Early intermittent hyperlipidaemia alters tissue macrophages to fuel atherosclerosis.

Hyperlipidaemia is a major risk factor of atherosclerotic cardiovascular disease (ASCVD). Risk of cardiovascular events depends on cumulative lifetime exposure to low-density lipoprotein cholesterol (LDL-C) and, independently, on the time course of exposure to LDL-C, with early exposure being associated with a higher risk1. Furthermore, LDL-C fluctuations are associated with ASCVD outcomes2-4. However, the precise mechanisms behind this increased ASCVD risk are not understood. Here we find that early intermittent feeding of mice on a high-cholesterol Western-type diet (WD) accelerates atherosclerosis compared with late continuous exposure to the WD, despite similar cumulative circulating LDL-C levels. We find that early intermittent hyperlipidaemia alters the number and homeostatic phenotype of resident-like arterial macrophages. Macrophage genes with altered expression are enriched for genes linked to human ASCVD in genome-wide association studies. We show that LYVE1+ resident macrophages are atheroprotective, and identify biological pathways related to actin filament organization, of which alteration accelerates atherosclerosis. Using the Young Finns Study, we show that exposure to cholesterol early in life is significantly associated with the incidence and size of carotid atherosclerotic plaques in mid-adulthood. In summary, our results identify early intermittent exposure to cholesterol as a strong determinant of accelerated atherosclerosis, highlighting the importance of optimal control of hyperlipidaemia early in life, and providing insights into the underlying biological mechanisms. This knowledge will be essential to designing effective therapeutic strategies to combat ASCVD.

Intravenous iron supplementation in heart failure patients induces temporary endothelial dysfunction with release of endothelial microvesicles.

Background: Intravenous iron supplementation is an established therapy for patients with heart failure (HF) and concomitant iron deficiency reducing the risk of HF hospitalization. However, concerns persist regarding potential adverse vascular effects, since iron may induce oxidative stress, inflammation, and apoptosis of endothelial cells. To assess endothelial health following ferric carboxymaltose (FCM) administration, we analyzed the profile of circulating endothelial microvesicles (EMVs) and endothelial progenitor cells (EPCs) in a cohort of 23 HF patients using flow cytometry. Results: Compared to healthy subjects, baseline levels of CD31+/CD41- EMVs were higher and EMVs featured a more apoptotic phenotype in HF patients. Following FCM administration, EMV levels showed a rapid but transient increase and displayed an altered phenotype profile with dominant augmentation of EMVs expressing inducible markers CD62E and CD54, indicating endothelial inflammatory activation and injury. Levels of circulating vasoregenerative CD45lowCD34+KDR+ EPCs were lower in HF patients and FCM application resulted in an early decrease of EPCs followed by substantial mobilization into the circulation after one week. Levels of EMVs and EPCs returned to baseline values within two and four weeks, respectively. HF patients with additional chronic kidney disease showed an elevated EMV/EPC ratio and diminished EPC mobilization, suggesting impaired vascular repair capacity. Providing a mechanistic link, in vitro experiments with cultured endothelial cells revealed that FCM dose-dependently promotes endothelial apoptosis, increases expression of adhesion molecules and CXCL12, and triggers generation of EMVs. Conclusion: Intravenous iron supplementation with FCM in HF patients induces a biphasic response with initial increased release of CD62E+ and CD54+ enriched EMVs and subsequent mobilization of EPCs, indicating endothelial dysfunction upon FCM and suggesting consecutive engagement of a defense program aimed to reconstitute vascular health.

LL-37-mediated activation of host receptors is critical for defense against group A streptococcal infection.

Group A Streptococcus (GAS) causes diverse human diseases, including life-threatening soft-tissue infections. It is accepted that the human antimicrobial peptide LL-37 protects the host by killing GAS. Here, we show that GAS extracellular protease ScpC N-terminally cleaves LL-37 into two fragments of 8 and 29 amino acids, preserving its bactericidal activity. At sub-bactericidal concentrations, the cleavage inhibits LL-37-mediated neutrophil chemotaxis, shortens neutrophil lifespan, and eliminates P2X7 and EGF receptors' activation. Mutations at the LL-37 cleavage site protect the peptide from ScpC-mediated splitting, maintaining all its functions. The mouse LL-37 ortholog CRAMP is neither cleaved by ScpC nor does it activate P2X7 or EGF receptors. Treating wild-type or CRAMP-null mice with sub-bactericidal concentrations of the non-cleavable LL-37 analogs promotes GAS clearance that is abolished by the administration of either P2X7 or EGF receptor antagonists. We demonstrate that LL-37-mediated activation of host receptors is critical for defense against GAS soft-tissue infections.

