CD14 mRNA Expression Research Articles

CD163+ macrophages are associated with a vulnerable plaque phenotype in human carotid plaques

Macrophages are a functionally heterogeneous group of immune cells abundant in atherosclerotic plaques. Macrophages expressing CD163 are associated with intraplaque hemorrhage and have previously been considered atheroprotective. However, in a recent study CD163-deficient atherosclerotic ApoE−/− mice exhibited smaller and less complex plaques, suggesting a proatherogenic role of CD163. Previous smaller studies on CD163+ macrophages and plaque stability in humans have yielded diverging results. Here we assessed the association of CD163+ cells to plaque vulnerability in a large cohort of human carotid plaques. CD163 protein expression was analyzed by immunohistochemistry in 200 human carotid plaques removed by endarterectomy from 103 patients with and 93 patients without cerebrovascular symptoms. Furthermore, CD163 mRNA expression was analyzed in 66 of the plaques. Both protein and mRNA expression of CD163 was higher in plaques from symptomatic patients and in plaques with high vulnerability index. CD163+ macrophages were primarily found in shoulder regions and in the center of the plaques. The present data show that CD163 is associated with increased plaque vulnerability in human carotid plaques, supporting the notion that CD163+ macrophages could contribute to clinical events.

Saccharomyces boulardii attenuates inflammatory response induced by Clostridium perfringens via TLR4/TLR15-MyD8 pathway in HD11 avian macrophages

Macrophages are professional phagocytic cells that play a critical role in initiating immune responses by presenting antigen and phagocytic clearance. The macrophages can be targeted for immunomodulation by beneficial microbes, such as probiotics. The aim of this study is to investigate the protective effect of Saccharomyces boulardii against Clostridium perfringens infection in avian macrophage cell line HD11. In this study, HD11 macrophages were prestimulated with S. boulardii for 6 h and then infected with C. perfringens for 3 h. Results showed that S. boulardii enhanced phagocytosis and bactericidal capacity against C. perfringens by HD11 cells. The S. boulardii effectively promoted the mRNA expression of CD80, CD83, and CD197 cell-surface molecules in C. perfringens-infected HD11 cells. Moreover, we found that prestimulation with S. boulardii reduced the mRNA expression of CD40, toll-like receptor [TLR] 4, and TLR15 induced by C. perfringens and thereby downregulated the mRNA expression of myeloid differentiation primary response 88, TNF receptor associated factor 6, nuclear factor kappa-B p65 subunit, and c-Jun N-terminal kinase genes in HD11 cells. The upregulation of cytokines (interleukin [IL]-6, tumor necrosis factor alpha, and IL-10) and inducible nitric oxide synthase mRNA expression in C. perfringens-infected HD11 cells were noticeably inhibited by S. boulardii pretreatment. Conclusively, these results might provide a new insight into the role of S. boulardii in regulating avian immune defense against C. perfringens invasion and immune escape.

Suppression of Inflammatory Mediators by Ethanol Extracts of <i>Pennisetum purpureum </i>S. (Napiergrass Taishigrass no. 2) in Activated RAW 264.7 Macrophages

Napiergrass Taishigrass no. 2 (NT2) is a new hybrid of napiergrass (Pennisetum purpureum S.), and is commonly added as an ingredient in bioorganic energy soup, a plant-based drink which is thought to have health-promoting effects. Thus, NT2 is hypothesized to possess biological functions. In this study, we explored the effects of NT2 ethanol extracts on in vitro antioxidant activities and on modulation of immune mediators in RAW 264.7 macrophages. Results showed that ethanol extracts contained significant amount of polyphenols and chlorophylls, and possessed in vitro ferric-reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidative activities. The ethanol extracts did not show immunomodulatory activities in macrophages without lipopolysaccharide (LPS, 100 ng/ml) treatment, but significantly suppressed the LPS-stimulated production of inflammatory mediators, including nitric oxide (NO), interleukin (IL)-6, and tumor necrosis factor (TNF)-α, and such effects were associated with decreased expression of inducible NO synthase (iNOS) protein and cluster of differentiation 14 (CD14) mRNA, suggesting that NT2 has anti-inflammatory activities. On the other hand, the extracts increased matrix metalloproteinase (MMP)-9 activity, but showed no effects on Toll-like receptor (TLR) expression. In conclusion, ethanol extracts of NT2 possessed in vitro antioxidative activities and exhibited anti-inflammatory activities by decreasing NO, IL-6, and TNF-α production in association with decreased CD14 mRNA expression in LPS-induced RAW 264.7 macrophages, indicating its potential roles on health promotion and prentive effects against chronic diseases and such effects were and it’s in vitro antioxidant activities, suggesting its potential roles on health promotion.

