Gene Expression In Macrophages Research Articles

Type III interferons are expressed in tuberculosis granulomas and promote an inflammatory phenotype in macrophages that differs from type I interferon

Abstract Humans and non-human primates express four subtypes of type III interferons (IFNλs; IFNλ1-IFNλ4). Unlike type I interferons, which have been thoroughly investigated in tuberculosis (TB), the role of IFNλs and their effects on immunity in TB remain unknown, especially at the disease site. To better understand the role of IFNλs in TB, we examined IFNλ1 and IFNλ4 expression in cynomolgus macaques with TB and investigated the effects of IFNα/β, IFNλ1 and IFNλ4 signaling on macaque macrophages. Using IHC, we identified differential IFNλ1 and IFNλ4 expression in macrophages and neutrophils, including IFNλ4 localization in the nuclei of epithelioid macrophages. CD206+ macrophages and CD3+ cells in the airway expressed IFNλR while CD11c+ macrophages in the lymphocyte cuff of the granuloma expressed IFNλR. To measure IFNλ1 and IFNλ4’s effect on macrophage gene expression and compare these cytokines against IFNα/β, we used NanoString transcriptional profiling and Ingenuity Pathway Analysis (IPA) on cytokine-stimulated macrophages to identify differentially regulated pathways. We found that IFNα/β upregulated the greatest number of ISGs, followed by IFNλ1, whereas IFNλ4 stimulation had minimal effect on gene expression. Interestingly, pro-inflammatory genes including IL-1β, IL-8, TLR1, BATF were upregulated by IFNλ1 while IL-1β and TLR1 were downregulated by IFNα/β. To determine the effects of IFNλs on anti-mycobacterial macrophage responses, we used a reporter Mtb strain to determine how IFNλ1 and IFNλ4 effect the viability of Mycobacterium tuberculosis. Our data suggest that IFNλs have non-redundant properties with type 1 interferons that may promote macrophage activation, inflammation, and antibacterial activity in TB.

Negative elongation factor complex enables macrophage inflammatory responses by controlling anti-inflammatory gene expression

Abstract Studies on macrophage gene expression have historically focused on events leading to RNA polymerase II recruitment and transcription initiation whereas the contribution of post-initiation steps to macrophage activation remains poorly understood. Here, we report widespread promoter-proximal RNA polymerase II pausing in resting macrophages, marked by broad co-localization of the negative elongation factor (NELF) complex and facilitated by PU.1. Upon inflammatory stimulation, over 60% of activated transcriptome is regulated by polymerase pause-release and a transient genome-wide NELF dissociation from chromatin, unexpectedly, independent of CDK9, a presumed NELF kinase. Genetic disruption of NELF in macrophages enhanced transcription of AP-1-encoding Fos and Jun and, consequently, AP-1 targets including Il10. Augmented expression of IL-10, a critical anti-inflammatory cytokine, in turn, attenuated production of pro-inflammatory mediators and, ultimately, macrophage-mediated inflammation in vivo. Together, these findings establish a previously unappreciated role of NELF in constraining transcription of inflammation inhibitors thereby enabling inflammatory macrophage activation.

Myeloid-specific deficiency of pregnane X receptor decreases atherosclerosis in LDL receptor-deficient mice

The pregnane X receptor (PXR) is a nuclear receptor that can be activated by numerous drugs and xenobiotic chemicals. PXR thereby functions as a xenobiotic sensor to coordinately regulate host responses to xenobiotics by transcriptionally regulating many genes involved in xenobiotic metabolism. We have previously reported that PXR has pro-atherogenic effects in animal models, but how PXR contributes to atherosclerosis development in different tissues or cell types remains elusive. In this study, we generated an LDL receptor-deficient mouse model with myeloid-specific PXR deficiency (PXRΔMyeLDLR-/-) to elucidate the role of macrophage PXR signaling in atherogenesis. The myeloid PXR deficiency did not affect metabolic phenotypes and plasma lipid profiles, but PXRΔMyeLDLR-/- mice had significantly decreased atherosclerosis at both aortic root and brachiocephalic arteries compared with control littermates. Interestingly, the PXR deletion did not affect macrophage adhesion and migration properties, but reduced lipid accumulation and foam cell formation in the macrophages. PXR deficiency also led to decreased expression of the scavenger receptor CD36 and impaired lipid uptake in macrophages of the PXRΔMyeLDLR-/- mice. Further, RNA-Seq analysis indicated that treatment with a prototypical PXR ligand affects the expression of many atherosclerosis-related genes in macrophages in vitro. These findings reveal a pivotal role of myeloid PXR signaling in atherosclerosis development and suggest that PXR may be a potential therapeutic target in atherosclerosis management.

