Microglia are yolk sac-derived macrophages residing in the parenchyma of brain and spinal cord, where they interact with neurons and other glial. After different conditioning paradigms and bone marrow (BM) or hematopoietic stem cell (HSC) transplantation, graft-derived cells seed the brain and persistently contribute to the parenchymal brain macrophage compartment. Here we establish that graft-derived macrophages acquire, over time, microglia characteristics, including ramified morphology, longevity, radio-resistance and clonal expansion. However, even after prolonged CNS residence, transcriptomes and chromatin accessibility landscapes of engrafted, BM-derived macrophages remain distinct from yolk sac-derived host microglia. Furthermore, engrafted BM-derived cells display discrete responses to peripheral endotoxin challenge, as compared to host microglia. In human HSC transplant recipients, engrafted cells also remain distinct from host microglia, extending our finding to clinical settings. Collectively, our data emphasize the molecular and functional heterogeneity of parenchymal brain macrophages and highlight potential clinical implications for HSC gene therapies aimed to ameliorate lysosomal storage disorders, microgliopathies or general monogenic immuno-deficiencies. Irradiation depletes brain microglia cells and induces replenishment of the pool by bone marrow (BM)-derived macrophage. Here the authors show, using mouse BM chimera, that BM-derived macrophages establish long-term residency in the brain, but remain distinct from resident microglia in their transcriptome and gene accessibility landscape.

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Engrafted parenchymal brain macrophages differ from microglia in transcriptome, chromatin landscape and response to challenge

Intracellular MAPK (mitogen-activated protein kinase) signalling cascades probably play an important role in the pathogenesis of cardiac and vascular disease. A substantial amount of basic science research has defined many of the details of MAPK pathway organization and activation, but the role of individual signalling proteins in the pathogenesis of various cardiovascular diseases is still being elucidated. In the present review, the role of the MAPKs ERK (extracellular signal-regulated kinase), JNK (c-Jun N-terminal kinase) and p38 MAPK in cardiac hypertrophy, cardiac remodelling after myocardial infarction, atherosclerosis and vascular restenosis will be examined, with attention paid to genetically modified murine model systems and to the use of pharmacological inhibitors of protein kinases. Despite the complexities of this field of research, attractive targets for pharmacological therapy are emerging.

MAPK signalling in cardiovascular health and disease: molecular mechanisms and therapeutic targets

Liver membranes from young beagle dogs were found to possess binding sites that resemble the low density lipo- protein (LDL) receptors originally described in cultured human fibroblasts. Treatment of the dogs with colestipol (a bile acid se- questrant) and mevinolin (a cholesterol synthesis inhibitor) pro- duced a 3-fold increase in LDL binding activity. This increase cor- related with a 2-fold increase in the fractional catabolic rate for intravenously administered human or canine "LI-labeled LDL, suggesting that the increased hepatic receptors were responsible for the enhanced clearance of LDL from plasma. The hepatic lipo- protein receptors of control and drug-treated dogs resembled hu- man fibroblast LDL receptors in that they bound apoprotein E- containing lipoproteins, such as very low density lipoproteins and a subfraction of high density lipoproteins (HDL ), with 10-fold higher affinity than the apoprotein B-containing lipoprotein LDL; failed to bind canine HDL and human HDL3 which are devoid of apoproteins B and E; failed to bind methylated LDL; required cal- cium; and were destroyed by Pronase. Treatment of dogs with mevinolin not only increased the fractional catabolic rate for LDL but also reduced the synthetic rate for the lipoprotein. The current data suggest that the liver of dogs contains functional LDL recep- tors that are susceptible to metabolic regulation and that a drug- induced increase in the activity of these receptors can contribute to a lowering of plasma levels of LDL-cholesterol.