The indirect antiangiogenic effect of IL-37 in the tumor microenvironment.

IL-37, a newly identified IL-1 family cytokine, has been shown to play an important role in inflammatory diseases, autoimmune diseases, and carcinogenesis. IL-37 has been suggested to suppress tumoral angiogenesis, whereas some publications showed that IL-37 promoted angiogenesis through TGF-ß signaling in both physiologic and pathologic conditions. Therefore, the function of IL-37 in tumoral angiogenesis is not clear and the underlying mechanism is not known. In this current study, we investigated the direct role of IL-37 on endothelial cells, as well as its indirect effect on angiogenesis through functioning on tumor cells both in vitro and in vivo. We found that IL-37 treatment directly promoted HUVEC migration and tubule formation, indicating IL-37 as a proangiogenic factor. Surprisingly, the supernatants from IL-37 overexpressing tumor cell line promoted HUVEC apoptosis and inhibited its migration and tubule formation. Furthermore, we demonstrated that IL-37 suppressed tumor angiogenesis in a murine orthotopic hepatocellular carcinoma model, suggesting its dominant antiangiogenesis role in vivo. Moreover, microarray and qPCR analysis demonstrated that IL-37 reduced the expressions of proangiogenic factors and increased the expressions of antiangiogenic factors by tumor cells. Matrix metalloproteinase (MMP)2 expression was significantly decreased by IL-37 in both cell lines and murine tumor models. MMP9 and vascular endothelial growth factor expressions were also reduced in murine tumors overexpressing IL-37, as well as in cell lines overexpressing IL-37 under hypoxic conditions. In conclusion, although IL-37 could exert direct proangiogenic effects on endothelial cells, it plays an antiangiogenic role via modulating proangiogenic and antiangiogenic factor expressions by tumor cells in the tumor microenvironment.

Two distinct interstitial macrophage populations coexist across tissues in specific subtissular niches.

Macrophages are a heterogeneous cell population involved in tissue homeostasis, inflammation, and various pathologies. Although the major tissue-resident macrophage populations have been extensively studied, interstitial macrophages (IMs) residing within the tissue parenchyma remain poorly defined. Here we studied IMs from murine lung, fat, heart, and dermis. We identified two independent IM subpopulations that are conserved across tissues: Lyve1loMHCIIhiCX3CR1hi (Lyve1loMHCIIhi) and Lyve1hiMHCIIloCX3CR1lo (Lyve1hiMHCIIlo) monocyte-derived IMs, with distinct gene expression profiles, phenotypes, functions, and localizations. Using a new mouse model of inducible macrophage depletion (Slco2b1 flox/DTR), we found that the absence of Lyve1hiMHCIIlo IMs exacerbated experimental lung fibrosis. Thus, we demonstrate that two independent populations of IMs coexist across tissues and exhibit conserved niche-dependent functional programming.

Hyaluronan Receptor LYVE-1-Expressing Macrophages Maintain Arterial Tone through Hyaluronan-Mediated Regulation of Smooth Muscle Cell Collagen.

The maintenance of appropriate arterial tone is critically important for normal physiological arterial function. However, the cellular and molecular mechanisms remain poorly defined. Here, we have shown that in the mouse aorta, resident macrophages prevented arterial stiffness and collagen deposition in the steady state. Using phenotyping, transcriptional profiling, and targeted deletion of Csf1r, we have demonstrated that these macrophages-which are a feature of blood vessels invested with smooth muscle cells (SMCs) in both mouse and human tissues-expressed the hyaluronan (HA) receptor LYVE-l. Furthermore, we have shown they possessed the unique ability to modulate collagen expression in SMCs by matrix metalloproteinase MMP-9-dependent proteolysis through engagement of LYVE-1 with the HA pericellular matrix of SMCs. Our study has unveiled a hitherto unknown homeostatic contribution of arterial LYVE-1+ macrophages through the control of collagen production by SMCs and has identified a function of LYVE-1 in leukocytes.