Mulberry Fruit Hot Water Extract Attenuates Adipose Tissue Inflammation with the Regulation of Macrophage Infiltration and M1/M2 Type Switching in Rats Fed a High-Fat Diet

Abstract Objectives This study aimed to investigate the effects of mulberry fruit extract (ME) on adipose tissue inflammation in rats fed a high-fat diet. Methods Male, Sprague-Dawley rats were divided into 4 groups and fed either a low-fat diet (LF, 10% kcal fat), high-fat diet (HF, 45% kcal fat), HF with 0.5% mulberry fruit extract (HF + LME), or HF with 1% mulberry fruit extract (HF + HME) for 14 weeks. Results The serum triglyceride (TG) and total cholesterol (TC) concentrations were significantly lower in the ME supplemented groups. In white adipose tissue (WAT), the HF + HME significantly down-regulated the mRNA expression of adipogenic genes, such as sterol regulatory element-binding protein-1c (SREBP-1c), peroxisome proliferator-activated receptor-γ (PPAR-γ), and adipocyte protein2 (aP2). The HF + HME significantly decreased both levels of mRNA and protein of pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein 1 (MCP1). The crown-like structure (CLS) and the mRNA expression of F4/80, a pan-macrophage marker, tended to be reduced in the ME supplemented groups. The ME supplemented groups down-regulated the mRNA expression of pro-inflammatory M1 macrophage markers, such as a cluster of differentiation (CD) 68, CD11c, and nitric oxide synthase 2 (NOS2). On the other hand, the mRNA expression of CD163 and arginase1 (ARG1), an anti-inflammatory M2 macrophage marker, was up-regulated by the HF + HME. Conclusions From these results, it is suggested that the ME might modulate the macrophage infiltration and phenotypic switching and alleviate adipose tissue inflammation in rats fed a high-fat diet. Funding Sources None.

Bacterial lipopolysaccharide induces the intracellular expression of trophoblastic specific CD74 isoform in human first trimester trophoblast cells: Correlation with unsuccessful early pregnancy

ObjectiveDuring first trimester of human pregnancy, the maternal system develops immunity against infection and to provide protection of allogeneic foetus from abortion. This study was undertaken to determine the role of trophoblast specific CD74 isoforms in first trimester trophoblast derived cells under normal and lipopolysaccharide (LPS) stimulated conditions. MethodsGene and protein of CD74 were determined in first trimester trophoblast derived cells, JEG-3 and ACH-3 P and also in human placenta by PCR, western blotting and immunoprecipitation. Effect of LPS mediated infection on the regulation of CD74 isoforms was studied intracellularly and also on the cells surface by flow cytometry. ResultsData demonstrated that JEG-3 and ACH-3 P cells under normal conditions have not expressed CD74 isoforms neither intracellularly or nor on the surface. These results were further validated directly in human placenta. However, treatment of these trophoblast cells with a bacterial LPS, significantly upregulated CD74 mRNA expression (p < 0.05). Furthermore, expression of CD74 on the surface was not detected even after stimulation with LPS. Interestingly, CD74 isoform at 35 kDa was significantly detected intracellularly upon stimulation with LPS (p < 0.05). These results were further confirmed by western blotting followed by immunoprecipitation. ConclusionsTo the best of our knowledge, this is the first study concluded that the bacterial LPS induce infection in the first trimester trophoblasts via intracellular upregulation of CD74. Data indicated that the lack of cell surface expression of trophoblastic specific isoforms of CD74 may provide protection for human pregnancy in the first trimester.

Baicalin prevents LPS-induced activation of TLR4/NF-κB p65 pathway and inflammation in mice via inhibiting the expression of CD14.

