Retention of the NLRP3 Inflammasome-Primed Neutrophils in the Bone Marrow Is Essential for Myocardial Infarction-Induced Granulopoiesis.

BACKGROUND: Acute myocardial infarction (MI) results in overzealous production and infiltration of neutrophils to the ischemic heart. This is mediated in part by granulopoiesis induced by the S100A8/A9-NLRP3-IL-1ß signaling axis in injury-exposed neutrophils. Despite the transcriptional upregulation of the NLRP3 (Nod Like Receptor Family Pyrin Domain-Containing 3) inflammasome and associated signaling components in neutrophils, the serum levels of IL-1ß (interleukin-1ß), the effector molecule in granulopoiesis, were not affected by MI, suggesting that IL-1ß is not released systemically. We hypothesize that IL-1ß is released locally within the bone marrow (BM) by inflammasome-primed and reverse-migrating neutrophils. METHODS: Using a combination of time-dependent parabiosis and flow cytometry techniques, we first characterized the migration patterns of different blood cell types across the parabiotic barrier. We next induced MI in parabiotic mice by permanent ligation of the left anterior descending artery and examined the ability of injury-exposed neutrophils to permeate the parabiotic barrier and induce granulopoiesis in noninfarcted parabionts. Last, using multiple neutrophil adoptive and BM transplant studies, we studied the molecular mechanisms that govern reverse migration and retention of the primed neutrophils, IL-1ß secretion, and granulopoiesis. Cardiac function was assessed by echocardiography. RESULTS: MI promoted greater accumulation of the inflammasome-primed neutrophils in the BM. Introducing a time-dependent parabiotic barrier to the free movement of neutrophils inhibited their ability to stimulate granulopoiesis in the noninfarcted parabionts. Previous priming of the NLRP3 inflammasome is not a prerequisite, but the presence of a functional CXCR4 (C-X-C-motif chemokine receptor 4) on the primed-neutrophils and elevated serum S100A8/A9 levels are necessary for homing and retention of the reverse-migrating neutrophils. In the BM, the primed-neutrophils secrete IL-1ß through formation of gasdermin D pores and promote granulopoiesis. Pharmacological and genetic strategies aimed at the inhibition of neutrophil homing or release of IL-1ß in the BM markedly suppressed MI-induced granulopoiesis and improved cardiac function. CONCLUSIONS: Our data reveal a new paradigm of how circulatory cells establish a direct communication between organs by delivering signaling molecules (eg, IL-1ß) directly at the sites of action rather through systemic release. We suggest that this pathway may exist to limit the off-target effects of systemic IL-1ß release.

Neutrophil-Derived S100A8/A9 Amplify Granulopoiesis After Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) triggers myelopoiesis, resulting in heightened production of neutrophils. However, the mechanisms that sustain their production and recruitment to the injured heart are unclear. METHODS: Using a mouse model of the permanent ligation of the left anterior descending artery and flow cytometry, we first characterized the temporal and spatial effects of MI on different myeloid cell types. We next performed global transcriptome analysis of different cardiac cell types within the infarct to identify the drivers of the acute inflammatory response and the underlying signaling pathways. Using a combination of genetic and pharmacological strategies, we identified the sequelae of events that led to MI-induced myelopoiesis. Cardiac function was assessed by echocardiography. The association of early indexes of neutrophilia with major adverse cardiovascular events was studied in a cohort of patients with acute MI. RESULTS: Induction of MI results in rapid recruitment of neutrophils to the infarct, where they release specific alarmins, S100A8 and S100A9. These alarmins bind to the Toll-like receptor 4 and prime the nod-like receptor family pyrin domain-containing 3 inflammasome in naïve neutrophils and promote interleukin-1ß secretion. The released interleukin-1ß interacts with its receptor (interleukin 1 receptor type 1) on hematopoietic stem and progenitor cells in the bone marrow and stimulates granulopoiesis in a cell-autonomous manner. Genetic or pharmacological strategies aimed at disruption of S100A8/A9 and their downstream signaling cascade suppress MI-induced granulopoiesis and improve cardiac function. Furthermore, in patients with acute coronary syndrome, higher neutrophil count on admission and after revascularization correlates positively with major adverse cardiovascular disease outcomes. CONCLUSIONS: Our study provides novel evidence for the primary role of neutrophil-derived alarmins (S100A8/A9) in dictating the nature of the ensuing inflammatory response after myocardial injury. Therapeutic strategies aimed at disruption of S100A8/A9 signaling or their downstream mediators (eg, nod-like receptor family pyrin domain-containing 3 inflammasome, interleukin-1ß) in neutrophils suppress granulopoiesis and may improve cardiac function in patients with acute coronary syndrome.