Assessment of the Effects of Silene roemeri and Silene sendtneri Ethanol Extracts on Human Monocytes/Macrophages

Immunomodulatory and immunocorrective therapies are important approaches to foster immunity. Biologically active substances (BAS) of plants, exhibiting a wide spectrum of pharmacological activity, can act as agents mobilizing the immune system. Plants belonging to the Caryophyllaceae family, including Silene sendtneri and S. roemeri, are rich in ecdysteroids and flavonoids, the compounds characterized by a large variety of biological activities with potential impact on the human immune system. In this study, the effect of native and chlorophyll-free plant extracts of S. sendtneri and S. roemeri on human monocyte-derived macrophages was examined. The production of cytokines was assessed using ELISA. Gene expression microarrays and RT-qPCR analysis were used to determine differentially expressed genes under the influence of the extracts. The results of the experiments showed that the extracts increased production of an M2-associated chemokine CCL-18 in macrophages. In addition, they induced the production of proinflammatory cytokines (TNF-α, IL-1β, and IL-6). The identified activity was detected both in native and chlorophyll-free extracts. Furthermore, plant extracts of S. sendtneri and S. roemeri influenced the expression of the CCL2, MMP9, VEGFA, and S100A9 genes in human macrophages. Overall, extracts of the Silene genus plants showed immunomodulatory effects by influencing macrophage activation in vitro.

TNF-α and IL-10 Control CXCL13 Expression in Human Macrophages

The chemokine CXCL13 controls the normal organization of secondary lymphoid tissues and the neogenesis of ectopic lymphoid structures in nonlymphoid organs, particularly the lungs. The progression and severity of idiopathic pulmonary fibrosis (IPF), a fatal and irreversible interstitial lung disease, is predicted by the circulating blood concentrations of CXCL13. Although CXCL13 is produced by pulmonary tissues, it has not been determined which cells are involved. This study examines CXCL13 production by lung tissue macrophages from patients with IPF and the signaling pathways controlling CXCL13 gene expression in human alveolar macrophages (AM) and monocyte-derived macrophages (MoDM). CXCL13 is found in CD68- and CD206-positive AM from patients with IPF, and the CXCL13 gene is induced in these macrophages and MoDM when they are stimulated with LPS. We found that TNF-α and IL-10 control optimal CXCL13 gene expression in MoDM and possibly in AM by activating the NF-κB and JAK/STAT pathways, respectively. We also found that blood TNF-α and CXCL13 concentrations are significantly correlated in patients with IPF, suggesting that TNF-α contributes to CXCL13 production in humans. In conclusion, the results of this study demonstrate that AM from patients with IPF produces CXCL13 and that the NF-κB and JAK/STAT pathways are required to induce the expression of this major chemokine.