Previous studies have shown that baicalin, an active ingredient of the Chinese traditional medicine Huangqin, attenuates LPS-induced inflammation by inhibiting the activation of TLR4/NF-κBp65 pathway, but how it affects this pathway is unknown. It has been shown that CD14 binds directly to LPS and plays an important role in sensitizing the cells to minute quantities of LPS via chaperoning LPS molecules to the TLR4/MD-2 signaling complex. In the present study we investigated the role of CD14 in the anti-inflammatory effects of baicalin in vitro and in vivo. Exposure to LPS (1 μg/mL) induced inflammatory responses in RAW264.7 cells, evidenced by marked increases in the expression of MHC II molecules and the secretion of NO and IL-6, and by activation of MyD88/NF-κB p65 signaling pathway, as well as the expression of CD14 and TLR4. These changes were dose-dependently attenuated by pretreatment baicalin (12.5-50 μM), but not by baicalin post-treatment. In RAW264.7 cells without LPS stimulation, baicalin dose-dependently inhibit the protein and mRNA expression of CD14, but not TLR4. In RAW264.7 cells with CD14 knockdown, baicalin pretreatment did not prevent inflammatory responses and activation of MyD88/NF-κB p65 pathway induced by high concentrations (1000 μg/mL) of LPS. Furthermore, baicalin pretreatment also inhibited the expression of CD14 and activation of MyD88/NF-κB p65 pathway in LPS-induced hepatocyte-derived HepG2 cells and intestinal epithelial-derived HT-29 cells. In mice with intraperitoneal injection of LPS and in DSS-induced UC mice, oral administration of baicalin exerted protective effects by inhibition of CD14 expression and inflammation. Taken together, we demonstrate that baicalin pretreatment prevents LPS-induced inflammation in RAW264.7 cells in CD14-dependent manner. This study supports the therapeutic use of baicalin in preventing the progression of LPS-induced inflammatory diseases.

Cyclic stretch induces vascular smooth muscle cell-derived connective tissue growth factor secretion and promotes endothelial progenitor cell differentiation and angiogenesis

Endothelial progenitor cells (EPCs) play an important role in endothelial repair following vascular injury and maintaining the integrity of endothelium, but the underlying molecular mechanisms regulating the differentiation of EPCs remain to be studied. In this study, the effects of cyclic stretch and the neighboring vascular smooth muscle cells (VSMCs) on the differentiation of EPCs, as well as the underlying mechanism were investigated in a co-culture model. Bone marrow-derived EPCs were cocultured with VSMCs, which were exposed to cyclic stretch with a magnitude of 5% (which mimics physiological mechanical stress) at a constant frequency of 1.25 Hz. The results indicated that stretched VSMCs modulated EPC differentiation into mature endothelial cell (ECs), with upregulation of EC markers (CD31, vWF and KDR) and promoted angiogenesis. Meanwhile, cyclic stretch unregulated the mRNA level and secretion level of connective tissue growth factor (CTGF). After treated with recombinant CTGF (r-CTGF) at the concentration of 20 ng/ml, the mRNA expression of CD31, vWF and KDR in EPCs were increased, EPC tube formation and the protein level of FZD8 and β-catenin were promoted. Small interfering RNA (siRNA)-mediated knockdown of CTGF in VSMCs inhibited cyclic stretch-induced EPC differentiation into ECs and attenuated EPC tube formation via modulation of FZD8/β-catenin signaling pathway in vitro. Consistently, in vivo matrigel plug assay demonstrated that r-CTGF treated EPCs enhanced angiogenesis. The present results indicate that cyclic stretch induces VSMC-derived CTGF secretion, which in turn activates FZD8 and β-catenin to promote co-cultured EPC differentiation into EC lineage and angiogenesis. Our findings provide insights into the mechanisms underlying the VSMC/EPC interactions and the pro-angiogenic function of cyclic stretch in modulating EPC differentiation. CTGF acts as a key intercellular mediator and may be as a potential therapeutic target for vascular injury.

Iron trafficking in patients with Indian Post kala-azar dermal leishmaniasis.