NKT Cell Hyporesponsiveness Leads to Unrestrained Accumulation of Marginal Zone B Cells in Hypercholesterolemic Apolipoprotein E-Deficient Mice.

Recently, the role of B cells in atherosclerosis has gained more attention but studies have mainly focused on B1 and follicular B cell subsets. Therefore, the contribution of marginal zone (MZ) B cells in experimental atherosclerosis remains elusive. In the current study, we examined the MZ B cell compartment in atherosclerotic apoE-deficient (apoE-/-) mice and found that hypercholesterolemia in these mice was associated with an increased number and percentage of MZ B cells. This aberrant accumulation of MZ B cells was not associated with alterations in their development or increased proliferation but was due to decreased apoptotic cell death. This decrease in MZ B cell death in apoE-/- mice was associated with the reduced capacity of invariant NKT (iNKT) cells to produce IFN-γ and IL-4 after activation. Lowering cholesterol plasma levels with ezetimibe in apoE-/- mice reversed iNKT function and MZ B cell accumulation. To elucidate the mechanism whereby iNKT cells control MZ B cell accumulation in apoE-/- mice, we performed an adoptive transfer of iNKT cells and found that only wild-type iNKT cells but not IFN-γ-/- iNKT cells reversed MZ B cell accumulation in apoE-/- recipient mice. Our findings reveal that lipid changes associated with atherosclerotic disease induce decreased production of IFN-γ by iNKT, which in turn leads to aberrant accumulation of MZ B cells. This study further extends the importance of iNKT cells in regulating MZ B cell compartment.

Neutrophils Self-Regulate Immune Complex-Mediated Cutaneous Inflammation through CXCL2.

Deposition of immune complexes (ICs) in tissues triggers acute inflammatory pathology characterized by massive neutrophil influx leading to edema and hemorrhage, and is especially associated with vasculitis of the skin, but the mechanisms that regulate this type III hypersensitivity process remain poorly understood. Here, using a combination of multiphoton intravital microscopy and genomic approaches, we re-examined the cutaneous reverse passive Arthus reaction and observed that IC-activated neutrophils underwent transmigration, triggered further IC formation, and transported these ICs into the interstitium, whereas neutrophil depletion drastically reduced IC formation and ameliorated vascular leakage in vivo. Thereafter, we show that these neutrophils expressed high levels of CXCL2, which further amplified neutrophil recruitment and activation in an autocrine and/or paracrine manner. Notably, CXCL1 expression was restricted to tissue-resident cell types, but IC-activated neutrophils may also indirectly, via soluble factors, modulate macrophage CXCL1 expression. Consistent with their distinct cellular origins and localization, only neutralization of CXCL2 but not CXCL1 in the interstitium effectively reduced neutrophil recruitment. In summary, our study establishes that neutrophils are able to self-regulate their own recruitment and responses during IC-mediated inflammation through a CXCL2-driven feed forward loop.

Interplay of metabolizing enzymes and transporter of xenobiotics.

1. Xenobiotics are metabolized and eliminated through the coordinated interplay of their metabolizing enzymes and transporters. However, these two activities in vitro are measured separately, with the addition of ATP as a pre-requisite. 2. We propose a human renal cell-line model which integrates the sulfate and glutathione conjugation of xenobiotics with the efflux of their respective conjugates. Sulfation and glutathionylation represent two major Phase II detoxification of xenobiotics in man. The reactions are catalyzed, respectively, by phenolsulfotransferase and glutathione-S-transferase followed by extrusion of their respective conjugates. 3. Using Ko-143, a specific inhibitor of breast cancer resistance protein (BCRP), an ATP-binding cassette (ABC) transporter, we identified this transporter to be responsible for the efflux of p-cresol sulfate, harmol sulfate and the glutathione conjugate of 1-chloro-2,4-dinitrobenzene. 4. The conjugation-cum-efflux was inhibited by oligomycin and uncouplers, which highlights the role of cellular mitochondria in providing ATP for the biosynthesis of their conjugating agents, 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and reduced glutathione as well as for the transport function of BCRP. 5. The human 786-O renal cell-line provides a "3-in-1" system linking ATP biosynthesis to metabolism of xenobiotics and their ultimate transport and elimination by BCRP; this integrated system was not apparent in other human cell-lines examined.

