Hwee Ying Lim

DC mobilization from the skin requires docking to immobilized CCL21 on lymphatic endothelium and intralymphatic crawling

Dermal DC mobilization requires docking to CCL21 on lymphatic endothelium

Lymphatic Vessels Are Essential for the Removal of Cholesterol from Peripheral Tissues by SR-BI-Mediated Transport of HDL

Removal of cholesterol from peripheral tissues to the bloodstream via reverse cholesterol transport (RCT) is a process of major biological importance. Here we demonstrate that lymphatic drainage is required for RCT. We have previously shown that hypercholesterolemia in mice is associated with impaired lymphatic drainage and increased lipid accumulation in peripheral tissues. We now show that restoration of lymphatic drainage in these mice significantly improves cholesterol clearance. Conversely, obstruction of lymphatic vessels in wild-type mice significantly impairs RCT. Finally, we demonstrate using silencing RNA interference, neutralizing antibody, and transgenic mice that removal of cholesterol by lymphatic vessels is dependent on the uptake and transcytosis of HDL by scavenger receptor class B type I expressed on lymphatic endothelium. Collectively, this study challenges the current view that lymphatic endothelium is a passive exchange barrier for cholesterol transport and provides further evidence for its interplay with lipid biology in health and disease.

Hypercholesterolemic mice exhibit lymphatic vessel dysfunction and degeneration.

Lymphatic vessels are essential for lipid absorption and transport. Despite increasing numbers of observations linking lymphatic vessels and lipids, little research has been devoted to address how dysregulation of lipid balance in the blood, ie, dyslipidemia, may affect the functional biology of lymphatic vessels. Here, we show that hypercholesterolemia occurring in apolipoprotein E-deficient (apoE(-/-)) mice is associated with tissue swelling, lymphatic leakiness, and decreased lymphatic transport of fluid and dendritic cells from tissue. Lymphatic dysfunction results in part from profound structural abnormalities in the lymphatic vasculature: namely, initial lymphatic vessels were greatly enlarged, and collecting vessels developed notably decreased smooth muscle cell coverage and changes in the distribution of lymphatic vessel endothelial hyaluronic acid receptor-1 (LYVE-1). Our results provide evidence that hypercholesterolemia in adult apoE(-/-) mice is associated with a degeneration of lymphatic vessels that leads to decreased lymphatic drainage and provides an explanation for why dendritic cell migration and, thus, immune priming, are compromised in hypercholesterolemic mice.

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 (MEFPTEN−/−) with hyper-activated Akt as a cell model, we observed a higher respiratory capacity in MEFPTEN−/− compared to the wildtype (MEFWT). The respiratory phenotype observed in MEFPTEN−/− was reproduced in MEFWT 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 MEFWT, 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 MEFPTEN−/− and MEFWT. Similarly, mRNA levels of the same subunits of the RCs detected in Western blots were not significantly different between MEFPTEN−/− and MEFWT 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 MEFPTEN−/−, 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.

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.

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.

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.

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

SUMMARY 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. Graphical Abstract Figure. No caption available. HighlightsLYVE‐1+ macrophages coat murine and human blood vessels harboring smooth muscle cellsDeficiency in LYVE‐1+ macrophages induces arterial stiffness and collagen depositionLYVE‐1+ macrophages degrade collagen on smooth muscle cells via pericellular MMP‐9LYVE‐1 on macrophage engages HA on smooth muscle for collagen degradation &NA; Macrophages are essential to maintain tissue homeostasis. Lim and colleagues demonstrate that perivascular LYVE‐1‐expressing macrophages prevent arterial stiffness by controlling the expression of collagen in vascular smooth muscle cells, a process dependent on the engagement of LYVE‐1 with hyaluronan on smooth muscle cells.