During infections involving intracellular pathogens, iron performs a double-edged function by providing the pathogen with nutrients, but also boosts the host's antimicrobial arsenal. Although the role of iron has been described in visceral leishmaniasis, information regarding its status in the dermal sequel, Post Kala-azar Dermal Leishmaniasis (PKDL) remains limited. Accordingly, this study aimed to establish the status of iron within monocytes/macrophages of PKDL cases. The intramonocytic labile iron pool (LIP), status of CD163 (hemoglobin-haptoglobin scavenging receptor) and CD71 (transferrin receptor, Tfr) were evaluated within CD14+ monocytes by flow cytometry, and soluble CD163 by ELISA. At the lesional sites, Fe3+ status was evaluated by Prussian blue staining, parasite load by qPCR, while the mRNA expression of Tfr (TfR1/CD71), CD163, divalent metal transporter-1 (DMT-1), Lipocalin-2 (Lcn-2), Heme-oxygenase-1 (HO-1), Ferritin, Natural resistance-associated macrophage protein (NRAMP-1) and Ferroportin (Fpn-1) was evaluated by droplet digital PCR. Circulating monocytes demonstrated elevated levels of CD71, CD163 and soluble CD163, which corroborated with an enhanced lesional mRNA expression of TfR, CD163, DMT1 and Lcn-2. Additionally, the LIP was raised along with an elevated mRNA expression of ferritin and HO-1, as also iron exporters NRAMP-1 and Fpn-1. In monocytes/macrophages of PKDL cases, enhancement of the iron influx gateways (TfR, CD163, DMT-1 and Lcn-2) possibly accounted for the enhanced LIP. However, enhancement of the iron exporters (NRAMP-1 and Fpn-1) defied the classical Ferritinlow/Ferroportinhigh phenotype of alternatively activated macrophages. The creation of such a pro-parasitic environment suggests incorporation of chemotherapeutic strategies wherein the availability of iron to the parasite can be restricted.

The role of CD68+ macrophage in classical Hodgkin lymphoma patients from Egypt

BackgroundCD68+ tumor-associated macrophages (TAM) play an important role in the progression of classical Hodgkin lymphoma (cHL). We assessed the role of CD20 and CD68 + TAM in a cohort of cHL patients from Egypt and correlated the number of CD68 + cells with patients’ characteristics, response to treatment, overall and progression free survival rates (OS & PFS).MethodsCD20 expression and CD68 + TAM numbers were assessed in representative tumor tissues obtained from 81 cHL patients using flowcytometry (FCM), immunohistochemistry (IHC), and Rt-PCR techniques.ResultsThe expression levels of CD68 protein by IHC was high in 27 (33.3%), moderate in 15 (18.5%), low in 15 (18.5%), and negative in 24 (29.6%) patients (p = 0.13). CD68-mRNA expression was high in 43/81(53.1%), and low in 38(46.9%) patients (p = 0.6). The number of CD68 + TAM (by FCM) was low (< 20 cells) in 42/81 (51.9%), and high (≥20 cells) in 39/81 (48.1%) patients (p = 0.74). CD68 expression (by FCM, IHC& Rt-PCR) associated significantly with poor response to treatment, decreased CD20 expression, reduced OS and PFS rates (p < 0.001 for all). CD68 expression (by Rt-PCR only) associated significantly with advanced disease stage (p = 0.04). The age of the patients, high CD20 expression & high CD68+ macrophage number were independent prognostic factors for OS (p= 0.02, p = 0.008 & p = 0.009; respectively). However, the age of the patient, high CD20, and high CD68+ macrophage expression (by FCM&IHC) were independent prognostic factors for DFS (p. = 0.004, p. = 0.01, p. = 0.007 and p. = 0.01; respectively).ConclusionCD68 + TAM expression (by Rt-PCR, FCM and/or IHC) can identify patients with poor response to treatment and reduced survival rates (OS& PFS). Assessment of CD68 + positive macrophages by FCM is superior to other methods (Rt-PCR and IHC) as a prognostic factor for DFS and OS rates.

Decreased mRNA Expressions of CD40L in Patients with Neuromyelitis Optica Spectrum Disorder.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease that preferentially affects central nerve system. Herein, we evaluated changes of CD40L and CD40 mRNA expressions in NMOSD and controls to explore their potential roles in development of NMOSD. The expressions of CD40L and CD40 mRNA in peripheral blood mononuclear cells (PBMCs) from patients with NMOSD and healthy controls were detected by quantitative real-time PCR (qPCR). Kruskal-Wallis tests were used to compare expression levels of CD40L and CD40 mRNA between groups, and Spearman correlation analysis was performed to evaluate correlation between mRNA expression levels and annual relapse rate (ARR) of NMOSD. A total of 71 patients with NMOSD and 42 gender- and age-matched healthy volunteers were recruited in our study. Compared with healthy controls, expression of CD40L mRNA was significantly decreased in untreated patients with NMOSD, and similar trends were observed also in CD40 mRNA expression although the difference was not significant. Other than that, immunosuppressants not only successfully increased CD40L and CD40 mRNA levels during remission of NMOSD, but also corrected the negative correlation between CD40L mRNA expression and annual relapse rate (ARR) of patients NMOSD. These results favored the long-term prognosis of NMOSD patients. Our results suggest that decreased expressions of CD40L mRNA may be involved in developing of NMOSD and the proper CD40L mRNA levels benefit to prevent attacks of NMOSD. Nevertheless, the relationship between protein and mRNA expressions of CD40L and their underlying roles in the pathogenesis of NMOSD remains to be further studied.

