Protein microarray analysis of expression of murine cytokines in spleen interfered with IFN-α and (or) LPS
ABSTRACT: 12 wild-type C57BL/6 (B6) mice were divided into 4 groups: control group, IFN-α group, LPS group, and IFN-α+ LPS group, every group contained 3 mice (n=3). IFN-α was administrated i.p. to IFN-α group and IFN-α+ LPS group once daily (QD) for 7 days at a medium dose of 105units/kg weight, PBS was administrated i.p. to control group and LPS group QD for 7 days at the same volume. LPS group and IFN-α+ LPS group were injected i.v. with 10 μg LPS for one mouse on the 8th day. Control group and IFN-α group were injected i.v. with PBS at the same volume. 6 h later, mice were sacrificed to harvest spleens for protein microarray experiment. Mouse Cytokine Antibody Array 3(62) was purchased from Ray Biotech, Norcross GA, US. Protein microarray of murine cytokines expression. Spleens from 12 mice (4 group) were treated as indicated in the summary. Equal amount total protein from each spleen was pooled prior to gene expression analysis.
Project description:Human hepatocyte chimeric mice were prepared and treated with hepatitis C virus (HCV) and/or interferon-alpha (IFN-α). To analyze the changes in gene expression, cDNA microarray analysis was performed with the collected human hepatocytes from the chimeric mouse livers. We consider that these results provide molecular insights into possible mechanisms used by HCV to evade innate immune responses, as well as novel therapeutic targets and a potential new indication for interferon therapy. A total of 15 human hepatocyte chimeric mice were prepared and divided into four experimental groups. Mice in group A were neither infected with HCV nor treated with IFN. Mice in group B were administered IFN-α 6 h before sacrifice but were not infected with HCV. The mice in groups C and D were both inoculated via the mouse tail vein with human serum containing HCV genotype 1b particles. At 8 weeks after inoculation, the mice in group D were administered IFN-α 6 h before sacrifice, but the mice in group C were not treated with IFN-α. The human hepatocytes in the mouse livers were collected after sacrifice and subjected to microarray analysis. After purification and reverse transcription of total RNA, cDNA was hybridized on Affymetrix GeneChip Human Gene U133 Plus 2.0 Arrays.
Project description:Transcriptional profiling of mouse primary embryonic fibroblasts (MEFs) from wild type (WT) and knockin littermates expressing STAT1F77A (KI). For both genotypes, untreated control cells were compared to cells treated with IFN-alpha or IFN-gamma. Two condition experiments; untreated versus IFN treated
Project description:Systemic acute inflammatory signals can cause profound anorexia by disrupting the physiological appetite regulation in the hypothalamic milieu. Conversely, obesity related chronic inflammation of the hypothalamus can disturb anorexigenic signals and promote abnormal body weight control. The aim of the present study was to compare the global hypothalamic endophenotype in C57/Bl6 mice exposed to a high-fat diet or with acute illness mediated by LPS. Ten-week old male C57/Bl6 mice (n=18) were randomly divided into four groups; the control 1 group (n =3) was fed a normal diet whereas the high-fat diet (HFD) group (n =6) was fed a high-fat diet for eight weeks. The control 2 group (n=3) received an intraperitoneal injection of saline whereas the LPS group (n=6) received an intraperitoneal injection of LPS. Mice were sacrificed 18-hr post-injection. Both control 2 and LPS groups were fed a normal diet for eight weeks before the injection. The hypothalamic regions were removed and analysed using a 2D LC-MS methodology. The proteomic analysis profiled 9,235 proteins (q<0.05) across all biological states, of which 522 proteins were found modulated in the HFD group and another 579 in the LPS group. The proteomic profiles demonstrated that the systemic acute inflammation linked with anorexia induced a negative feedback loop of appetite control in the hypothalamus, suggesting an effort to re-establish homeostasis. By contrast, the chronic inflammation associated with obesity initiated a “perpetual cycle” of positive feedback enhancement of appetite regulation further exacerbating positive energy balance.