The NADL strain of bovine viral diarrhea virus induces the secretion of IL-1β through caspase 1 in bovine macrophages

Bovine viral diarrhea virus (BVDV) infects different cell types including antigen-presenting cells such as macrophages. The infection induces pro-inflammatory cytokines like interleukin 1 beta (IL-1β), which is necessary to trigger a successful inflammatory response against infections. Several authors have reported differences between IL-1β gene expression and protein detection in BVDV-infected macrophages. These patterns may be related to inflammasome assembly, which promote the formation of active caspase 1 in order to produce mature IL-1β molecules. Our goal was to assess BVDV ability to induce the release of IL-β through a caspase 1 dependent pathway in bovine macrophages. We infected peripheral blood monocyte-derived macrophages using BVDV NADL strain at 0.001, 0.1, 2 and 10 multiplicities of infection (MOI) and we measured IL-1β at different times 2, 6, 12, 24, 48, 72 h.We found an increase of 1140–2154 pg for a MOI of 10:1 and 2:1 respectively. To inhibit caspase 1, we used either carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Z-VAD) or carbobenzoxy-tyr-valyl-alanyl-aspartyl-[O-methyl]- fluoromethylketone (Y-VAD). We found decreased IL-1β secretion 2154 pg/ml to 854 pg/ml IL-1β secretion using Y-VAD and we observed decrease from 2154 pg/ml to 22.33 pg/ml with Z-VAD, and this inhibition was followed by diminished viral replication from 2.25 × 107 to 2.1 × 105 CCID50, which suggests that caspase 1-dependent secretion of the IL-1β active molecule is important for viral replication.This is the first report showing that BVDV infected-bovine macrophages trigger the caspase 1 dependent pathway for IL-1β activation and that activation increases viral replication.

Computational Approach to Identifying Universal Macrophage Biomarkers.

Macrophages engulf and digest microbes, cellular debris, and various disease-associated cells throughout the body. Understanding the dynamics of macrophage gene expression is crucial for studying human diseases. As both bulk RNAseq and single cell RNAseq datasets become more numerous and complex, identifying a universal and reliable marker of macrophage cell becomes paramount. Traditional approaches have relied upon tissue specific expression patterns. To identify universal biomarkers of macrophage, we used a previously published computational approach called BECC (Boolean Equivalent Correlated Clusters) that was originally used to identify conserved cell cycle genes. We performed BECC analysis using the known macrophage marker CD14 as a seed gene. The main idea behind BECC is that it uses massive database of public gene expression dataset to establish robust co-expression patterns identified using a combination of correlation, linear regression and Boolean equivalences. Our analysis identified and validated FCER1G and TYROBP as novel universal biomarkers for macrophages in human and mouse tissues.

AlteredExpressionofUnfoldedProteinResponseGenesinMacrophagesfrom Behcet’s Disease

Endoplasmic reticulum (ER) stress triggers the unfolded protein response (UPR), which has been correlated with enhancedproduction of inflammatory cytokines. Given the important pathogenic roles of macrophages and inflammatory responses in theetiopathogenesis of Behcet’s disease (BD), this study aimed to assess the mRNA expression pattern of genes involved in theUPR pathway in macrophages from smoker and non-smoker BD patients. This case-control study was conducted between 2015and 2016 in Shariati Hospital, Tehran, Iran. Monocytes were enriched from obtained whole blood samples of 10 smokers and 10non-smoker BD patients as well as 10 healthy individuals. Using macrophage-colony stimulating factor (M-CSF), separatedmonocytes were differentiated into macrophages. After total RNA purification and cDNA synthesis, quantification analysis ofUPR genes, including activating transcription factor (ATF) 4, ATF6, X-box binding protein 1 (XBP1), binding immunoglobulinprotein (BIP), C/EBP homologous protein (CHOP), homocysteine-inducible, endoplasmic reticulum stress-inducible, ubiquitin-like domain member 1 (HERP), and growth arrest and DNA damage-inducible protein (GADD34), was performed using SYBRgreen master mix and real-time PCR. Among the measured genes, HERP mRNA was overexpressed in macrophages from BDpatients in comparison with healthy macrophages. HERP and GADD34 genes were upregulated in smoker BD patients comparedwith non-smoker BD patients as well as healthy subjects. Cigarette smoke can induce UPR gene expression in BD patients. Thealtered UPR gene expression in BD macrophages may contribute to BD pathogenesis.