Regulatory role for hepatic low density lipoprotein receptors in vivo in the dog (cholesterol synthesis inhibitor/mevinolin/bile acid sequestrant/colestipol/catabolism of plasma lipoproteins)

Liposomes in ultrasonic drug and gene delivery

Human glioblastomas harbour a subpopulation of glioblastoma stem cells that drive tumorigenesis. However, the origin of intratumoural functional heterogeneity between glioblastoma cells remains poorly understood. Here we study the clonal evolution of barcoded glioblastoma cells in an unbiased way following serial xenotransplantation to define their individual fate behaviours. Independent of an evolving mutational signature, we show that the growth of glioblastoma clones in vivo is consistent with a remarkably neutral process involving a conserved proliferative hierarchy rooted in glioblastoma stem cells. In this model, slow-cycling stem-like cells give rise to a more rapidly cycling progenitor population with extensive self-maintenance capacity, which in turn generates non-proliferative cells. We also identify rare 'outlier' clones that deviate from these dynamics, and further show that chemotherapy facilitates the expansion of pre-existing drug-resistant glioblastoma stem cells. Finally, we show that functionally distinct glioblastoma stem cells can be separately targeted using epigenetic compounds, suggesting new avenues for glioblastoma-targeted therapy.

Fate mapping of human glioblastoma reveals an invariant stem cell hierarchy

Macrophages are central to the initiation and progression of atherosclerosis and thus can be very appropriate targets for therapy. Cell adhesion molecules mediating monocytes recruitment to the endothelium are attractive therapy targets and their inhibitors are in clinical trials. Macrophage scavenger receptors like SR-A and CD-36 mediate foam cell formation by facilitating the uptake of modified lipids. Peroxisome proliferator-activated receptors (PPAR), liver X receptor (LXR)-mediated signaling, mitogen-activated protein kinase (MAPK) induced phosphorylation events seem to play an important role in this phenomenon. Proteins affecting macrophage cholesterol metabolism and transport, including ATP-binding cassette (ABC) A1, ABCG1, acyl-CoA:cholesterol acyltransferase (ACAT), apolipoprotein A-1 (ApoA-1), neutral cholesteryl ester hydrolase (NCEH) also regulate foam cell formation and are being developed as therapeutic targets by many pharmaceutical companies. Macrophage proliferation and apoptosis are important events controlling inflammatory response, plaque vulnerability, and destabilization. Free cholesterol (FC) activates the macrophage endoplasmic reticulum (ER) stress pathway and apoptosis. Free radicals and nitric oxide also modulate macrophage foam cell formation and apoptosis. Various antioxidants like AGI-1067 and BO-653 are in clinical trials for atherosclerosis treatment. Macrophage matrix metalloproteinase's (MMP's) play a significant role in weakening and rupture of plaques. Efforts are on to develop isoform specific MMP inhibitor. CD-14, MMP-3, ABCA1, Toll-like receptor-4 (TLR-4), lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), arachidonate lipoxygenase-15 (ALOX-15), and Connexin37 polymorphisms and macrophage dysfunction signify their importance in atherosclerosis. Deciphering the role of macrophages in regulating dyslipidemia and inflammation during atherosclerosis is important for developing them as therapeutic targets.

Macrophages: An elusive yet emerging therapeutic target of atherosclerosis

The recent failure of candidate drugs like cholesterol ester transfer protein (CETP) and acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors calls for a revised approach for screening anti-atherosclerotic drugs and development of new models of atherosclerosis. For this it is important to understand the mechanism of the disease in a particular model. Models simultaneously showing hyperlipidemia, inflammation and associated complications of diabetes and hypertension will serve the purpose better as they mimic the actual clinical condition. Besides this, analyzing candidate molecules in vivo, in vitro and at various levels of atherosclerosis progression is important. Models based on various cells and process involved in atherosclerosis should be used for screening candidate molecules. The challenge lies in bridging the gap between genetically friendly small animal and human-like bigger animal models. Sequencing of the mouse and human genome, development of a single nucleotide polymorphism (SNP) database and in silico quantitative trait loci (QTL) linkage analysis may enhance the understanding of atherosclerosis and help develop new therapeutic targets.