Project description:Interferon (IFN)-alpha causes high rates of depression and fatigue, and is used to investigate the impact of innate immune cytokines on brain and behavior. However, little is known about transcriptional profiles of circulating immune cells during chronic IFN-alpha administration. Accordingly, genome-wide transcriptional profiling was performed on peripheral blood mononuclear cells from 21 patients with chronic hepatitis C virus either awaiting IFN-alpha therapy (n=10) or after 12 weeks of IFN-alpha treatment (n=11). Significance analysis of microarray data identified 252 up-regulated gene transcripts, the majority of which were related to IFN-alpha/antiviral or innate-immune/inflammatory signaling. Of these upregulated genes, 2'-5'-oligoadenylate synthetase 2 (OAS2) was the only gene that was differentially expressed in patients that developed IFN-alpha-induced depression/fatigue, and correlated with depression and fatigue scores at 12 weeks of IFN-alpha administration. Promoter-based bioinformatic and cellular origin analyses revealed IFN-alpha-induced increases in genes bearing transcription factor binding motifs (TFBMs) related to myeloid differentiation, IFN-alpha signaling, API and CREB/ATF family of transcription pathways, with changes derived primarily from monocytes and plasmacytoid dendritic cells. Patients with high depression/fatigue scores demonstrated up-regulation of genes bearing TFBMs for myeloid differentiation, IFN-alpha and AP1 signaling, and down regulation of TFBMs for CREB/ATF-related transcription factors. Cellular origin analyses indicated a shift toward genes derived from CD8+T and NK cells in subjects with high depression/fatigue scores. These results reveal an antiviral and inflammatory transcriptional profile after 12 weeks IFN-alpha, accompanied by increased OAS2 expression, decreased CREB/ATF transcriptional control, and a shift from monocyte-derived genes to those of cytotoxic lymphocytes in IFN-alpha-induced depression/fatigue. Total RNA was isolated from the peripheral blood mononuclear cells (PBMC) obtained at 12 weeks from HCV patients treated with IFN-alpha plus ribavirin (n=11) and untreated HCV patients awaiting IFN-alpha/ribavirin therapy (control subjects, n=10).
Project description:Hepatitis B virus (HBV) causes both acute and chronic liver inflammation. Approximately 600,000 CHB patients each year die of HBV-related diseases such as cirrhosis and liver cancer. Therefore, CHB remains a global health concern. Although there have been anti-HBV agents for treating CHB, they have some limitations including viral-drug resistance and adverse effects. Type III IFN or IFN-λ is promising to use as anti-HBV agents because of its anti-viral activities like type I IFNs. In addition, the expression of its receptor, IFNLR1, is limited only in epithelial cells including hepatocytes. Thus, treatment with IFN-lambda results in less side effects compared to IFN-alpha treatment. IFN-lambdas have been shown to inhibit the replication of several viruses including IAV, DENV, EMCV, HIV, HCV, and HBV; however, there have been no studies on the effects of IFN-λ3, the highest activity among other subtypes, on HBV replication. Therefore, this study aims to determine antiviral activities of IFN-λ3 against HBV replication and to investigate its molecular mechanism responsible for suppressing HBV propagation. The results showed that HBV transcripts and amount of intracellular HBV DNA were decreased in HepG2.2.15 cells, stable HBV-transfected hepatoblastoma cell line, treated with IFN-λ3 in a dose-dependent manner. This indicated that IFN-λ3 could inhibit HBV replication. Next, we performed quantitative proteomics to investigate the proteome changes in HepG2.2.15 treated with IFN-λ3. The proteins that changed their expressions were involved in several biological processes such as defense to viral infection, immune responses, cell-cell adhesion, transcription, translation, and metabolism. We further confirmed the proteomics results by immunoblotting assay. Consistent with MS data, it found that the expression of OAS3, SAMHD1 and STAT1 were increased as a result of IFN-λ3 stimulation. These results indicated that proteomics results were reproducible and reliable. Finally, we proposed 3 possible mechanisms involved in suppressing HBV replication including i.) IFN-λ3 induced anti-viral proteins affecting many steps in HBV life cycle ii.) IFN-λ3 promoted antigen processing and antigen presentation and iii.) IFN-λ3 rescued RIG-I signaling to promote both type I and type III IFN production.