Exogenous IL‐4 Promotes Myocardial Infarction Repair by Turning off Pro‐Inflammation in Neutrophils while Stimulating Anti‐Inflammation in Macrophages to Induce Neutrophil Phagocytosis

Macrophages and neutrophils are primary leukocytes involved in the inflammatory response to myocardial infarction (MI). While IL‐4 is an in vitro stimulus for anti‐inflammation, the MI myocardium does not express IL‐4. We hypothesized that continuous exogenous IL‐4 infusion starting 24 h after MI would promote a polarization switch in inflammatory cells towards a reparative phenotype. C57BL/6J male mice (3–6 months of age) were subcutaneously infused with either saline (n=17) or IL‐4 (20 ng/g/day; n=17) and evaluated at MI day 3. Macrophages and neutrophils were isolated ex vivo from the infarct region and examined for pro‐inflammatory genes (Ccl3, Ccl5, Il1b, Il12a, and Tnfa) and anti‐inflammatory genes (Arg1, Ym1, Mrc1, Il10, and Tgfb, Il6). Exogenous IL‐4 decreased pro‐inflammatory Ccl3, IL‐12a, Tnfa, and Tgfb1 in neutrophils while increasing anti‐inflammatory Arg1 and Ym1 in macrophages (all p<0.05); none of the other markers were different. Tissue clearance by IL‐4 treated neutrophils was not different, while phagocytosis of neutrophils, but not myocytes, doubled in IL‐4 treated macrophages (p<0.05). Because macrophage cell physiology was influenced by IL‐4, whole transcriptome analysis of macrophages isolated from the infarct regions of IL‐4 (n=10) or saline (n=11) treated mice was performed by RNA‐sequencing. Of the 24,341 genes sequenced, 2,042 genes were differentially expressed with IL‐4 stimulation (all p<0.05). Pdgfc gene expression was ranked first by p value, increasing from 1.8±0.3 in saline treated macrophages to 5.4±0.2 FPKM units in macrophages stimulated with IL‐4 (p=1×10−9). Importantly, changes in macrophage physiology and transcriptome occurred before differences in cardiac physiology evaluated by echocardiography, which allowed the isolation of effects in cell phenotype not induced by global myocardial effects. Bone marrow derived monocytes stimulated with mouse recombinant PDGF‐CC protein (10 μg/ml) or PDGF‐CC blocking antibody (10 μg/ml) revealed no effect on Arg1 or Ym1 expression, indicating the in vivo effect of IL‐4 to stimulate macrophage gene expression was independent of PDGF‐CC. Altogether, our results indicate that exogenous IL‐4 promotes inflammation resolution by turning off pro‐inflammation in neutrophils while stimulating anti‐inflammation in macrophages to induce neutrophil phagocytosis.Support or Funding InformationNational Institutes of Health under Award Numbers HL075360, HL129823, and HL137319, Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development under Award Numbers 5I01BX000505

Depletion of microRNA-451 in response to allergen exposure accentuates asthmatic inflammation by regulating Sirtuin2.

The incidence of asthma has increased from 5.5% to near 8% of the population, which is a major health concern. The hallmarks of asthma include eosinophilic airway inflammation that is associated with chronic airway remodeling. Allergic airway inflammation is characterized by a complex interplay of resident and inflammatory cells. MicroRNAs (miRNAs) are small noncoding RNAs that function as posttranscriptional modulators of gene expression. However, the role of miRNAs, specifically miR-451, in the regulation of allergic airway inflammation is unexplored. Our previous findings showed that oxidant stress regulates miR-451 gene expression in macrophages during an inflammatory process. In this paper, we examined the role of miR-451 in regulating macrophage phenotype using an experimental poly-allergenic murine model of allergic airway inflammation. We found that miR-451 contributes to the allergic induction of CCL17 in the lung and plays a key role in proasthmatic macrophage activation. Remarkably, administration of a Sirtuin 2 (Sirt2) inhibitor diminished alternate macrophage activation and markedly abrogated triple-allergen [dust mite, ragweed, Aspergillus fumigatus (DRA)]-induced lung inflammation. These data demonstrate a role for miR-451 in modulating allergic inflammation by influencing allergen-mediated macrophages phenotype.

