Monocytes/macrophages are activated in several autoimmune diseases, including systemic sclerosis (scleroderma; SSc), with increased expression of interferon (IFN)-regulatory genes and inflammatory ...cytokines, suggesting dysregulation of the innate immune response in autoimmunity. In this study, we investigated whether the lytic form of Epstein-Barr virus (EBV) infection (infectious EBV) is present in scleroderma monocytes and contributes to their activation in SSc.
Monocytes were isolated from peripheral blood mononuclear cells (PBMCs) depleted of the CD19+ cell fraction, using CD14/CD16 negative-depletion. Circulating monocytes from SSc and healthy donors (HDs) were infected with EBV. Gene expression of innate immune mediators were evaluated in EBV-infected monocytes from SSc and HDs. Involvement of Toll-like receptor (TLR)8 in viral-mediated TLR8 response was investigated by comparing the TLR8 expression induced by infectious EBV to the expression stimulated by CL075/TLR8/agonist-ligand in the presence of TLR8 inhibitor in THP-1 cells.
Infectious EBV strongly induced TLR8 expression in infected SSc and HD monocytes in vitro. Markers of activated monocytes, such as IFN-regulated genes and chemokines, were upregulated in SSc- and HD-EBV-infected monocytes. Inhibiting TLR8 expression reduced virally induced TLR8 in THP-1 infected cells, demonstrating that innate immune activation by infectious EBV is partially dependent on TLR8. Viral mRNA and proteins were detected in freshly isolated SSc monocytes. Microarray analysis substantiated the evidence of an increased IFN signature and altered level of TLR8 expression in SSc monocytes carrying infectious EBV compared to HD monocytes.
This study provides the first evidence of infectious EBV in monocytes from patients with SSc and links EBV to the activation of TLR8 and IFN innate immune response in freshly isolated SSc monocytes. This study provides the first evidence of EBV replication activating the TLR8 molecular pathway in primary monocytes. Immunogenicity of infectious EBV suggests a novel mechanism mediating monocyte inflammation in SSc, by which EBV triggers the innate immune response in infected cells.
Pathogens have been implicated in the initiation and/or promotion of systemic sclerosis (scleroderma, SSc); however, no evidence was found to substantiate the direct contribution to this disease in ...past years. Recently, significant advances have been made in understanding the role of the innate immune system in SSc pathogenesis, supporting the idea that pathogens might interact with host innate immune-regulatory responses in SSc. In light of these findings, we review the studies that identified the presence of pathogens in SSc, along with studies on pathogens implicated in driving the innate immune dysregulation in SSc. The goal of this review is to illustrate how these pathogens, specifically viruses, may play important role both as triggers of the innate immune system, and critical players in the development of SSc disease.
Endothelin 1 (ET-1) is a key regulator of vascular homeostasis. We have recently reported that the presence of Human antigen class I, HLA-B35, contributes to human dermal microvascular endothelial ...cell (HDMEC) dysfunction by upregulating ET-1 and proinflammatory genes. Likewise, a Toll-like receptor 3 (TLR3) ligand, Poly(I:C), was shown to induce ET-1 expression in HDMECs. The goal of this study was to determine the molecular mechanism of ET-1 induction by these two agonists. Because HLA-B35 expression correlated with induction of Binding Immunoglobulin Protein (BiP/GRP78) and several heat shock proteins, we first focused on ER stress and unfolded protein response (UPR) as possible mediators of this response. ER stress inducer, Thapsigargin (TG), HLA-B35, and Poly(I:C) induced ET-1 expression with similar potency in HDMECs. TG and HLA-B35 activated the PERK/eIF2α/ATF4 branch of the UPR and modestly increased the spliced variant of XBP1, but did not affect the ATF6 pathway. Poly(I:C) also activated eIF2α/ATF4 in a protein kinase R (PKR)-dependent manner. Depletion of ATF4 decreased basal expression levels of ET-1 mRNA and protein, and completely prevented upregulation of ET-1 by all three agonists. Additional experiments have demonstrated that the JNK and NF-κB pathways are also required for ET-1 upregulation by these agonists. Formation of the ATF4/c-JUN complex, but not the ATF4/NF-κB complex was increased in the agonist treated cells. The functional role of c-JUN in responses to HLA-B35 and Poly(I:C) was further confirmed in ET-1 promoter assays. This study identified ATF4 as a novel activator of the ET-1 gene. The ER stress/UPR and TLR3 pathways converge on eIF2α/ATF4 during activation of endothelial cells.