Association of soluble CD40 levels with -1 C>T CD40 polymorphism and chronic kidney disease in systemic lupus erythematosus.

BackgroundCD40 is a transmembrane protein mainly expressed on the antigen‐presenting cells surface. CD40 plays a crucial role in immunoglobulin class switching and antibodies production. Genetic polymorphisms in the CD40 gene have been associated with increased risk of systemic lupus erythematosus (SLE) in several populations. This study aimed to evaluate the association of CD40 polymorphisms (−1 C > T, rs1883832 and 6,048 G > T, rs4810485) with SLE susceptibility, as well as with mRNA expression and soluble CD40 (sCD40) levels.MethodsThe study included 293 patients with SLE and 294 control subjects (CS). Genotyping was performed by PCR‐RFLP method. CD40 mRNA expression was determined by quantitative real‐time PCR, and ELISA quantified sCD40 levels.ResultsThe CD40 polymorphisms −1 C > T and 6,048 G > T were associated with SLE susceptibility. There was no difference between CD40 mRNA expression and CD40 polymorphisms. The sCD40 levels were lower in SLE patients with TT haplotype, whereas higher sCD40 levels were associated with damage and impaired renal function according to SLICC and KDIGO. The sCD40 levels were negatively correlated with eGFR.ConclusionThe CD40 gene polymorphisms increase the risk of SLE in the western Mexican population. The sCD40 levels are associated with −1 C > T polymorphism and chronic kidney disease.

Assessment of CD40 and CD40L expression in rheumatoid arthritis patients, association with clinical features and DAS28.

The predominance of the effector mechanisms by CD4 + T cells is a characteristic of inflammatory autoimmune diseases such as rheumatoid arthritis (RA). The CD40/CD40L costimulatory pathway contributes to these pathogenic mechanisms by promoting autoantibody production and inflammation. Aberrant expression of CD40 and CD40L in RA patients has been shown, the latter prevailing in females. However, contrasting results have emerged regarding the clinical associations of these findings. We determined the association of CD40 and CD40L expression with the clinical activity evaluated through DAS28 in RA patients. A total of 38 female RA patients and 10 age- and sex-matched control subjects were included. CD40 and CD40L mRNA expression was quantified by real-time qPCR, cell surface proteins were determined by flow cytometry, and protein soluble forms were determined by ELISA. The expansion of a CD4 + T cell subpopulation expressing CD40 was identified in the RA group. In addition, high frequencies of CD4 + CD40L + T cells expressing high levels of CD40L, increased levels of sCD40L and overexpression of CD40L mRNA were observed in these patients. Moreover, there was a gradual increase in CD40L when data were stratified according to DAS28, except for very active patients. No correlation was observed between the levels of mRNA, cell surface protein and soluble protein of CD40 and CD40L with the clinical features of RA patients. There is an altered expression of CD40L in female RA patients in association with clinical activity assessed by DAS28, these findings support the evidence that suggests CD40L as a marker of clinical activity.

Synergistic effect of Dermatophagoides pteronyssinus allergens and dexamethasone on the expression of CD163 by peripheral blood mononuclear cells

BackgroundThe CD163 is a marker of monocyte/macrophage anti-inflammatory function. Its soluble form (sCD163) also exert anti-inflammatory activities including inhibition of T cell proliferation. ObjectiveTo evaluate the effect of dexamethasone (Dx) and Dermatophagoides pteronyssinus (Dp) on ex vivo production of sCD163 by peripheral blood mononuclear cells (PBMCs). MethodsPBMCs from 26 allergic asthma patients (AAs) and 12 non-atopic healthy controls (HCs) were cultured with Dp, Dx, Dp + Dx or without any stimulation for up to 144 h (T144). Concentration of sCD163, interleukin (IL) -6 and IL-10 in PMBC culture supernatants was evaluated using ELISA. The mRNA expression of CD163 by PBMCs was estimated using quantitative PCR (qPCR). ResultsAt T144 the median concentration of CD163 in unstimulated PBMC cultures of AAs was greater than that in HCs (p = 0.008). Concomitant application of Dp and Dx resulted in a synergistic effect reflected by a dramatic increase of sCD163 concentration both in HCs (p = 0.0002) and AAs (p < 0.0001). Also a synergistic effect of Dp and Dx on CD163 mRNA expression was seen at T24 and T48 but not at T6 or T12. Among asthmatic patients the effect of Dx on sCD163 production was attenuated in severe in comparison to mild-to-moderate AAs (p = 0.0007). Moreover, Dp-induced production of IL-6 but not IL-10 was inhibited by Dx (p < 0.0001). Inhibition of IL-10 decreased sCD163 concentration by more than 50%. ConclusionsDx-triggered upregulation of anti-inflammatory CD163 expression by monocytes is synergistic with endogenous mechanisms involved in the resolution of Dp-induced inflammation. This effect is impaired in severe asthma patients.