Matrix-Targeting Immunotherapy Controls Tumor Growth and Spread by Switching Macrophage Phenotype.

The interplay between cancer cells and immune cells is a key determinant of tumor survival. Here, we uncovered how tumors exploit the immunomodulatory properties of the extracellular matrix to create a microenvironment that enables their escape from immune surveillance. Using orthotopic grafting of mammary tumor cells in immunocompetent mice and autochthonous models of breast cancer, we discovered how tenascin-C, a matrix molecule absent from most healthy adult tissues but expressed at high levels and associated with poor patient prognosis in many solid cancers, controls the immune status of the tumor microenvironment. We found that, although host-derived tenascin-C promoted immunity via recruitment of proinflammatory, antitumoral macrophages, tumor-derived tenascin-C subverted host defense by polarizing tumor-associated macrophages toward a pathogenic, immune-suppressive phenotype. Therapeutic monoclonal antibodies that blocked tenascin-C activation of Toll-like receptor 4 reversed this phenotypic switch in vitro and reduced tumor growth and lung metastasis in vivo, providing enhanced benefit in combination with anti-PD-L1 over either treatment alone. Combined tenascin-C:macrophage gene-expression signatures delineated a significant survival benefit in people with breast cancer. These data revealed a new approach to targeting tumor-specific macrophage polarization that may be effective in controlling the growth and spread of breast tumors.

Ablation of Myeloid Cell MRP8 Ameliorates Nephrotoxic Serum-induced Glomerulonephritis by Affecting Macrophage Characterization through Intraglomerular Crosstalk

Toll-like receptor 4 (TLR4) and one of its endogenous ligands myeloid-related protein 8 (MRP8 or S100A8), especially expressed in macrophages, play an important role in diabetic nephropathy and autoimmune disorders. However, detailed mechanisms and consequence of MRP8 expression remain unknown, partly due to embryonic lethality of MRP8 knockout mice. In this study, Myeloid lineage cell-specific MRP8 knockout mice were generated, and nephrotoxic serum-induced glomerulonephritis was developed. Mice with conditional ablation of MRP8 gene in myeloid cells exhibited less severe histological damage, proteinuria and inflammatory changes compared to control mice. Mechanism of MRP8 upregulation was investigated using cultured cells. Co-culture of macrophages with mesangial cells or mesangial cell-conditioned media, but not with proximal tubules, markedly upregulated MRP8 gene expression and inflammatory M1 phenotype in macrophages, which was attenuated in MRP8-deleted bone marrow-derived macrophages. Effects of MRP8 deletion was further studied in the context of macrophage-inducible C-type lectin (Mincle), which is critically involved in maintenance of M1 phenotype of macrophages. MRP8 ablation in myeloid cells suppressed the induction of Mincle expression on macrophages in glomerulonephritis. Thus, we propose that intraglomerular crosstalk between mesangial cells and macrophages plays a role in inflammatory changes in glomerulonephritis, and MRP8-dependent Mincle expression in macrophage may be involved in the process.

Targeting tumor-associated macrophage (TAM) mediated inhibition of T-cell migration in prostate cancer using epigenetic modifying agents.

