TREML4 Promotes Inflammatory Programs in Human and Murine Macrophages and Alters Atherosclerosis Lesion Composition in the Apolipoprotein E Deficient Mouse.

The Triggering Receptor Expressed on Myeloid cells-like 4 (TREML4) is a member of the TREM receptor family, known modulators of inflammatory responses. We have previously found that TREML4 expression positively correlates with human coronary arterial calcification (CAC). However, the role of TREML4 in the pathogenesis of cardiovascular disease remains incompletely defined. Since macrophages play a key role in inflammatory conditions, we investigated if activated macrophages selectively expressed TREML4 and found that carriage of either one of the eQTL SNP's previously associated with increased TREML4 expression conferred higher expression in human inflammatory macrophages (M1) compared to alternatively activated macrophages (M2). Furthermore, we found that TREML4 expression in human M1 dysregulated several inflammatory pathways related to leukocyte activation, apoptosis and extracellular matrix degradation. Similarly, murine M1 expressed substantial levels of Treml4, as did oxLDL treated macrophages. Transcriptome analysis confirmed that murine Treml4 controls the expression of genes related to inflammation and lipid regulation pathways, suggesting a possible role in atherosclerosis. Analysis of Apoe-/-/Treml4-/- mice showed reduced plaque burden and lesion complexity as indicated by decreased stage scores, macrophage content and collagen deposition. Finally, transcriptome analysis of oxLDL-loaded murine macrophages showed that Treml4 represses a specific set of genes related to carbohydrate, ion and amino acid membrane transport. Metabolomic analysis confirmed that Treml4 deficiency may promote a beneficial relationship between iron homeostasis and glucose metabolism. Together, our results suggest that Treml4 plays a role in the development of cardiovascular disease, as indicated by Treml4-dependent dysregulation of macrophage inflammatory pathways, macrophage metabolism and promotion of vulnerability features in advanced lesions.

Orally administered ß-glucan attenuates the Th2 response in a model of airway hypersensitivity.

ß-Glucan is a polysaccharide that can be extracted from fungal cell walls. Wellmune WGP(®), a preparation of ß-1,3/1,6-glucans, is a dietary supplement that has immunomodulating properties. Here we investigated the effect WGP had on a mouse model of asthma. OVA-induced asthma in mice is characterized by infiltration of eosinophils into the lung, production of Th2 cytokines and IgE. Daily oral administration of WGP (400 µg) significantly reduced the influx of eosinophils into the lungs of OVA-challenged mice compared to control mice. In addition, WGP inhibited pulmonary production of Th2 cytokines (IL-4, IL-5, IL-13), however serum IgE levels were unaffected by WGP treatment. These data indicate that WGP could potentially be useful as an oral supplement for some asthma patients, however, it would need to be combined with therapies that target other aspects of the disease such as IgE levels. As such, further studies that examine the potential of WGP in combination with other therapies should be explored.

IFN-γ causes aplastic anemia by altering hematopoietic stem/progenitor cell composition and disrupting lineage differentiation.

Aplastic anemia (AA) is characterized by hypocellular marrow and peripheral pancytopenia. Because interferon gamma (IFN-γ) can be detected in peripheral blood mononuclear cells of AA patients, it has been hypothesized that autoreactive T lymphocytes may be involved in destroying the hematopoietic stem cells. We have observed AA-like symptoms in our IFN-γ adenylate-uridylate-rich element (ARE)-deleted (del) mice, which constitutively express a low level of IFN-γ under normal physiologic conditions. Because no T-cell autoimmunity was observed, we hypothesized that IFN-γ may be directly involved in the pathophysiology of AA. In these mice, we did not detect infiltration of T cells in bone marrow (BM), and the existing T cells seemed to be hyporesponsive. We observed inhibition in myeloid progenitor differentiation despite an increase in serum levels of cytokines involved in hematopoietic differentiation and maturation. Furthermore, there was a disruption in erythropoiesis and B-cell differentiation. The same phenomena were also observed in wild-type recipients of IFN-γ ARE-del BM. The data suggest that AA occurs when IFN-γ inhibits the generation of myeloid progenitors and prevents lineage differentiation, as opposed to infiltration of activated T cells. These results may be useful in improving treatment as well as maintaining a disease-free status.

Integrative DNA, RNA, and protein evidence connects TREML4 to coronary artery calcification.

