Project description:Identify IL-2 mediated genes in Kit225 cells. IL-2 withdrawn cells were treated with IL-2 or left untreated for 3 hours and then subjected to gene expression microarray analysis.
Project description:We performed an absolute quantification of IL-10RA and IL-10RB on the human cell lines U937, HUT78, THP1, K562. To identify suitable peptides to use as hevay peptide spike-ins label-free proteomic analysis was performed on the HEK-Blue IL-10 cells (hkb-il10v2_Invivogen) cell line.
Project description:We used Affymetrix expression arrays to determine changes in gene expression associated with activation of human NK cells mediated through treatment with cytokines IL-2, IL-12 and IL-18 over a 24 hour period.
Project description:We used Affymetrix expression arrays to determine changes in gene expression associated with activation of human NK cells mediated through treatment with cytokines IL-2, IL-12 and IL-18 over a 24 hour period. Human natural killer cells were isolated via negative selection from PBMCs of healthy donors. Cells were found to be > 95% CD3-CD56+. RNA was harvested at time of isolation or after 24 hour stimulation from 8 x 10^6 cells per condition. For stimulations, cells were incubated at 37C (5% CO2) in RPMI, suuplemented with 10% Fetal Bovine Serum at 1.5 x 10^6 cells/ml. Cytokine stimualtions were conducted with IL-2 (100U/mL), IL-12 (10ng/mL) and IL-18 (100ng/mL) from 2 male donors (N = 4). Expression analysis was carried out to determine transcriptional changes associated with 24 hr stimulation relative to freshly isolated cells.
Project description:Interleukin-6 (IL-6) plays an important role in progressive bone loss in rheumatoid arthritis (RA). However, the molecular mechanisms through which IL-6 propels RA synovial fibroblasts (RASFs) to contribute to bone loss are not fully understood. In the present study, we investigated the effects of IL-6 and IL-6 receptor (IL-6/IL-6R induced trans-signaling in human RASFs. Treatment of human RASFs with IL-6 and IL-6 receptor (IL-6/IL-6R, 100 ng/ml each) alone or in combination with M-CSF (2 ng/ml) and RANKL (10 ng/ml) for 12 days resulted in the phenotypic changes to osteoclast-like features as determined by increase in TRAP staining and enhanced pit formation by ~3-fold in bone resorption assay in vitro. IL-6/IL-6R induced a dose-dependent increase in the expression of transcription factor Ets2 and enhanced its nuclear translocation in human RASFs. Interestingly, the untargeted proteomics analysis of the conditioned media from IL6/IL6R activated RASFs using Mass-Spectrometry (MS) technique and Trans-Proteomic Pipeline tool showed the production of 113 analytes unique to IL-6/IL-6R stimulation. Further analysis of the proteomics data using STRING database for pathway analysis showed the impact of IL-6/IL-6R on immunometabolic reprogramming of human RASFs towards osteoclast-like phenotype, which was partly mediated through Ets2. These preliminary studies identified a novel role of IL-6/IL-6R-mediated trans signaling in the metabolic reprogramming in RASFs that could potentially be targeted by inhibiting Ets2 to limit their role in bone resorption in RA.
Project description:Macrophage activation triggers intense genetic and immunometabolic reprograming that support distinct macrophage functions, and mitochondria are central to this process. To date, the extent to which they are remodeled and how this impacts macrophage function remains unclear. Here, we integrate transcriptomics with whole-cell and highly purified mitochondrial proteomics to profile LPS/IFNγ- andIL-4/IL-13-stimulated bone-marrow–derived macrophages (BMDM). We uncover a striking disconnect between mitochondrial transcripts and proteins and identify a STAT6-dependent increase in mitochondrial gene expression and intramitochondrial translation as hallmarks of IL-4/IL-13 stimulation. Pharmacological blockade of mitochondrial translation or individual respiratory chain complexes impairs activation, while ATP synthase inhibition uniquely triggers mitochondrial hyperpolarization and the integrated stress response, halting IL-4/IL-13 activation. Restoring mitochondrial membrane potential or inhibiting the stress response rescues effector synthesis. Together, we establish mitochondrial gene expression and membrane potential as regulators of macrophage immunometabolism and identify mitochondrial translation as a critical, drug-sensitive determinant of IL-4/IL-13 macrophage function.
Project description:To investigate the effect of IL-6 on gene expression in IL-6-dependent MM cell lines, we have employed whole genome microarray expression profiling as a discovery platform to identify genes with the potential to be associated with the cell proliferation of MM cells. ANBL-6 and FLAM-76 cells were incubated for 48 h in the absense of IL-6, and then the cells were treated with IL-6 (10 ng/mL) for 0, 6, 12, and 24 h in vitro.
Project description:IL-6, a proinflammtory cytokine produced by antigen presenting cells and non-hematopoietic cells in response to external stimuli, acts as an important bridge between the innate and adaptive immune responses. IL-6 together with IL-4 can promote Th2 polarization, while in combination with TGFbeta mediates Th17 differentiation. We examined early changes in gene expression in mouse CD4+ T cells induced by IL-6. Keywords: adaptive immune response