Increased expression of CD40 and CD40L in acute coronary syndrome patients: association with genetic variants in western Mexican population

Abstract Acute coronary syndrome (ACS) belongs to the clinical entities characterized by obstruction of a coronary artery. In its etiology, inflammation contributes significantly trough the activation of immune cells by co-stimulatory molecules as CD40 and CD40L. The purpose of this work was to explore the changes of mRNA and circulating soluble levels of CD40-CD40L in ACS patients and association with genetic variants in a western Mexican population. 320 ACS patients and 300 individuals with similar age than cases without ischemic cardiopathy were recruited. Genotype determination was made using TaqMan SNP genotyping assays. CD40 and CD40L mRNA expression in whole blood samples were quantified using TaqMan Gene Expression Assays. Soluble levels were measured in plasma by enzyme-linked immunosorbent assay. We did not find evidence of association between on CD40 (rs1883832, rs4810485 and rs11086998) and CD40L (rs3092952 and rs3092920) genetic variants and susceptibility to ACS nor changes in their mRNA expression (p&amp;gt;0.05), although rs1883832 and rs4810485 affected significantly sCD40 circulating levels (p=0.005; p=0.015, respectively). Circulating soluble levels were higher in ACS patients; specifically sCD40L was higher in male patients with unstable angina compared to female patients (p=0.016). Our results showed genetic variants affect soluble levels of CD40 although the CD40 and CD40L polymorphisms are not associated with susceptibility to ACS in western Mexican population. CD40L mRNA expression, sCD40 and sCD40L plasma levels were higher in ACS patients even when they are under pharmacological treatment, which suggests an active role of the CD40-CD40L system in the immunopathogenesis of acute coronary syndrome.

Overexpression of scavenger receptor and infiltration of macrophage in epicardial adipose tissue of patients with ischemic heart disease and diabetes

BackgroundOxidized low-density lipoproteins and scavenger receptors (SRs) play an important role in the formation and development of atherosclerotic plaques. However, little is known about their presence in epicardial adipose tissue (EAT). The objective of the study was to evaluate the mRNA expression of different SRs in EAT of patients with ischemic heart disease (IHD), stratifying by diabetes status and its association with clinical and biochemical variables.MethodsWe analyzed the mRNA expression of SRs (LOX-1, MSR1, CXCL16, CD36 and CL-P1) and macrophage markers (CD68, CD11c and CD206) in EAT from 45 patients with IHD (23 with type 2 diabetes mellitus (T2DM) and 22 without T2DM) and 23 controls without IHD or T2DM.ResultsLOX-1, CL-P1, CD68 and CD11c mRNA expression were significantly higher in diabetic patients with IHD when compared with those without T2DM and control patients. MSR1, CXCL16, CD36 and CD206 showed no significant differences. In IHD patients, LOX-1 (OR 2.9; 95% CI 1.6–6.7; P = 0.019) and CD68 mRNA expression (OR 1.7; 95% CI 0.98–4.5; P = 0.049) were identified as independent risk factors associated with T2DM. Glucose and glycated hemoglobin were also shown to be risk factors.ConclusionsSRs mRNA expression is found in EAT. LOX-1 and CD68 and were higher in IHD patients with T2DM and were identified as a cardiovascular risk factor of T2DM. This study suggests the importance of EAT in coronary atherosclerosis among patients with T2DM.

A Multiparameter Flow Cytometry Analysis Panel to Assess CD163 mRNA and Protein in Monocyte and Macrophage Populations in Hyperinflammatory Diseases.

