA-sequencing (n=6), ATAC-sequencing (n=6) and ChIP-sequencing (n=6) for determination of hyperglycaemia-induced trained immunity. Bone marrow transplantation from mice with (n=9) or without (n=6) diabetes into [normoglycaemic] Ldlr -/- mice was used to assess its functional significance in vivo . Evidence of hyperglycaemia-induced trained immunity was sought in human peripheral blood mononuclear cells (PBMCs) from patients with diabetes (n=8) compared with case controls (n=16) and in human atherosclerotic plaque macrophages excised by laser capture microdissection. Results: In macrophages, high extracellular glucose promoted pro-inflammatory gene expression and pro-atherogenic functional characteristics, through glycolysis-dependent mechanisms. Bone marrow-derived macrophages (BMDM) from diabetic mice, retained these characteristics, even when cultured in physiological glucose, indicating hyperglycaemia-induced trained immunity. Bone marrow transplantation from diabetic mice into [normoglycaemic] Ldlr -/- mice increased aortic root atherosclerosis, confirming a disease-relevant and persistent form of trained innate immunity. Integrated ATAC-seq, ChIP-seq and RNA-seq analyses of haematopoietic stem cells and BMDM revealed a pro-inflammatory "priming effect" in diabetes. The pattern of open chromatin implicated transcription factor, RUNX1, while transcriptomes of atherosclerotic plaque macrophages and peripheral leukocytes in patients with type 2 diabetes were enriched for RUNX1 targets, consistent with a potential role in human disease. Pharmacological inhibition of RUNX1 in vitro inhibited the trained phenotype. Conclusions: Hyperglycaemia-induced trained immunity may explain why targeting elevated glucose is ineffective in reducing macrovascular risk in diabetes and suggests new targets for disease prevention and therapy.

Transcriptionally activated monocytes are recruited to the heart after acute myocardial infarction (AMI). After AMI in mice and humans, the number of extracellular vesicles (EVs) increased acutely. In humans, EV number correlated closely with the extent of myocardial injury. We hypothesized that EVs mediate splenic monocyte mobilization and program transcription following AMI. Some plasma EVs bear endothelial cell (EC) integrins, and both proinflammatory stimulation of ECs and AMI significantly increased VCAM-1–positive EV release. Injected EC-EVs localized to the spleen and interacted with, and mobilized, splenic monocytes in otherwise naive, healthy animals. Analysis of human plasma EV-associated miRNA showed 12 markedly enriched miRNAs after AMI; functional enrichment analyses identified 1,869 putative mRNA targets, which regulate relevant cellular functions (e.g., proliferation and cell movement). Furthermore, gene ontology termed positive chemotaxis as the most enriched pathway for the miRNA-mRNA targets. Among the identified EV miRNAs, EC-associated miRNA-126-3p and -5p were highly regulated after AMI. miRNA-126-3p and -5p regulate cell adhesion– and chemotaxis-associated genes, including the negative regulator of cell motility, plexin-B2. EC-EV exposure significantly downregulated plexin-B2 mRNA in monocytes and upregulated motility integrin ITGB2. These findings identify EVs as a possible novel signaling pathway by linking ischemic myocardium with monocyte mobilization and transcriptional activation following AMI.

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Aims Acute myocardial infarction rapidly increases blood neutrophils (<2 h). Release from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 h after injury, and after neutrophil elevation. This suggests that additional non-chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 min) release of extracellular vesicles (EVs), which have emerged as an important means of cell–cell signalling and are thus a potential mechanism for communicating with remote tissues. Methods and results Here, we show that injury to the myocardium rapidly mobilizes neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to arrival at the injured tissue. Time course analysis of plasma-EV composition revealed a rapid and selective increase in EVs bearing VCAM-1. These EVs, which were also enriched for miRNA-126, accumulated preferentially in the spleen where they induced local inflammatory gene and chemokine protein expression, and mobilized splenic-neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing, we generated VCAM-1-deficient EC-EVs and showed that its deletion removed the ability of EC-EVs to provoke the mobilization of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Conclusions Our findings show a novel EV-dependent mechanism for the rapid mobilization of neutrophils to peripheral blood from a splenic reserve and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.