Project description:We performed a phase I/II, randomized, double-blind, placebo-controlled dose-escalation study to examine the safety, immunogenicity, and biological effects of active immunization with interferon alpha-Kinoid (IFN-K) in systemic lupus erythematosus (SLE) patients. Women 18-50 years of age with mild to moderate SLE were immunized with three (n=10) or four doses (n=9) of 30, 60, 120, 240 microgram IFN-K or saline. Anti-IFNalpha antibodies were detected in all IFN-K-immunized patients. Transcriptomic analysis separated patients at baseline into type I IFN signature-positive and -negative groups. IFN-K induced higher anti-IFNalpha titers in signature-positive than in signature-negative patients and, in signature-positive patients, reduced the expression of IFN-induced genes. The decrease in IFN score correlated with the anti-IFNalpha antibody titers, and with baseline IFN score. IFN-K or placebo was administered at day 0, day 7, day 28 in all patients. Half the subjects received a fourth injection at day 84 (see treatment protocol)
Project description:G-protein coupled receptors (GPCRs) have diverse roles in physiological processes, including immunity. Gs-coupled GPCRs increase while Gi-coupled ones decrease intracellular cAMP. Previous studies suggest that, in epithelial cells, Gs-coupled GPCRs enhance whereas Gi-coupled GPCRs suppress pro-inflammatory immune responses. In order to examine the issue, we chose beta2 adrenergic receptor and GPR40 as representatives of Gs- and Gi- coupled GPCRs, respectively, and examined their effects on TNF-alpha and IFN-gamma-(TNF-alpha + IFN-gamma) induced gene expression by HaCaT. We used microarrays to detail the global changes of gene expression induced by a beta2 adrenergic receptor agonist terbutaline or GPR40 agonist GW9508 pre-treatment in TNF-alpha + IFN-gamma - stimulated HaCaT cells. HaCaT cells were pre-treated with terbutaline or GW9508, TNF-alpha + IFN-gamma were then added, and cultured for another 24 h. Cells were then used for RNA extraction and hybridization on Affymetrix microarrays. We sought to clarify changes in gene expression after 1) TNF-alpha + IFN-gamma, 2) TNF-alpha + IFN-gamma + terbutaline, and 3) TNF-alpha + IFN-gamma + GW9508 treatment. To this end, we set 4 groups of samples; 1) unstimulated group, 2) TNF-alpha + IFN-gamma-stimulated group, 3) TNF-alpha + IFN-gamma + terbutaline-stimulated group, and 4) TNF-alpha + IFN-gamma + GW9508-stimulated group. In each group, HaCaT cells were stimulated in triplicate wells (n=3).
Project description:We used a transcriptomic approach to systematically compare the response to inflammatory stimuli for two murine cardiomyocyte models. Our results indicate that murine neonatal cardiomyocytes (NNC) are able to exhibit a pronounced response upon inflammatory stimuli, while HL-1 cells were predominantly recruiting transcripts corresponding to Jak-Stat-signalling. RNA from murine HL-1 cardiomyocytes and isolated neonatal cardiomyocytes (NNC) stimulated by infectious (PCI) serum or LPS or a cytokine mix (CM) were extracted (n=4 each group) and subjected to microarray analysis for comparison of transcriptomic responses.
Project description:mRNA microarray analysis of bone marrow derived macrophages treated under four conditions, including Naïve (N). Bone marrow derived macrophages (BMDM) were derived from the bone marrow of mice and cultured in the presence of PAO, IFN-gamma, or lipopolysaccharide (LPS). Profiled groups include Naive, LPS, IFN, PAO. Compared each of the groups (PAO, LPS, IFN) with Naïve group.