Transcription profiling of primary human bronchial epithelial cells stimulated with IL-22 and IL-17
ABSTRACT: Primary HBE cells were stimulated with IL-22 and IL-17, and gene expression was studied using an Affymetrix platform microarray, in order to investigate which genes may be upregulated or downregulated in response to these cytokines. Of particular interest was the host defense genes such as antimicrobial peptides, which have been shown to be upregulated by IL-22 and IL-17 in skin keratinocytes. Experiment Overall Design: There were 4 conditions to this study (media, IL-22, IL-17 and IL-22+17) and there were 3 biological replicates of each condition. Experiment Overall Design: Gene expression study using one timepoint of 24 hours after stimulating these primary cells with the above conditions
Project description:Primary HBE cells were stimulated with IL-22 and IL-17, and gene expression was studied using an Affymetrix platform microarray, in order to investigate which genes may be upregulated or downregulated in response to these cytokines. Of particular interest was the host defense genes such as antimicrobial peptides, which have been shown to be upregulated by IL-22 and IL-17 in skin keratinocytes. Keywords: cytokine effect Overall design: There were 4 conditions to this study (media, IL-22, IL-17 and IL-22+17) and there were 3 biological replicates of each condition. Gene expression study using one timepoint of 24 hours after stimulating these primary cells with the above conditions
Project description:Microarray analysis using the HumanHT-12 v4 Expression BeadChip was utilized to determine the biological function of IL-22. The data indicate an extensive effect of IL-22 on many major molecular functions including activation of antimicrobial genes and downregulation of immune-associated pathways. Functional studies performed in-vitro using human salivary gland cells treated with IL-22 indicated a direct effect of IL-22 on cell cycling, specifically reducing cellular proliferation at the G2-M phase by activation of STAT3. Total RNAs were obtained from salivary gland cells stimulated with recombinant IL-22 for 45 minutes. Differential expression analyses were conducted using the LIMMA package from the Bioconductor project. MTT assay, flow cytometry and western blotting were used to identify the function of IL-22 on human salivary gland cells.
Project description:In this study, we sought to determine how IL-17 and TNF influence normal human melanocytes, either alone, or with both cytokines together. We reveal a dichotomous effect of IL-17 and TNF, which not only elicit essential mitogenic cytokines but also suppress melanogenesis by down-regulating genes of melanogenesis pathway Comparison of one batch of primary human melanocyte line cultured in serum free media, treated with TNF and/or IL-17, for either 24 or 48 hours.
Project description:Global kinase activity induced by cytokines IL-32g and IL-17A/F were determined using peptide arrays representing phophorylation targets The objective of this study was to identify common and unique phosphorylation targets of pro-inflammatory cytokines IL-32 and IL-17. These cytokines are associated with the pathogenesis and severity of chronic inflammatory disorders, therefore signaling intermediates of these cytokines could be beneficial as alternate theraputic targets that may likely influence different inflammatory pathways. Phosphorylation of proteins is a critical mechanism in the regulation of cellular processes. This process is meticulously regulated by enzymes known as kinases, which are increasingly being identified as drug targets for a variety of diseases. In this study we interrogated kinase activities (kinome) induced in the presence of the human recombinant cytokines IL-32g and IL-17A/F employing peptide arrays representing 300 peptides, printed in triplicate, representing select phosphorylation events. Human macrophage-like THP-1 cells were used for this comparative kinome analysis. Macrophage-like THP-1 cells were stimulated with either IL-32g (20 ng/ml) or IL-17A/F (20 ng/ml) for 15 min, and the peptide arrays were used to comprehensively analyze protein phosphorylation profiles in the presence of these cytokines. Two independet biological experiments were performed and the kinoem analysis was done in triplicates for each. The phosphorylations of the peptides on the array were quantified in the cytokine-treated cells relative to the un-stimulated control cells. Differentially phosphorylated targets were defined as greater than 1.5 fold increase or decrease (p < 0.06) in phosphorylation compared to un-stimulated control cells.