Mononuclear phagocytes (MNPs) are vital for maintaining intestinal homeostasis but, in response to acute microbial stimulation, can also trigger immunopathology, accelerating recruitment of Ly6Chi monocytes to the gut. The regulators that control monocyte tissue adaptation in the gut remain poorly understood. Interferon regulatory factor 5 (IRF5) is a transcription factor previously shown to play a key role in maintaining the inflammatory phenotype of macrophages. Here, we investigate the impact of IRF5 on the MNP system and physiology of the gut at homeostasis and during inflammation. We demonstrate that IRF5 deficiency has a limited impact on colon physiology at steady state but ameliorates immunopathology during Helicobacter hepaticus–induced colitis. Inhibition of IRF5 activity in MNPs phenocopies global IRF5 deficiency. Using a combination of bone marrow chimera and single-cell RNA-sequencing approaches, we examined the intrinsic role of IRF5 in controlling colonic MNP development. We demonstrate that IRF5 promotes differentiation of Ly6Chi monocytes into CD11c+ macrophages and controls the production of antimicrobial and inflammatory mediators by these cells. Thus, we identify IRF5 as a key transcriptional regulator of the colonic MNP system during intestinal inflammation.

Interferon regulating factor 5 (IRF5) is a multifunctional regulator of immune responses, and has a key pathogenic function in gut inflammation, but how IRF5 is modulated is still unclear. Having performed a kinase inhibitor library screening in macrophages, here we identify protein-tyrosine kinase 2-beta (PTK2B/PYK2) as a putative IRF5 kinase. PYK2-deficient macrophages display impaired endogenous IRF5 activation, leading to reduction of inflammatory gene expression. Meanwhile, a PYK2 inhibitor, defactinib, has a similar effect on IRF5 activation in vitro, and induces a transcriptomic signature in macrophages similar to that caused by IRF5 deficiency. Finally, defactinib reduces pro-inflammatory cytokines in human colon biopsies from patients with ulcerative colitis, as well as in a mouse colitis model. Our results thus implicate a function of PYK2 in regulating the inflammatory response in the gut via the IRF5 innate sensing pathway, thereby opening opportunities for related therapeutic interventions for inflammatory bowel diseases and other inflammatory conditions.

Mononuclear phagocytes (MNPs) play a key role in maintaining intestinal homeostasis but also in triggering immunopathology in response to acute microbial stimulation, which induces the recruitment of masses of Ly6C hi monocytes to the gut. The regulators that control monocyte tissue adaptation in the gut remain poorly understood. Interferon Regulatory Factor 5 (IRF5) is a transcription factor previously shown to play a key role in maintaining the inflammatory phenotype of macrophages. Here we investigate the impact of IRF5 on the MNP system and physiology of the gut at homeostasis and during inflammation. We demonstrate that IRF5 deficiency has a limited impact on colon physiology at steady state, but ameliorates immunopathology during Helicobacter hepaticus induced colitis. Inhibition of IRF5 activity in MNPs phenocopies global IRF5 deficiency. Using a combination of bone marrow chimera and single cell RNA-sequencing approaches we compare the differentiation trajectories of wild type and IRF5 deficient monocytes in a shared inflammatory environment and demonstrate that IRF5 stipulates a choice in monocyte differentiation towards macrophages. Specifically, IRF5 promotes the generation of pathogenic CD11c + macrophages and controls the production of inflammatory mediators by these cells. Thus, we identify IRF5 as a key transcriptional controller of pathogenic monocyte differentiation in the gut.

Acute myocardial infarction rapidly increases blood neutrophils (<2 hours). Release of neutrophils from bone marrow, in response to chemokine elevation, has been considered their source, but chemokine levels peak up to 24 hours after injury, and after neutrophil elevation. This suggests that additional non chemokine-dependent processes may be involved. Endothelial cell (EC) activation promotes the rapid (<30 minutes) release of extracellular vesicles (EVs), which are enriched in vascular cell adhesion molecule-1 (VCAM-1) and miRNA-126, and are thus a potential mechanism for communicating with remote tissues. Here, we show that injury to the myocardium rapidly mobilises neutrophils from the spleen to peripheral blood and induces their transcriptional activation prior to their arrival at injured tissue. Ischemic myocardium leads to the generation and release of EC-derived-EVs bearing VCAM-1. EC-EV delivery to the spleen alters inflammatory gene and chemokine protein expression, and mobilises neutrophils to peripheral blood. Using CRISPR/Cas9 genome editing we generated VCAM-1-deficient EV and showed that its deletion removed the ability of EC-EV to provoke the mobilisation of neutrophils. Furthermore, inhibition of miRNA-126 in vivo reduced myocardial infarction size in a mouse model. Our findings show a novel mechanism for the rapid mobilisation of neutrophils to peripheral blood from a splenic reserve, independent of classical chemokine signalling, and establish a proof of concept for functional manipulation of EV-communications through genetic alteration of parent cells.