Objective
Pulmonary arterial hypertension (PAH), a common complication of limited cutaneous systemic sclerosis (lcSSc), is associated with alterations of markers of inflammation and vascular damage ...in peripheral blood mononuclear cells (PBMCs). Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) have been implicated in autoimmune and inflammatory diseases. The goal of this study was to assess whether markers of ER stress and the UPR are present in PBMCs from lcSSc patients with PAH.
Methods
PBMCs were purified from 36 healthy controls, 32 lcSSc patients with PAH, and 34 lcSSc patients without PAH. Gene expression in healthy control PBMCs stimulated with thapsigargin was analyzed by DNA microarray. Genes were validated by quantitative real‐time reverse transcription–polymerase chain reaction in PBMCs from healthy controls and lcSSc patients.
Results
Several ER stress/UPR genes, including BiP, activating transcription factor 4 (ATF‐4), ATF‐6, and a spliced form of X‐box binding protein 1, were up‐regulated in PBMCs from lcSSc patients, with the highest levels in patients with PAH. Thapsigargin up‐regulated heat‐shock proteins (HSPs) and interferon (IFN)–regulated genes in PBMCs from healthy controls. Selected HSP genes (particularly DnaJB1) and IFN‐related genes were also found at significantly elevated levels in PBMCs from lcSSc patients, while IFN regulatory factor 4 expression was significantly decreased. There was a positive correlation between DnaJB1 and severity of PAH (measured by pulmonary artery pressure) (r = 0.56, P < 0.05) and between ER stress markers and interleukin‐6 levels (r = 0.53, P < 0.0001) in PBMCs from lcSSc patients.
Conclusion
This study demonstrates an association between select ER stress/UPR markers and lcSSc with PAH, suggesting that ER stress and the UPR may contribute to the altered function of circulating immune cells in lcSSc.
To characterise global chemokine expression in systemic sclerosis (SSc) skin in order to better understand the relationship between chemokine expression and vascular inflammation in this disease.
We ...investigated chemokine mRNA expression in the skin through quantitative PCR analysis comparing patients with diffuse cutaneous (dcSSc) or limited cutaneous (lcSSc) disease with healthy controls. We tested correlations between the most regulated chemokines and vascular inflammation and macrophage recruitment. CCL19 expression was examined in human primary immune cells treated with innate immune activators.
The chemokines, CCL18, CCL19 and CXCL13, were upregulated in dcSSc skin, and CCL18 in lcSSc skin. Expression of CCL19 in dcSSc skin correlated with markers of vascular inflammation and macrophage recruitment. Immunofluorescence data showed CCL19 colocalisation with CD163 macrophages in dcSSc skin. In vitro studies on human primary cells demonstrated that CCL19 expression was induced after toll-like receptor activation of peripheral blood mononuclear cells and separated populations of CD14 monocytes.
CCL18, CCL19 and CXCL13-chemoattractants for macrophage and T cell recruitment-were three of six chemokines with the highest expression in dcSSc skin. Increased CCL19 expression in the skin suggests a role for CCL19 in the recruitment of immune cells to the peripheral tissue. Induction of CCL19 in macrophages but not structural cells indicates a role for skin-resident or recruited immune cells in perivascular inflammation. This study demonstrates that CCL19 is a sensitive marker for the perivascular inflammation and immune cell recruitment seen in dcSSc skin disease.
HLA-B*35 is associated with increased risk of developing pulmonary hypertension in SSc patients. We previously reported that HLA-B*35 induces endothelial cell dysfunction via activation of ER ...stress/UPR and upregulation of the inflammatory response. Because PBMCs from lcSSc-PAH patients are also characterized by activation of ER stress/UPR and inflammation, the goal of this study was to assess whether the presence of HLA-B*35 contributes to those characteristics.