CD163 facilitates regulation and resolution of inflammation and removal of free hemoglobin and is highly expressed in myeloid cells from patients with inflammatory disorders, such as systemic juvenile idiopathic arthritis (SJIA) and macrophage activation syndrome (MAS). Our recent studies indicate that regulation of CD163 mRNA expression is a key functional property of polarized monocytes and macrophages and is mediated at the transcriptional and posttranscriptional level, including via microRNAs. The goal of the current study is to develop a multiparameter flow cytometry panel incorporating detection of CD163 mRNA for polarized monocyte and macrophage populations in disorders such as SJIA and MAS. THP-1 cells and CD14+ human monocytes were stained using fluorochrome-conjugated Abs to myeloid surface markers, along with CD163 mRNA. Staining for mRNA could reliably detect CD163 expression while simultaneously detecting different macrophage populations using Abs targeting CD14, CD64, CD80, CD163, and CD209. This approach was found to be highly sensitive for increased mRNA expression when macrophages were polarized with IL-10 [M(IL-10)], with a strong signal over a broad range of IL-10 concentrations, and showed distinct kinetics of CD163 mRNA and protein induction upon IL-10 stimulation. Finally, this panel demonstrated clear changes in polarization markers in unstimulated monocytes from patients with SJIA and MAS, including upregulated CD163 mRNA and increased CD64 expression. This approach represents a robust and sensitive system for RNA flow cytometry, useful for studying CD163 expression as part of a multimarker panel for human monocytes and macrophages, with broad applicability to the pathogenesis of hyperinflammatory diseases.

Pro-inflammatory cytokine expression studies of subclinical and clinical endometritis in endometrial tissues of buffaloes.

The objective of the study was to investigate the role of pro-inflammatory cytokines in the pathogenesis of endometritis in buffaloes, which can lead to early diagnosis of endometritis. The expression of CD14, TLR-4, and IL-1β encoding gene in endometrium of buffaloes with subclinical endometritis were 1.87, 2.71, and 3.48-fold, while samples with clinical endometritis showed only 2.90, 4.76, and 8.02-fold for CD14, TLR-4, and IL-1β, respectively, though numerical value of expression was higher in subclinical and clinical endometritis but it was at par with control. The expression profile for IL-6, IL-8, and TNF-α mRNA for subclinical endometritis were 11.95, 14.87, and 12.95-fold while for clinical endometritis, values were 18.17, 37.97, and 28.83-fold for IL-6, IL-8, and TNF-α mRNA, respectively, which were significantly higher in subclinical and clinical endometritis as compared to apparently normal samples at follicular phase. In the luteal group, the subclinical endometritis sample showed 2.13, 6.43, 14.52, 22.38, 18.64, and 2.82-fold respectively, for CD14, IL-1β IL-6, IL-8, TNF-α, and TLR-4 mRNA. Except TLR-4, the values of expression were significantly higher when compared with apparently normal. Whereas, the expression values of clinical endometritic uteri were 4.01, 7.15, 17.20, 45.55, 52.58, and 32.95-fold respectively, for CD14, TLR-4, IL-1β, IL-6, IL-8, and TNF-α mRNA which were significantly higher as compared to apparently normal uteri. An increase in the mRNA expression of CD14, TLR-4, and pro-inflammatory cytokines having higher diagnostic importance in uterine inflammation.

Astragaloside IV alleviates heart failure by promoting angiogenesis through the JAK-STAT3 pathway

Context: Heart failure (HF) is one of the most serious diseases worldwide. Astragaloside IV (ASI) is widely used for the treatment of cardiovascular disease in China.Objective: To evaluate the protective effect of ASI on the HF in a Sprague–Dawley rat model of left coronary artery ligation, and investigate the angiogenesis-related mechanisms.Materials and methods: Left coronary artery was ligated to induce a rat model of HF, and the rats were treated with vehicle (saline) or different doses of ASI (0.1, 0.3 and 1 mg/kg/day) by oral gavage for 6 weeks. Cardiac function was evaluated by echocardiography. Infarct size was determined by triphenyltetrazolium chloride staining. Cardiac vascular density was analyzed by microangiography. Real-time PCR, Western blot and chromatin immunoprecipitation were performed to investigate the mechanisms.Results: ASI treatment improved the body weight and survival rate of HF rats, as well as the cardiac function of HF rats, with significantly improved ejection fraction (75.27 ± 5.75% vs. 36.26 ± 4.14%) and fractional shortening (45.39 ± 3.66% vs. 17.88 ± 1.32%). ASI reduced the infarct size of the HF rats by 47%. ASI promoted angiogenesis, with increased vascular density (2.08-fold) and induced mRNA expression of CD31 (1.81-fold) and VEGF (2.70-fold) in the ischemic heart. Furthermore, ASI induced the phosphorylation of JAK (1.89-fold) and STAT3 (2.95-fold), as well as the activity of VEGF promoter which was regulated by STAT3.Discussion and conclusions: ASI alleviated HF by promoting angiogenesis through JAK-STAT3 pathway, providing novel alternative strategies to prevent HF in the future.