Metabolic signatures of renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is characterized by the constitutive up-regulation of the hypoxia inducible factor-1. One of its target enzymes, pyruvate dehydrogenase (PDH) kinase 1 (PDHK1) showed increased protein expression in tumor as compared to patient-matched normal tissues. PDHK1 phosphorylated and inhibited PDH whose enzymatic activity was severely diminished, depriving the TCA cycle of acetylCoA. We and others have shown a decrease in the protein expressions of all respiratory complexes alluding to a compromise in oxidative phosphorylation (OXPHOS). On the contrary, we found that key parameters of OXPHOS, namely ATP biosynthesis and membrane potential were consistently measurable in mitochondria isolated from ccRCC tumor tissues. Interestingly, an endogenous mitochondrial membrane potential (MMP) was evident when ADP was added to mitochondria isolated from ccRCC but not in normal tissues. In addition, the MMP elicited in the presence of ADP by respiratory substrates namely malate/glutamate, succinate, α-ketoglutarate and isocitrate was invariably higher in ccRCC. Two additional hallmarks of ccRCC include a loss of uncoupling protein (UCP)-2 and an increase in UCP-3. Based on our data, we proposed that inhibition of UCP3 by ADP could contribute to the endogenous MMP observed in ccRCC and other cancer cells.

A three-dimensional atlas of human dermal leukocytes, lymphatics, and blood vessels.

Dendritic cells (DCs), macrophages (Mφ), and T cells are major components of the skin immune system, but their interstitial spatial organization is poorly characterized. Using four-channel whole-mount immunofluorescence staining of the human dermis, we demonstrated the three-dimensional distribution of CD31(+) blood capillaries, LYVE-1(+) lymphatics, discrete populations of CD11c(+) myeloid DCs, FXIIIa(+) Mφ, and lymphocytes. We showed phenotypic and morphological differences in situ between DCs and Mφ. DCs formed the first dermal cellular layer (0-20 µm beneath the dermoepidermal junction), Mφ were located deeper (40-60 µm), and CD3(+) lymphocytes were observed throughout (0-60 µm). Below this level, DCs, T cells, and the majority of Mφ formed stable perivascular sheaths. Whole-mount imaging revealed the true extent of dermal leukocytes previously underestimated from cross-section views. The total area of apical dermis (0-30 µm) contained approximately 10-fold more myeloid DCs than the entire blood volume of an average individual. Surprisingly, <1% of dermal DCs occupied lymphatics in freshly isolated skin. Dermal DCs rapidly accumulated within lymphatics, but Mφ remained fixed in skin explants cultured ex vivo. The leukocyte architecture observed in normal skin was distorted in inflammation and disease. These studies illustrate the micro-anatomy of dermal leukocytes and provide further insights into their functional organization.

PTEN/Akt signaling controls mitochondrial respiratory capacity through 4E-BP1.