166 Background: Cytotoxic T lymphocytes (CTLs) perform vital anti-tumor functions and are critical to the efficacy of many anticancer therapies. In prostate cancer, the characteristic paucity of activated CTLs within the tumor microenvironment (TME) may be a key factor in disease progression and likely underlies the limited role for immune checkpoint inhibitors (ICIs) in prostate cancer treatment. In this study, we utilized novel microfluidic technologies to evaluate whether TAMs may be driving the exclusion of T cells from the prostate TME and whether the immunosuppressive functions of TAMs could be modified by epigenetic modifying agents. Methods: Primary macrophages and autologous T cells were derived from peripheral blood samples of prostate cancer patients at the University of Wisconsin. Mono-, co-, and tri-culture systems of macrophages, T cells, and 22RV1 cells (androgen-dependent prostate cancer cell line) were cultured in 2D and 3D in microfluidic cell culture platforms. Culture systems were treated with the EZH2 inhibitors (EZH2i) DZNep or EPZ-6438 or left untreated. Macrophages were also treated with M1 (IFN-g) and M2 (IL-4) polarizing cytokines. Systems were analyzed for T cell migration as well as mRNA and protein expression in each cell population. Results: Autologous macrophages inhibited activated T cell migration towards tumor cells in a multi-cellular microscale TME. T cell migration was restored through treatment with EZH2i. Gene expression analysis identified that EZH2i altered macrophage gene expression in the unpolarized and M1/M2 polarized states. In particular, there was increased expression of genes involved in T cell recruitment/chemotaxis, including CXCL10, CXCL11, CXCL12, following EZH2i treatment. Conclusions: We used novel microfluidic technologies to model and analyze multicellular TMEs using primary, patient-derived cells. We demonstrate that TAM-mediated suppression of T cell migration is mediated, in part, through epigenetic pathways, which can be targeted with EZH2i. Treatment with EZH2i, alone or in combination other therapies such as ICIs, may enhance cytotoxic T cell migration and activity in primary prostate cancer.

Cxcr4 distinguishes HSC-derived monocytes from microglia and reveals monocyte immune responses to experimental stroke.

Monocyte-derived and tissue-resident macrophages are ontogenetically distinct components of the innate immune system. Assessment of their respective functions in pathology is complicated by changes to the macrophage phenotype during inflammation. Here we find that Cxcr4-CreER enables permanent genetic labeling of hematopoietic stem cells (HSCs) and distinguishes HSC-derived monocytes from microglia and other tissue-resident macrophages. By combining Cxcr4-CreER-mediated lineage tracing with Cxcr4 inhibition or conditional Cxcr4 ablation in photothrombotic stroke, we find that Cxcr4 promotes initial monocyte infiltration and subsequent territorial restriction of monocyte-derived macrophages to infarct tissue. After transient focal ischemia, Cxcr4 deficiency reduces monocyte infiltration and blunts the expression of pattern recognition and defense response genes in monocyte-derived macrophages. This is associated with an altered microglial response and deteriorated outcomes. Thus, Cxcr4 is essential for an innate-immune-system-mediated defense response after cerebral ischemia. We further propose Cxcr4-CreER as a universal tool to study functions of HSC-derived cells.

Regulation of 8-Hydroxydaidzein in IRF3-Mediated Gene Expression in LPS-Stimulated Murine Macrophages.

Cytokines and chemokines are transcriptionally regulated by inflammatory transcription factors such as nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and interferon regulatory factor (IRF)-3. A daidzein derivative compound, 8-hydroxydaidzein (8-HD), isolated from soy products, has recently gained attention due to various pharmacological benefits, including anti-inflammatory activities. However, regulation of the inflammatory signaling mechanism for 8-HD is still poorly understood, particularly with respect to the IRF-3 signaling pathway. In this study, we explored the molecular mechanism of 8-HD in regulating inflammatory processes, with a focus on the IRF-3 signaling pathway using a lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid [Poly (I:C)] stimulated murine macrophage cell line (RAW264.7). The 8-HD downregulated the mRNA expression level of IRF-3-dependent genes by inhibiting phosphorylation of the IRF-3 transcription factor. The inhibitory mechanism of 8-HD in the IRF-3 signaling pathway was shown to inhibit the kinase activity of IKKε to phosphorylate IRF-3. This compound can also interfere with the TRIF-mediated complex formation composed of TRAF3, TANK, and IKKε leading to downregulation of AKT phosphorylation and reduction of IRF-3 activation, resulted in inhibition of IRF-3-dependent expression of genes including IFN-β, C-X-C motif chemokine 10 (CXCL10), and interferon-induced protein with tetratricopeptide repeats 1 (IFIT1). Therefore, these results strongly suggest that 8-HD can act as a promising compound with the regulatory function of IRF-3-mediated inflammatory responses.