Bone Marrow-Derived Cells Restore Functional Integrity of the Gut Epithelial and Vascular Barriers in a Model of Diabetes and ACE2 Deficiency.

RATIONALE: There is incomplete knowledge of the impact of bone marrow cells on the gut microbiome and gut barrier function. OBJECTIVE: We postulated that diabetes mellitus and systemic ACE2 (angiotensin-converting enzyme 2) deficiency would synergize to adversely impact both the microbiome and gut barrier function. METHODS AND RESULTS: Bacterial 16S rRNA sequencing and metatranscriptomic analysis were performed on fecal samples from wild-type, ACE2-/y, Akita (type 1 diabetes mellitus), and ACE2-/y-Akita mice. Gut barrier integrity was assessed by immunofluorescence, and bone marrow cell extravasation into the small intestine was evaluated by flow cytometry. In the ACE2-/y-Akita or Akita mice, the disrupted barrier was associated with reduced levels of myeloid angiogenic cells, but no increase in inflammatory monocytes was observed within the gut parenchyma. Genomic and metatranscriptomic analysis of the microbiome of ACE2-/y-Akita mice demonstrated a marked increase in peptidoglycan-producing bacteria. When compared with control cohorts treated with saline, intraperitoneal administration of myeloid angiogenic cells significantly decreased the microbiome gene expression associated with peptidoglycan biosynthesis and restored epithelial and endothelial gut barrier integrity. Also indicative of diabetic gut barrier dysfunction, increased levels of peptidoglycan and FABP-2 (intestinal fatty acid-binding protein 2) were observed in plasma of human subjects with type 1 diabetes mellitus (n=21) and type 2 diabetes mellitus (n=23) compared with nondiabetic controls (n=23). Using human retinal endothelial cells, we determined that peptidoglycan activates a noncanonical TLR-2 (Toll-like receptor 2) associated MyD88 (myeloid differentiation primary response protein 88)-ARNO (ADP-ribosylation factor nucleotide-binding site opener)-ARF6 (ADP-ribosylation factor 6) signaling cascade, resulting in destabilization of p120-catenin and internalization of VE-cadherin as a mechanism of deleterious impact of peptidoglycan on the endothelium. CONCLUSIONS: We demonstrate for the first time that the defect in gut barrier function and dysbiosis in ACE2-/y-Akita mice can be favorably impacted by exogenous administration of myeloid angiogenic cells.

Characterizing the Retinal Phenotype in the High-Fat Diet and Western Diet Mouse Models of Prediabetes.

We sought to delineate the retinal features associated with the high-fat diet (HFD) mouse, a widely used model of obesity. C57BL/6 mice were fed either a high-fat (60% fat; HFD) or low-fat (10% fat; LFD) diet for up to 12 months. The effect of HFD on body weight and insulin resistance were measured. The retina was assessed by electroretinogram (ERG), fundus photography, permeability studies, and trypsin digests for enumeration of acellular capillaries. The HFD cohort experienced hypercholesterolemia when compared to the LFD cohort, but not hyperglycemia. HFD mice developed a higher body weight (60.33 g vs. 30.17g, p < 0.0001) as well as a reduced insulin sensitivity index (9.418 vs. 62.01, p = 0.0002) compared to LFD controls. At 6 months, retinal functional testing demonstrated a reduction in a-wave and b-wave amplitudes. At 12 months, mice on HFD showed evidence of increased retinal nerve infarcts and vascular leakage, reduced vascular density, but no increase in number of acellular capillaries compared to LFD mice. In conclusion, the HFD mouse is a useful model for examining the effect of prediabetes and hypercholesterolemia on the retina. The HFD-induced changes appear to occur slower than those observed in type 2 diabetes (T2D) models but are consistent with other retinopathy models, showing neural damage prior to vascular changes.