Akt, a serine/threonine kinase has been shown to stimulate glycolysis in cancer cells but its role in mitochondrial respiration is unknown. Using PTEN-knockout mouse embryonic fibroblasts (MEF(PTEN-/-)) with hyper-activated Akt as a cell model, we observed a higher respiratory capacity in MEF(PTEN-/-) compared to the wildtype (MEF(WT)). The respiratory phenotype observed in MEF(PTEN-/-) was reproduced in MEF(WT) by gene silencing of PTEN which substantiated its role in regulating mitochondrial function. The increased activities of the respiratory complexes (RCs) I, III and IV were retained in the same relative proportions as those present in MEF(WT), alluding to a possible co-ordinated regulation by PTEN/Akt. Using LY294002 (a PI3K inhibitor) and Akt inhibitor IV, we showed that the regulation of enzyme activities and protein expressions of the RCs was dependent on PI3K/Akt. There was insignificant difference in the protein expressions of mitochondrial transcription factor: peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) and its downstream targets, the nuclear respiratory factor 1 (NRF-1) and mitochondrial transcription factor A (mtTFA) between MEF(PTEN-/-) and MEF(WT). Similarly, mRNA levels of the same subunits of the RCs detected in Western blots were not significantly different between MEF(PTEN-/-) and MEF(WT) suggesting that the regulation by Akt on mitochondrial function was probably not via gene transcription. On the other hand, a decrease of total 4E-BP1 with a higher expression of its phosphorylated form relative to total 4E-BP1 was found in MEF(PTEN-/-), which inferred that the regulation of mitochondrial respiratory activities by Akt was in part through this protein translation pathway. Notably, gene silencing of 4E-BP1 up-regulated the protein expressions of all RCs and the action of 4E-BP1 appeared to be specific to these mitochondrial proteins. In conclusion, PTEN inactivation bestowed a bioenergetic advantage to the cells by up-regulating mitochondrial respiratory capacity through the 4E-BP1-mediated protein translation pathway.

Expansion of cortical and medullary sinuses restrains lymph node hypertrophy during prolonged inflammation.

During inflammation, accumulation of immune cells in activated lymph nodes (LNs), coupled with a transient shutdown in lymphocyte exit, results in dramatic cellular expansion. Counter-regulatory measures to restrain LN expansion must exist and may include re-establishment of lymphocyte egress to steady-state levels. Indeed, we show in a murine model that egress of lymphocytes from LNs was returned to steady-state levels during prolonged inflammation following initial retention. This restoration in lymphocyte egress was supported by a preferential expansion of cortical and medullary sinuses during late inflammation. Cortical and medullary sinus remodeling during late inflammation was dependent on temporal and spatial changes in vascular endothelial growth factor-A distribution. Specifically, its expression was restricted to the subcapsular space of the LN during early inflammation, whereas its expression was concentrated in the paracortical and medullary regions of the LN at later stages. We next showed that this process was mostly driven by the synergistic cross-talk between fibroblastic reticular cells and interstitial flow. Our data shed new light on the biological significance of LN lymphangiogenesis during prolonged inflammation and further underscore the collaborative roles of stromal cells, immune cells, and interstitial flow in modulating LN plasticity and function.

Respiratory competent mitochondria in human ovarian and peritoneal cancer.

Impaired respiration was proposed by Warburg to be responsible for aerobic glycolysis in cancer cells. However, intact mitochondria isolated from human ovarian and peritoneal cancer tissues exhibit substantive oxidative phosphorylating activities in terms of membrane potential, ATP biosynthesis and oxygen consumption. The specific activities of succinate, malate and glutamate dehydrogenases are comparable to reported values for human skeletal muscle, heart and liver but the rate of ATP production is one order of magnitude lower compared to human skeletal muscle. It was concluded that the TCA cycle is functional in these ovarian cancer tissues which contain OXPHOS competent mitochondria.

Uncoupling of oxidative phosphorylation by curcumin: implication of its cellular mechanism of action.

Curcumin is a phytochemical isolated from the rhizome of turmeric. Recent reports have shown curcumin to have antioxidant, anti-inflammatory and anti-tumor properties as well as affecting the 5'-AMP activated protein kinase (AMPK), mTOR and STAT-3 signaling pathways. We provide evidence that curcumin acts as an uncoupler. Well-established biochemical techniques were performed on isolated rat liver mitochondria in measuring oxygen consumption, F(0)F(1)-ATPase activity and ATP biosynthesis. Curcumin displays all the characteristics typical of classical uncouplers like fccP and 2,4-dinitrophenol. In addition, at concentrations higher than 50 microM, curcumin was found to inhibit mitochondrial respiration which is a characteristic feature of inhibitory uncouplers. As a protonophoric uncoupler and as an activator of F(0)F(1)-ATPase, curcumin causes a decrease in ATP biosynthesis in rat liver mitochondria. The resulting change in ATP:AMP could disrupt the phosphorylation status of the cell; this provides a possible mechanism for its activation of AMPK and its downstream mTOR and STAT-3 signaling.