Tensor decomposition of stimulated monocyte and macrophage gene expression profiles identifies neurodegenerative disease-specific trans-eQTLs.

Recent human genetic studies suggest that cells of the innate immune system have a primary role in the pathogenesis of neurodegenerative diseases. However, the results from these studies often do not elucidate how the genetic variants affect the biology of these cells to modulate disease risk. Here, we applied a tensor decomposition method to uncover disease associated gene networks linked to distal genetic variation in stimulated human monocyte and macrophage gene expression profiles. We report robust evidence that some disease associated genetic variants affect the expression of multiple genes in trans. These include a Parkinson’s disease locus influencing the expression of genes mediated by a protease that controls lysosomal function, and Alzheimer’s disease loci influencing the expression of genes involved in type 1 interferon signaling, myeloid phagocytosis, and complement cascade pathways. Overall, we uncover gene networks in induced innate immune cells linked to disease associated genetic variants, which may help elucidate the underlying biology of disease.

In silico identification of novel transcription factors associated with CYP27B1 transcriptional regulation in LPS-challenged mononuclear phagocytes

Renal and extrarenal production of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)2D), is catalyzed by CYP27B1, an enzyme also called 1-α-hydroxylase. The overproduction of 1,25(OH)2D has been described in granulomatous diseases. High circulating concentrations of 1,25(OH)2D can lead to hypercalcemia. The aim of this work was to characterize the transcriptional regulation of CYP27B1 in human mononuclear phagocytes exposed to LPS due to its relevance to understanding the hypercalcemia and ectopic calcifications associated with chronic inflammatory diseases such as tuberculosis and other granulomatous diseases. The human CYP27B1 promoter analysis identified binding sites for published TF, SNPs, novel putative TFBS and conserved sites compared to mice. Then, using microarray data, a meta-analysis was performed to obtain a global view of the gene expression in LPS-challenged dendritic cells, monocytes and macrophages. Finally, two experiments, GSE40885 and time series GSE19765, were analyzed in-depth using differential expression analysis which permitted the identification of TF co-expressed with CYP27B1. This work allowed us to formulate a CYP27B1 transcriptional regulation model for LPS-challenged monocytes/macrophages. The importance of two TF families, NFKB and CEBPB, was confirmed. Data also suggests that PLAGL2 and STAT4 which are novel TF could participate in the CYP27B1 transcriptional regulation in cells exposed to LPS. These TF, in turn, would be interacting with regions that present polymorphisms in the general population which might explain the pathological phenotypes associated with altered vitamin D metabolism.

Regulation of Cell Viability and Anti-inflammatory Tristetraprolin Family Gene Expression in Mouse Macrophages by Cottonseed Extracts

Bioactive plant extracts have been used for the prevention and treatment of various diseases. One of the major classes of bioactive compounds is plant polyphenols. Cottonseed ethanol extracts were determined by HPLC-MS analysis to be essentially free of toxic gossypol. The objective of this study was to investigate the effect of cottonseed ethanol extracts on the cytotoxicity and regulation of anti-inflammatory tristrataprolin (TTP) family gene expression in mouse cells. MTT, qPCR and immunoblotting assays tested the effects of cottonseed extracts in mouse RAW264.7 macrophages and 3T3-L1 adipocytes. No cytotoxicity effect was observed in macrophages treated with extracts from the coat or kernel of glanded and glandless cottonseed. Similarly, the viability of mouse adipocytes was not affected by cottonseed extracts. In contrast, gossypol and lipopolysaccharides were toxic to macrophages but not adipocytes under high concentration or long time treatment. Cottonseed extracts exhibited modest effect on TTP family gene expression in macrophages but glandless cottonseed coat extract significantly increased TTP mRNA and protein levels with a magnitude similar to cinnamon and green tea polyphenol extract and insulin. These results demonstrated that cottonseed extracts are harmless towards the mouse cells and that glandless cottonseed coat extract stimulates TTP gene expression. We propose that glandless cottonseed is a safe source of plant polyphenols with anti-inflammatory property.