PBMCs were purified from healthy controls (n = 49 HC) and lcSSc patients, (n = 44 with PAH, n = 53 without PAH). PBMCs from each group were stratified for the presence of HLA-B*35. Global changes in gene expression in response to HLA-B*35, HLA-B*8 or empty lentivirus were investigated by microarray analysis in HC PBMCs. Total RNA was extracted and qPCR was performed to measure gene expression.
ER stress markers, in particular the chaperones BiP and DNAJB1 were significantly elevated in PBMC samples carrying the HLA-B*35 allele. IL-6 expression was also significantly increased in HLA-B*35 lcSSc PBMCs and positively correlated with ER stress markers. Likewise, HMGB1 was increased in HLA-B*35-positive lcSSc PBMCs. Global gene expression analysis was used to further probe the role of HLA-B*35. Among genes downregulated by HLA-B*35 lentivirus were genes related to complement (C1QB, C1QC), cell cycle (CDNK1A) and apoptosis (Bax, Gadd45). Interestingly, complement genes (C1QC and C1QB) showed elevated expression in lcSSc without PAH, but were expressed at the low levels in lcSSc-PAH. The presence of HLA-B*35 correlated with the decreased expression of the complement genes. Furthermore, HLA-B*35 correlated with decreased expression of cyclin inhibitors (p21, p57) and pro-apoptotic genes (Bax, Gadd45) in lcSSc B35 subjects. FYN, a tyrosine kinase involved in proliferation of immune cells, was among the genes that were positively regulated by HLA-B*35. HLA-B*35 correlated with increased levels of FYN in lcSSc PBMCs.
Our study demonstrates that HLA-B*35 contributes to the dysregulated expression of selected ER stress, inflammation and proliferation related genes in lcSSc patient PBMCs, as well as healthy individuals, thus supporting a pathogenic role of HLA-B*35 in the development of PAH in SSc patients.
Abstract only
Objective
Previous studies have implicated ER stress and UPR in autoimmune and inflammatory diseases.The goal of this study was to assess whether markers of ER stress/UPR are present in ...PBMCs from limited cutaneous systemic sclerosis (lcSSc) patients with pulmonary arterial hypertension (PAH).
Methods
PBMCs were purified from healthy controls (HC n=36) and lcSSc patients (n=38 with PAH and n=39 without PAH).Gene expression in HC PBMCs stimulated with ER stress inducer, thapsigargin (TG) was analyzed by DNA microarray.Expression levels of select ER stress/UPR genes and inflammation genes were analyzed by qPCR.
Results
ER stress/UPR genes, including BiP, transcription factors ATF4 and ATF6, and a spliced form of XBP1 were upregulated in lcSSc PBMCs, with the highest levels in patients with PAH.TG induced expression of HSPs and IFN‐regulated genes in control PBMCs.Selected HSP genes, particularly DNAJB1, and IFN‐related genes were significantly elevated in lcSSc PBMCs.There was a positive correlation between DNAJB1 and severity of PAH disease (PAP) (r = 0.59, p<0.05) and between BiP and IL‐6 levels (r = 0.64, p< 0.0001) in lcSSc PBMCs.
Conclusion
These studies demonstrate a possible association between ER stress/UPR gene activation and lcSSc‐PAH suggesting that ER stress/UPR contribute to the interferon signature and altered function of circulating immune cells in patients with lcSSc.
Experimental evidence has shown that probucol, a hypocholesterolemic agent, is also able to increase glutathione peroxidase (GPx) activity. However, there is a lack of knowledge about the ...mechanism(s) involved in this event. In this study, in vitro experiments with purified GPx1 from bovine erythrocytes and cultured SH-SY5Y neuroblastoma cells, as well as in silico studies with GPx1, were performed in order to elucidate mechanisms mediating the stimulatory effect of probucol on GPx activity and to investigate the relevance of this event in terms of susceptibility against peroxide-induced cytotoxicity. In vitro experiments with purified GPx1 showed a direct stimulatory effect of probucol on the activity of GPx1, which was related to an increase in
V
max
with no changes in
K
M
. Probucol also increased GPx activity in cultured SH-SY5Y neuroblastoma cells, while the levels of GPx1 expression were not changed, corroborating the results found with the purified enzyme. In addition, probucol rendered SH-SY5Y cells more resistant to hydroperoxide-induced cytotoxicity, and this event was abolished in GPx1 knocked-down cells. In silico studies with GPx1 pointed to a potential binding site for probucol at the close vicinity of the GSH pocket. Collectively, the results presented herein indicate that GPx1 plays a central role in the probucol-induced protective effects against peroxide toxicity. This highlights a novel target (GPx1) and a new mechanism of action (direct activation) for an “old drug.” The relevance of such results for in vivo conditions deserves further investigation.
