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TNF time course study

ABSTRACT: TNF time course series of HelA tet off cells cultured in presence or absence of Dox TNF is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the Nuclear Factor-kB (NF-kB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kB dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kB inhibitor to systematically identify NF-kB dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kB signaling was analyzed. Methods: Cell culture, treatment and transfection-The human cervical epithelioid carcinoma cell line HeLa expressing tTA (HeLa Tet-Off) pBI-EGFP- IkBa Mut was constructed earlier (23). HeLa Tet-Off were grown in medium containing 90 % Dulbecco’s Modified Eagle’s Medium (DMEM), 10 % heat-inactivated fetal bovine serum (FBS), 4mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mg/ml G418, 100 units /ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5 % CO2. For TNF stimulation, freshly isolated cells were split into two cultures, one group maintained in Dox, the other without for 7 d, a time at which IkBa Mut expression was maximal. Thereafter, recombinant human (rh) TNFa (25 ng/ml, final concentration) was added directly to the culture medium for indicated times prior to harvest. Oligonucleotide probe based microarray-Hu95Av2 GeneChip (Affymetrix Inc, Santa Clara, CA) containing 12,626 sequenced human genes were hybridized according to the manufacturer’s recommendations and washed using both non-stringent (1 M NaCl, 25 °C) and stringent (1 M NaCl, 50 °C) conditions prior to staining with phycoerythrin streptavidin (10 mg/ml final). Arrays were scanned using a Gene Array Scanner (Hewlett Packard) and analyzed using the Gene Chip Analysis Suite 5 software (Affymetrix Inc). For each gene, 16-20 probe pairs are immobilized as ~25-mer oligonucleotides that hybridize throughout the mRNA; each probe pair is represented as a perfect match (PM) oligonucleotide and a mismatch (MM) oligonucleotide as hybridization control. The the Absolute Call [e.g., the gene is detected (“present”) or not (“absent”)] and the Signal Intensity (measure of mRNA abundance) is determined. Four independent experiments were performed identically including control (0 h ), 1, 3 and 6 h TNF stimulation (25 ng/ml) in the presence or absence of Doxycyline (2 mg/ml) in growth medium. These are indicated as 180, 181, 220 and 266 series, and are considered replicates of each other. Keywords: time-course

ORGANISM(S): Homo sapiens

PROVIDER: GSE2624 | GEO | 2005/07/04

SECONDARY ACCESSION(S): PRJNA92135

REPOSITORIES: GEO

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Project description:TNF time course series of HelA tet off cells cultured in presence or absence of Dox; TNF is a pro-inflammatory cytokine that controls expression of inflammatory genetic networks. Although the Nuclear Factor-kB (NF-kB) pathway is crucial for mediating cellular TNF responses, the complete spectrum of NF-kB dependent genes is unknown. In this study, we used a tetracycline-regulated cell line expressing an NF-kB inhibitor to systematically identify NF-kB dependent genes. A microarray data set generated from a time course of TNF stimulation in the presence or absence of NF-kB signaling was analyzed. Methods: Cell culture, treatment and transfection-The human cervical epithelioid carcinoma cell line HeLa expressing tTA (HeLa Tet-Off) pBI-EGFP- IkBa Mut was constructed earlier (23). HeLa Tet-Off were grown in medium containing 90 % Dulbecco's Modified Eagle's Medium (DMEM), 10 % heat-inactivated fetal bovine serum (FBS), 4mM L-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 100 mg/ml G418, 100 units /ml penicillin G sodium, and 100 mg/ml streptomycin sulfate in a humidified atmosphere of 5 % CO2. For TNF stimulation, freshly isolated cells were split into two cultures, one group maintained in Dox, the other without for 7 d, a time at which IkBa Mut expression was maximal. Thereafter, recombinant human (rh) TNFa (25 ng/ml, final concentration) was added directly to the culture medium for indicated times prior to harvest. Oligonucleotide probe based microarray-Hu95Av2 GeneChip (Affymetrix Inc, Santa Clara, CA) containing 12,626 sequenced human genes were hybridized according to the manufacturer's recommendations and washed using both non-stringent (1 M NaCl, 25Â°C) and stringent (1 M NaCl, 50Â°C) conditions prior to staining with phycoerythrin streptavidin (10 mg/ml final). Arrays were scanned using a Gene Array Scanner (Hewlett Packard) and analyzed using the Gene Chip Analysis Suite 5 software (Affymetrix Inc). For each gene, 16-20 probe pairs are immobilized as ~25-mer oligonucleotides that hybridize throughout the mRNA; each probe pair is represented as a perfect match (PM) oligonucleotide and a mismatch (MM) oligonucleotide as hybridization control. The the Absolute Call [e.g., the gene is detected (present) or not (absent)] and the Signal Intensity (measure of mRNA abundance) is determined. Four independent experiments were performed identically including control (0 h), 1, 3 and 6 h TNF stimulation (25 ng/ml) in the presence or absence of Doxycyline (2 mg/ml) in growth medium. These are indicated as 180, 181, 220 and 266 series, and are considered replicates of each other.