Project description:We sought to provide a comprehensive evaluation of the effects of TNF-α and IL-17 on the keratinocyte gene profile in order to identify genes that might be co-regulated by these cytokines. We then sought to determine how genes that were synergistically activated by both cytokines relate to the psoriasis transcriptome. Here, we identified 160 unique genes that were synergistically up-regulated by IL-17 and TNF-α and 188 unique genes where the two cytokines had at least an additive effect. Among highly up-regulated genes were those involved in neutrophil and lymphocyte chemotaxis, inflammation, and epidermal differentiation. Synergistically up-regulated genes included some of the highest expressed genes in lesional psoriatic skin with an impressive correlation between IL-17/TNF-α induced genes and the psoriasis gene signature. In conclusion, keratinocytes may be key drivers of pathogenetic inflammatory circuits in psoriasis through integrating responses to TNF-α and IL-17. This may explain high efficacy of targeting psoriasis with either anti-TNF-α or agents that block Th17 T-cells/IL-17 and has important implications for the development of new therapeutic agents. Comparison of keratinocyte responses to IL-17, TNF-α (1 ng mL-1 and 10 ng mL-1), and the combination of both cytokines in psoriasis.
Project description:NK cells are innate immune cells that recognize and kill foreign, virally-infected and tumor cells without the need for prior immunization. NK expansion following viral infection is IL-2 or IL-15-dependent. To identify Runx3 responsive genes, NK cells were isolated from spleen of WT and Runx3-/- mice . Ten samples (5 WT and 5 Runx3-/-) of freshly isolated NK cells were separately obtained from individual mice. Cells were cultured for 7 days with IL-2 or IL-15.
Project description:In this study we aim to determine the role of IL-4/STAT6 in gene expression in human keratinocytes using RNA-sequencing approach. Human keratinocytes were cultured for 2 or 5 days with calcium chloride to induce terminal differentiation as determined by the expression of epidermal differentiation complex genes. The cells were then stimulated with IL-4 for 3 and 24 hours, or along the 5 days culture period. We observed that IL-4 inhibits fully differentiation of keratinocytes, induces genes involved with production of inflammatory mediators, and reduces the healing capacity of human keratinocytes. Moreover, STAT6 controlled important genes involved with calcium binding, inflammation and epidermis development. Human keratinocytes were differentiated with calcium chloride for 2 days and incubated with media alone or 20ng/ml of recombinant human IL-4 for 3 and 24 hours. Human keratinocytes were differentiated with calcium chloride for 5 days with or wihout recombinant human IL-4 (20ng/ml). Keratinocytes transfected with control or STAT6 siRNA were differentiated with calcium chloride for 2 days and then stimulated with recombinant huma IL-4 for 24 hours.
Project description:RORγt+ innate lymphoid cells (ILC) are crucial players of innate immune responses and represent a major source of IL-22, which has an important role in mucosal homeostasis. The signals required by RORγt+ ILC to express IL-22 and other cytokines, including TNF, have only partially been elucidated. Here we show that RORγt+ ILC can directly sense the environment by the engagement of the activating receptor NKp44. NKp44 triggering in RORγt+ ILC selectively activates a coordinated pro-inflammatory program, including TNF, while cytokine stimulation induces preferentially IL-22 expression. However, combined engagement of NKp44 and cytokine receptors results in a strong synergistic effect. These data support the concept that NKp44+ RORγt+ ILC can be activated without cytokines and are able to switch between IL-22 or TNF production, depending on the triggering stimulus. Transcriptome analysis of CD56+CD127hi tonsil ILC, Ex vivo and after stimulation with aNKp44, IL-1/IL-7/IL-23 or aNKp44/IL-1/IL-7/IL-23 for 3.5h. RNA was extracted and pooled from 2 donors each, Amplified and labeled according to manufacturer´s instructions (GeneChipU133plus2® , Affymetrix). The analysis was performed in triplicates.
Project description:IL-17A and IL-22 induced several inflammation-induced genes and anti-microbial molecules including Pla2g2a and Lcn2 on HepG2 cells when the cells were treated with the cytokines before Listeria monocytogenes infection. HepG2 cells were treated with 50 ng/ml IL-17A and 10 ng/ml IL-22 before L. monocytogenes in vitro infection at MOI 10. Reference cells were not treated with the cytokines before the infection. Two independent experiments were carried out.
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.