The progressive increase in nitrate’s (NO3−) presence in surface and groundwater enhances environmental and human health risks. The aim of this work is the fabrication and characterization of ...sensitive, real-time, low-cost, and portable amperometric sensors for low NO3− concentration detection in water. Copper (Cu) micro-flowers were electrodeposited on top of carbon screen-printed electrodes (SPCEs) via cyclic voltammetry (with voltage ranging from −1.0 V to 0.0 V at a scan rate of 0.1 V s−1). The obtained sensors exhibited a high catalytic activity toward the electro-reduction in NO3−, with a sensitivity of 44.71 μA/mM. They had a limit of detection of 0.87 µM and a good dynamic linear concentration range from 0.05 to 3 mM. The results were compared to spectrophotometric analysis. In addition, the devices exhibited good stability and a maximum standard deviation (RSD) of 5% after ten measurements; reproducibility, with a maximum RSD of 4%; and repeatability after 10 measurements with the RSD at only 5.63%.
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by synaptic loss and cognitive impairments. The presence of extracellular senile plaques (mainly composed of amyloid-β (Aβ) ...peptide) is an important molecular hallmark in AD and neuronal damage has been attributed, at least in part, to Aβ-mediated toxicity. Although the molecular mechanisms involved in the pathogenesis of AD are not yet completely understood, several lines of evidence indicate that oxidative stress and cholesterol dyshomeostasis play crucial roles in mediating the synaptic loss and cognitive deficits observed in AD patients. This study evaluated the effects of Probucol, a phenolic lipid-lowering agent with anti-inflammatory and antioxidant properties, on biochemical parameters related to oxidative stress and synaptic function (hippocampal glutathione and synaptophysin levels; glutathione peroxidase, glutathione reductase and acetylcholinesterase activities; lipid peroxidation), as well as on behavioral parameters related to the cognitive function (displaced and new object recognition tasks) in Aβ-exposed mice. Animals were treated with a single intracerebroventricular (i.c.v.) injection of aggregated Aβ1–40 (400pmol/site) and, subsequently, received Probucol (10mg/kg, i.p.) once a day, during the following 2weeks. At the end of treatments, Aβ1–40-exposed animals showed a significant impairment on learning-memory ability, which was paralleled by a significant decrease in hippocampal synaptophysin levels, as well as by an increase in hippocampal acetylcholinesterase activity. Importantly, Probucol treatment blunted the deleterious effects of Aβ1–40 on learning-memory ability and hippocampal biochemistry. Although Aβ1–40 treatment did not change hippocampal glutathione levels and glutathione peroxidase (GPx) and glutathione reductase (GR) activities, Aβ1–40-exposed animals showed increased hippocampal lipid peroxidation and this event was completely blunted by Probucol treatment. These findings reinforce and extend the notion of the hazardous effects of Aβ1–40 toward hippocampal synaptic homeostasis and cognitive functions. In addition, the present results indicate that Probucol is able to counteract the cognitive and biochemical impairments induced by i.c.v. Aβ1–40 administration in mice. The study is the first to report the protective effects of Probucol (a “non-statin cholesterol-lowering drug”) against Aβ1–40-induced synaptic and behavioral impairments, rendering this compound a promising molecule for further pharmacological studies on the search for therapeutic strategies to treat or prevent AD.
► Probucol is a “non-statin” cholesterol-lowering drug. ► This study evaluated the effects of Probucol against Aβ1–40-induced synaptic and behavioral impairments in mice. ► i.c.v. Aβ1–40 administration caused cognitive and synaptic impairments. ► Probucol treatment blunted the deleterious effects of Aβ1–40.
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