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Models to Study Atherosclerosis: A Mechanistic Insight

In monocytic cells, Toll-like receptor 4 (TLR4)- and TLR2-induced reactive oxygen species (ROS) cause oxidative stress and inflammatory response; however, the mechanism is not well understood. The present study investigated the role of interleukin-1 receptor-associated kinase (IRAK), extracellular signal-regulated kinase (ERK), p67phox and Nox-2 in TLR4- and TLR2-induced ROS generation during interleukin-1 beta (IL-1β) transcription, processing, and secretion. An IRAK1/4 inhibitor, U0126, PD98059, an NADPH oxidase inhibitor (diphenyleneiodonium (DPI)), and a free radical scavenger (N-acetyl cysteine (NAC))-attenuated TLR4 (lipopolysaccharide (LPS))- and TLR2 (Pam3csk4)-induced ROS generation and IL-1β production in THP-1 and primary human monocytes. An IRAK1/4 inhibitor and siRNA-attenuated LPS- and Pam3csk4-induced ERK-IRAK1 association and ERK phosphorylation and activity. LPS and Pam3csk4 also induced IRAK1/4-, ERK- and ROS-dependent activation of activator protein-1 (AP-1), IL-1β transcription, and IL-1β processing because significant inhibition in AP-1 activity, IL-1β transcription, Pro- and mature IL-β expression, and caspase-1 activity was observed with PD98059, U0126, DPI, NAC, an IRAK1/4 inhibitor, tanshinone IIa, and IRAK1 siRNA treatment. IRAK-dependent ERK-p67phox interaction, p67phox translocation, and p67phox-Nox-2 interaction were observed. Nox-2 siRNA significantly reduced secreted IL-1β, IL-1β transcript, pro- and mature IL-1β expression, and caspase-1 activity indicating a role for Nox-2 in LPS- and Pam3csk4-induced IL-1β production, transcription, and processing. In the present study, we demonstrate that the TLR4- and TLR2-induced IRAK-ERK pathway cross-talks with p67phox-Nox-2 for ROS generation, thus regulating IL-1β transcription and processing in monocytic cells.

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The IRAK-ERK-p67phox-Nox-2 axis mediates TLR4, 2-induced ROS production for IL-1β transcription and processing in monocytes.

The role of IL-1R-associated kinase (IRAK)1 and its interaction with protein kinase C (PKC)δ in monocytes to regulate IL-1β production has not been reported so far. The present study thus investigates such mechanisms in the THP1 cell line and human monocytes. PMA treatment to THP1 cells induced CD11b, TLR2, TLR4, CD36, IRAK1, IRAK3, and IRAK4 expression, IRAK1 kinase activity, PKCδ and JNK phosphorylation, AP-1 and NF-κB activation, and secretory IL-1β production. Moreover, PMA-induced IL-1β production was significantly reduced in the presence of TLR2, TLR4, and CD11b Abs. Rottlerin, a PKCδ-specific inhibitor, significantly reduced PMA-induced IL-1β production as well as CD11b, TLR2 expression, and IRAK1-JNK activation. In PKCδ wild-type overexpressing THP1 cells, IRAK1 kinase activity and IL-1β production were significantly augmented, whereas recombinant inactive PKCδ and PKCδ small interfering RNA significantly inhibited basal and PMA-induced IRAK1 activation and IL-1β production. Endogenous PKCδ-IRAK1 interaction was observed in quiescent cells, and this interaction was regulated by PMA. IRAK1/4 inhibitors, their small interfering RNAs, and JNK inhibitor also attenuated PMA-induced IL-1β production. NF-κB activation inhibitor and SN50 peptide inhibitor, however, failed to affect PMA-induced IL-1β production. A similar role of IRAK1 in IL-1β production and its regulation by PKCδ was evident in the primary human monocytes, thus signifying the importance of our finding. To our knowledge, the results obtained demonstrate for the first time that IRAK1 and PKCδ functionally interact to regulate IL-1β production in monocytic cells. A novel mechanism of IL-1β production that involves TLR2, CD11b, and the PKCδ/IRAK1/JNK/AP-1 axis is thus being proposed.

Rajiv Lochan Tiwari

Papers

Macrophages: An elusive yet emerging therapeutic target of atherosclerosis

Models to Study Atherosclerosis: A Mechanistic Insight

The IRAK-ERK-p67phox-Nox-2 axis mediates TLR4, 2-induced ROS production for IL-1β transcription and processing in monocytes.

IL-1R–Associated Kinase-1 Mediates Protein Kinase Cδ-Induced IL-1β Production in Monocytes

Macrophages: An Elusive Yet Emerging Therapeutic Target of Atherosclerosis