Advanced Glycated apoA-IV Loses Its Ability to Prevent the LPS-Induced Reduction in Cholesterol Efflux-Related Gene Expression in Macrophages.

We addressed how advanced glycation (AGE) affects the ability of apoA-IV to impair inflammation and restore the expression of genes involved in cholesterol efflux in lipopolysaccharide- (LPS-) treated macrophages. Recombinant human apoA-IV was nonenzymatically glycated by incubation with glycolaldehyde (GAD), incubated with cholesterol-loaded bone marrow-derived macrophages (BMDMs), and then stimulated with LPS prior to measurement of proinflammatory cytokines by ELISA. Genes involved in cholesterol efflux were quantified by RT-qPCR, and cholesterol efflux was measured by liquid scintillation counting. Carboxymethyllysine (CML) and pyrraline (PYR) levels, determined by Liquid Chromatography-Mass Spectrometry (LC-MS/MS), were greater in AGE-modified apoA-IV (AGE-apoA-IV) compared to unmodified-apoA-IV. AGE-apoA-IV inhibited expression of interleukin 6 (Il6), TNF-alpha (Tnf), IL-1 beta (Il1b), toll-like receptor 4 (Tlr4), tumor necrosis factor receptor-associated factor 6 (Traf6), Janus kinase 2/signal transducer and activator of transcription 3 (Jak2/Stat3), nuclear factor kappa B (Nfkb), and AGE receptor 1 (Ddost) as well as IL-6 and TNF-alpha secretion. AGE-apoA-IV alone did not change cholesterol efflux or ABCA-1 levels but was unable to restore the LPS-induced reduction in expression of Abca1 and Abcg1. AGE-apoA-IV inhibited inflammation but lost its ability to counteract the LPS-induced changes in expression of genes involved in macrophage cholesterol efflux that may contribute to atherosclerosis.

Signature of genome wide gene expression in classical swine fever virus infected macrophages and PBMCs of indigenous vis-a-vis crossbred pigs

The genetic basis of differential host immune response vis-à-vis transcriptome profile was explored in PBMCs of indigenous (Ghurrah) and crossbred pigs after classical swine fever vaccination and in monocyte derived macrophages (MDMs) challenged with virulent classical swine fever (CSF) virus. The humoral immune response (E2 antibody) was higher (74.87%) in crossbred than indigenous pigs (58.20%) at 21st days post vaccination (21dpv). The rate of reduction of ratio of CD4+/CD8+ was higher in crossbred pigs than indigenous pigs at 7th days post vaccination (7dpv). The immune genes IFIT1, IFIT5, RELA, NFKB2, TNF and LAT2 were up regulated at 7dpv in RNA seq data set and was in concordance during qRT-PCR validation. The Laminin Subunit Beta 1 (LAMB1) was significantly (p ≤ 0.05) down-regulated in MDMs of indigenous pigs and consequently a significantly (p ≤ 0.01) higher copy number of virulent CSF virus was evidenced in macrophages of crossbred pigs than indigenous pigs. Activation of LXR:RXR pathway at 60 h post infection (60hpi) in MDMs of indigenous versus crossbred pigs inhibited nuclear translocation of NF-κB, resulted into transrepression of proinflammatory genes. But it helped in maintenance of HDL level by lowering down cholesterol/LDL level in MDMs of indigenous pigs. The key immune genes (TLR2, TLR4, IL10, IL8, CD86, CD54, CASP1) of TREM1 signaling pathway were upregulated at 7dpv in PBMCs but those genes were downregulated at 60hpi in MDMs indigenous pigs. Using qRT-PCR, the validation of differentially expressed, immunologically important genes (LAMB1, OAS1, TLR 4, TLR8 and CD86) in MDMs revealed that expression of these genes were in concordance with RNA-seq data.