Project description:To test the efficacy of TNFR-Fc and anti-TWEAK mAb treatment alone and in combination Tumor necrosis factor (TNF)-alpha is a major effector in various inflammatory conditions. TNF-like weak inducer of apoptosis (TWEAK) is a member of the TNF superfamily that promotes inflammatory tissue damage through its receptor, FGF-inducible molecule 14 (Fn14). Since both TWEAK and TNF-alpha have been shown to mediate pathological responses through inter-dependent or independent pathways by in vitro, the potential interplay of these pathways was investigated in a mouse colitis model. Acute colitis was induced by rectal injection of trinitrobenzene sulfonic acid (TNBS), with administration of control IgG, TNF receptor (TNFR)-Ig chimeric protein, anti-TWEAK monoclonal antibody, or the combination of TNFR-Ig and anti-TWEAK antibody. On day 4, disease severity was evaluated and gene expression profiling was analyzed using whole colon tissue. Levels of transcript of TWEAK, Fn14 and NF-kB-related molecules were measured in purified colon epithelial cells (ECs). NF-kB activation was investigated with Western blot and immunohistochemical analysis. As a result, activation of the canonical, but not noncanonical NF-kB pathway was the hallmark of inflammatory responses in this model. Inflammation induced upregulation of Fn14 only in ECs but not in other cell types. Combination treatment of TNFR-Ig and anti-TWEAK antibody synergistically reduced disease severity in comparison with the control antibody or single agent treatment. Gene expression profile of the colon indicated downregulation of canonical NF-kB pathway with combination treatment. In conclusion, synergistic activation of canonical NF-kB by TWEAK and TNF-alpha is critical for the induction of inflammatory tissue damage in acute inflammation. TNBS colitis was induced by intrarectal administration of a 2 % solution of TNBS in phosphate-buffered saline (PBS): ethanol (1:1). For acute inflammatory responses, 70 M-NM-<g/g body weight of TNBS was given on day 0 and animals sacrificed on day 4. One hour prior to administration of TNBS, groups of mice were injected i.p. with the control IgG2a mAb (anti-human CD20) (10 mg/kg), TNFR-Ig (0.3 mg/kg), anti-TWEAK (mP2D10, 10 mg/kg), the combination of TNFR-Fc (0.3 mg/kg) and anti-TWEAK mP2D10 (10 mg/kg), or were untreated. For single agent and combination treatments groups, 0.3 mg/kg TNFR-Ig was employed, since 1 mg/kg of TNFR-Ig markedly ameliorated TNBS colitis but 0.3 mg/kg was much less effective as monitored by the effect on colon length and body weight. Colon tissue was rolled and snap-frozen in liquid nitrogen. Specimens for RNA extraction were cut from the frozen rolled colon. A set of experiments using 5-8 mice for each experimental group was performed twice, labelled 7 and 10 in the CEL files. Total number of mice for each experimental condition was as follows; untreated TNBS colitis group, 8; control antibody, 15; TNFR-Ig, 12; anti-TWEAK mAb, 14; combination of TNFR-Ig and anti-TWEAK mAb, 15.

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TNF time course study

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