S100A8/A9 in Myocardial Infarction.

S100A8/A9 represents a novel biomarker and therapeutic target in sterile inflammatory diseases. Among the various S100 proteins, S100A8 and S100A9 have been shown to be the most important of all the damage-associated molecular pattern (DAMP) proteins in sterile inflammatory conditions such as diabetes, cardiovascular disease, autoimmune disorders, etc. We present here methods to quantify S100A8/A9 expression in various tissues in mouse models of myocardial infarction (MI) using flow cytometry (FC), immunofluorescence, quantitative real-time polymerase chain reaction (q-RT-PCR), and enzyme-linked immunosorbent assays (ELISA).

Exercise Mediated Nrf2 Signaling Protects the Myocardium From Isoproterenol-Induced Pathological Remodeling.

Although exercise derived activation of Nrf2 signaling augments myocardial antioxidant signaling, the molecular mechanisms underlying the benefits of moderate exercise training (MET) in the heart remain elusive. Here we hypothesized that exercise training stabilizes Nrf2-dependent antioxidant signaling, which then protects the myocardium from isoproterenol-induced damage. The present study assessed the effects of 6 weeks of MET on the Nrf2/antioxidant function, glutathione redox state, and injury in the myocardium of C57/BL6J mice that received isoproterenol (ISO; 50 mg/kg/day for 7 days). ISO administration significantly reduced the Nrf2 promoter activity (p < 0.05) and downregulated the expression of cardiac antioxidant genes (Gclc, Nqo1, Cat, Gsr, and Gst-µ) in the untrained (UNT) mice. Furthermore, increased oxidative stress with severe myocardial injury was evident in UNT+ISO when compared to UNT mice receiving PBS under basal condition. Of note, MET stabilized the Nrf2-promoter activity and upheld the expression of Nrf2-dependent antioxidant genes in animals receiving ISO, and attenuated the oxidative stress-induced myocardial damage. Echocardiography analysis revealed impaired diastolic ventricular function in UNT+ISO mice, but this was partially normalized in the MET animals. Interestingly, while there was a marginal reduction in ubiquitinated proteins in MET mice that received ISO, the pathological signs were attenuated along with near normal cardiac function in response to exercise training. Thus, moderate intensity exercise training conferred protection against ISO-induced myocardial injury by augmentation of Nrf2-antioxidant signaling and attenuation of isoproterenol-induced oxidative stress.

Genetic Deletion of Syndecan-4 Alters Body Composition, Metabolic Phenotypes, and the Function of Metabolic Tissues in Female Mice Fed A High-Fat Diet.

Syndecans are transmembrane proteoglycans that, like integrins, bind to components of the extracellular matrix. Previously, we showed significant associations of genetic variants in the Syndecan-4 (SDC4) gene with intra-abdominal fat, fasting plasma glucose levels, and insulin sensitivity index in children, and with fasting serum triglyceride levels in healthy elderly subjects. An independent study also reported a correlation between SDC4 and the risk of coronary artery disease in middle-aged patients. Here, we investigated whether deletion of Sdc4 promotes metabolic derangements associated with diet-induced obesity by feeding homozygous male and female Sdc4-deficient (Sdc4-/-) mice and their age-matched wild-type (WT) mice a high-fat diet (HFD). We found that WT and Sdc4-/- mice gained similar weight. However, while no differences were observed in males, HFD-fed female Sdc4-/- mice exhibited a higher percentage of body fat mass than controls and displayed increased levels of plasma total cholesterol, triglyceride, and glucose, as well as reduced whole-body insulin sensitivity. Additionally, they had an increased adipocyte size and macrophage infiltration in the visceral adipose tissue, and higher triglyceride and fatty acid synthase levels in the liver. Together with our previous human genetic findings, these results provide evidence of an evolutionarily conserved role of SDC4 in adiposity and its complications.