Dysregulation of T cell immunoglobulin and mucin domain 3 (TIM-3) signaling in peripheral immune cells is associated with immune dysfunction in autistic children

Evidence suggests that immune dysregulation is associated with autism spectrum disorder (ASD). T cell immunoglobulin and mucin domain-3 (TIM-3) has a critical role in several inflammatory disorders; however, the role of TIM-3 signaling has not been demonstrated in ASD. In the present study, we assessed the role of TIM-3 signaling in children with ASD. We expected that increased numbers of TIM-3+ cells could alter immune function in children with ASD. We revealed production of TIM-3 on CD3+, CD4+, CD8+, CD11a+,b+, CD14+, CD62P+, and CXCR5+ PBMCs in children with ASD and typically developing (TD) controls using immunofluorescent staining. We further demonstrated the production of IL-1β, IFN-γ, IL-17 A, and Foxp3 in TIM-3+ PBMCs of TD controls and individuals with ASD. We also observed the mRNA expression levels of TIM-3, CD11a,b, CD14, IL-1β and IFN-γ using RT-PCR. We further assessed the protein levels of TIM-3, IL-1β, CXCR5, and IFN-γ using western blotting. The results showed that children with ASD had increased numbers of CD3+TIM-3+, CD4+TIM-3+, CD8+TIM-3+, CD11a,b+TIM-3+, CD14+TIM-3+, CD62P+TIM-3+ and CXCR5+TIM-3+ cells compared with TD controls. Our results further showed that children with ASD had increased IL-1β+TIM-3+, IFN-γ+TIM-3+, and IL-17+TIM-3+, and decreased Foxp3+TIM-3+ production compared with that in TD controls. Our results indicated that children with ASD significantly induced TIM-3, CD11a,b, CD14, CXCR5, IL-1β and IFN-γ mRNA and protein expression levels compared with TD controls. The results suggested that detection of TIM-3 signaling could contribute to the early diagnoses of ASD.

Macrophage populations show an M1-to-M2 transition in an experimental model of coronal pulp tissue engineering with mesenchymal stem cells.

To assess M1/M2 macrophage phenotypes in a coronal pulp regeneration model in rats, under the hypothesis that there are dynamic M1/M2 phenotype changes during the different stages of the pulp regeneration. The maxillary first molars of Wistar rats were pulpotomized, and biodegradable hydrogel-made scaffolds carrying rat bone marrow mesenchymal stem cells were implanted in the pulp chamber. After 3, 7 and 14days, samples were processed for (i) histological analysis and double immunoperoxidase staining for CD68 (a general macrophage marker) and one of either CCR7 (an M1 marker), CD163 (an M2 marker) or CD206 (an M2 marker); (ii) real-time PCR for AIF1 (an M1 marker), CD163, CD206, IL-10 and TNF-α mRNA expression; and (iii) Western blotting for the detection of CD68, CCR7 and CD206 proteins. Histological analysis of the implanted region revealed sparse cellular distribution at 3days, pulp-like tissue with a thin dentine bridge-like structure at 7days, and dentine bridge-like mineralized tissue formation and resorption of most scaffolds at 14days. CCR7+ macrophages had the highest density at 3days, and then significantly decreased until 14days (P<0.05). In contrast, M2 marker (CD163 or CD206) expressing macrophages had the lowest density at 3days and significantly increased until 14days (P<0.05). AIF1 and TNF-α mRNA levels, and CD68 and CCR7 protein levels were highest at 3days. CD163 and CD206 mRNA levels, and CD206 protein levels increased with time and showed the highest at 14days. IL-10 mRNA was highest at 3days, decreased at 7days and increased at 14days. Macrophages in the regenerating pulp tissue underwent a distinct transition from M1-dominant to M2-dominant, suggesting that the M1-to-M2 transition of macrophages plays an important role in creating a favourable microenvironment necessary for pulp tissue regeneration.