Coronary artery calcification (CAC) is a heritable and definitive morphologic marker of atherosclerosis that strongly predicts risk for future cardiovascular events. To search for genes involved in CAC, we used an integrative transcriptomic, genomic, and protein expression strategy by using next-generation DNA sequencing in the discovery phase with follow-up studies using traditional molecular biology and histopathology techniques. RNA sequencing of peripheral blood from a discovery set of CAC cases and controls was used to identify dysregulated genes, which were validated by ClinSeq and Framingham Heart Study data. Only a single gene, TREML4, was upregulated in CAC cases in both studies. Further examination showed that rs2803496 was a TREML4 cis-eQTL and that the minor allele at this locus conferred up to a 6.5-fold increased relative risk of CAC. We characterized human TREML4 and demonstrated by immunohistochemical techniques that it is localized in macrophages surrounding the necrotic core of coronary plaques complicated by calcification (but not in arteries with less advanced disease). Finally, we determined by von Kossa staining that TREML4 colocalizes with areas of microcalcification within coronary plaques. Overall, we present integrative RNA, DNA, and protein evidence implicating TREML4 in coronary artery calcification. Our findings connect multimodal genomics data with a commonly used clinical marker of cardiovascular disease.

Environmental factors determine DAP12 deficiency to either enhance or suppress immunopathogenic processes.

DNAX-activation protein 12 (DAP12), a transmembrane adapter, plays a major role in transducing activation signals in natural killer cells and various myeloid cells. Quantitative RT-PCR detected in normal mouse eyes considerable levels of DAP12 and multiple DAP12-coupled receptors, in particular TREM-1, Clec5a and SIRPb1. The role of DAP12 and its receptors in experimental autoimmune diseases has been controversial. Here, we analysed the effect of DAP12 deficiency on the capacity of mice to mount immunopathogenic cellular responses to the uveitogenic ocular antigen and interphotoreceptor retinoid-binding protein (IRBP), and to develop experimental autoimmune uveitis (EAU). Surprisingly, sequential analysis of EAU in mice deficient in DAP12 in two different animal facilities at first revealed enhanced disease as compared with wild-type mice, but when these mice were re-derived into a second, cleaner, animal facility, the response of control mice was essentially unchanged, whereas the DAP12 null mice were markedly hyporesponsive relative to controls in the new facility. Accordingly, when stimulated in vitro with IRBP, lymphocytes from the DAP12-deficient mice housed in the two facilities proliferated and produced opposite profiles of pro-inflammatory and anti-inflammatory cytokines, compared with their controls. These findings therefore demonstrate that the effects of DAP12 deficiency on development of autoimmune disease are dramatically affected by environmental factors.

LAB/NTAL facilitates fungal/PAMP-induced IL-12 and IFN-γ production by repressing ß-catenin activation in dendritic cells.

Fungal pathogens elicit cytokine responses downstream of immunoreceptor tyrosine-based activation motif (ITAM)-coupled or hemiITAM-containing receptors and TLRs. The Linker for Activation of B cells/Non-T cell Activating Linker (LAB/NTAL) encoded by Lat2, is a known regulator of ITAM-coupled receptors and TLR-associated cytokine responses. Here we demonstrate that LAB is involved in anti-fungal immunity. We show that Lat2-/- mice are more susceptible to C. albicans infection than wild type (WT) mice. Dendritic cells (DCs) express LAB and we show that it is basally phosphorylated by the growth factor M-CSF or following engagement of Dectin-2, but not Dectin-1. Our data revealed a unique mechanism whereby LAB controls basal and fungal/pathogen-associated molecular patterns (PAMP)-induced nuclear ß-catenin levels. This in turn is important for controlling fungal/PAMP-induced cytokine production in DCs. C. albicans- and LPS-induced IL-12 and IL-23 production was blunted in Lat2-/- DCs. Accordingly, Lat2-/- DCs directed reduced Th1 polarization in vitro and Lat2-/- mice displayed reduced Natural Killer (NK) and T cell-mediated IFN-γ production in vivo/ex vivo. Thus our data define a novel link between LAB and ß-catenin nuclear accumulation in DCs that facilitates IFN-γ responses during anti-fungal immunity. In addition, these findings are likely to be relevant to other infectious diseases that require IL-12 family cytokines and an IFN-γ response for pathogen clearance.

Negative regulation of myeloid-derived suppressor cells in cancer.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with suppressive function on immune response. In this review, we discuss recent studies about mechanisms of expansion and suppressive function of MDSCs during inflammation, infection and autoimmune diseases, as well as pro-angiogenic and pro-metastatic functions of these cells in tumor development. Further, we focus on novel studies of MDSCs and therapeutic approaches to eliminate these cells in cancer.

Simultaneous on-chip DC dielectrophoretic cell separation and quantitative separation performance characterization.

Through integration of a MOSFET-based microfluidic Coulter counter with a dc-dielectrophoretic cell sorter, we demonstrate simultaneous on-chip cell separation and sizing with three different samples including 1) binary mixtures of polystyrene beads, 2) yeast cells of continuous size distribution, and 3) mixtures of 4T1 tumor cells and murine bone marrow cells. For cells with continuous size distribution, it is found that the receiver operator characteristic analysis is an ideal method to characterize the separation performance. The characterization results indicate that dc-DEP separation performance degrades as the sorting throughput (cell sorting rate) increases, which provides insights into the design and operation of size-based microfluidic cell separation.

Rgs2 mediates pro-angiogenic function of myeloid derived suppressor cells in the tumor microenvironment via upregulation of MCP-1.

BACKGROUND: Tumor growth is intimately linked with stromal interactions. Myeloid derived suppressor cells (MDSCs) are dramatically elevated in cancer patients and tumor bearing mice. MDSCs modulate the tumor microenvironment through attenuating host immune response and increasing vascularization. METHODOLOGY/PRINCIPAL FINDINGS: In searching for molecular mediators responsible for pro-tumor functions, we found that regulator of G protein signaling-2 (Rgs2) is highly increased in tumor-derived MDSCs compared to control MDSCs. We further demonstrate that hypoxia, a common feature associated with solid tumors, upregulates the gene expression. Genetic deletion of Rgs2 in mice resulted in a significant retardation of tumor growth, and the tumors exhibit decreased vascular density and increased cell death. Interestingly, deletion of Rgs2 in MDSCs completely abolished their tumor promoting function, suggesting that Rgs2 signaling in MDSCs is responsible for the tumor promoting function. Cytokine array profiling identified that Rgs2-/- tumor MDSCs produce less MCP-1, leading to decreased angiogenesis, which could be restored with addition of recombinant MCP-1. CONCLUSION: Our data reveal Rgs2 as a critical regulator of the pro-angiogenic function of MDSCs in the tumor microenvironment, through regulating MCP-1 production.

Heterozygous deficiency of delta-catenin impairs pathological angiogenesis.

Vascular and neuronal networks share a similar branching morphology, and emerging evidence implicates common mechanisms in the formation of both systems. delta-Catenin is considered a neuronal catenin regulating neuron cell-cell adhesion and cell motility. Here, we report expression of delta-catenin in vascular endothelium, and show that deletion of only one allele of delta-catenin is sufficient to impair endothelial cell motility and vascular assembly in vitro and pathological angiogenesis in vivo, thereby inhibiting tumor growth and wound healing. In contrast, deletion of one or both allele of delta-catenin had no effects on hormone-induced physiological angiogenesis in the uterus. Molecular analysis confirmed a gene dosage effect of delta-catenin on Rho GTPase activity. Moreover, we show that inflammatory cytokines, but not angiogenic factors, regulate delta-catenin expression, and the levels of delta-catenin positively correlate to human lung cancers. Collectively, our data suggest that inflammation, commonly associated with disease conditions, induces delta-catenin expression that specifically regulates pathological, and not physiological, angiogenesis. Because only pathological angiogenesis is sensitive to decreased levels of delta-catenin, this may provide a good target for antiangiogenic therapy.

Endothelial cell adhesion molecules and cancer progression.

The role of cell adhesion molecules (CAMs), such as intercellular cell adhesion molecule-1 (ICAM-1), vascular endothelial cell adhesion molecule-1 (VCAM-1), E-selectin, and P-selectin, has been studied extensively in the process of inflammation. These molecules are responsible for recruiting leukocytes onto the vascular endothelium before extravasation to the injured tissues. Some circulating cancer cells have been shown to extravasate to a secondary site using a process similar to inflammatory cells. The most studied ligands for CAMs expressed on cancer cells, sialyl Lewis (a/x) antigens, are shown to be involved in adhesion to endothelial cells by binding to E-selectin. This process, shared by inflammatory cells and cancer cells, may partially explain the link between inflammation and tumorigenesis. Furthermore, this process may elucidate the therapeutic benefit of anti-inflammatory drugs in cancer treatment. The complexity of the tumor microenvironment has been revealed in the past decade. Currently, intense investigation is aimed at various aspects of the tumor microenvironment in addition to the tumor cells themselves. Here, we review the role of CAMs in extravasation of circulating cancer cells, a key step in metastasis.