hek bluetm htlr9 reporter assay  (InvivoGen)

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: PS ASO innate immunogenicity varies with cell system, treatment duration, and concentration. ( A ) Model PS-ASOs used for this study. “o”indicates a phosphodiester linkage; all other linkages are phosphonothioate. Constrained Ethyl (cEt) and MOE 2’ modifications are indicated for each PS-ASO. Fast-acting or slow-acting TLR9 agonism is listed as defined by Pollak et al. 2022 (, ). ( B ) Pulse treatment in the BJAB cell line (left) and the THP1-TLR9 cell line (right). Relative qRT-PCR levels of CCL22 mRNA following a 1, 2, 3, or 4 h incubation with 1.6 µM of the indicated PS ASOs in serum-free RPMI media. RNA lysates were collected 24 h following treatment. ( C ) Continuous treatment in the BJAB cell line (left) and the THP1-TLR9 cell line (right). Relative qRT-PCR levels of CCL22 following a 2-, 4-, 8-, or 24-h incubation with 1.6 µM of indicated PS ASOs in serum-free RPMI media. RNA lysates were collected immediately following treatment. ( D ) Dose response in BJAB cell line (left) or THP1-TLR9 cell line (right). Cells were treated for 2 h with varying concentrations (0.064, 0.32, 1.6, or 8 µM) of PS ASOs. RNA lysates were collected 24 h after treatment. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Immunopeptidomics, Concentration Assay, Quantitative RT-PCR, Incubation

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Differential kinetics of cytokine secretion following PS ASO stimulation in BJAB and THP1-TLR9 cells. THP1-TLR9 (left) and BJAB cells (right) were treated with 1.6 μM of the indicated PS ASOs for 2 h, before supernatants were collected for cytokine analysis of TNF-α ( A ) and IL-10 ( B ). THP1-TLR9 cells were also assessed for levels of secreted IL-1β ( C ) and IL-6 ( D ). Values were interpolated to a standard curve of known protein concentration, and are expressed as mean at each time point. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Protein Concentration

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Trp degradation enzymes, IDO1 and IL4I1 , are upregulated in immune cells stimulated with PS ASOs. ( A ) Correlation of tryptophan oxidation enzymes and TLR9 activation in BJAB cells (top) and THP1-TLR9 cells (bottom) after treatment with 1.6 µM of the indicated PS ASOs for 2 h. Relative qRT-PCR levels of CCL22 and IL4I1/IDO1 mRNA were measured at 8, 24, 48, and 72 h post-treatment. Spearman’s rank correlation was used to assess the association between CCL22 mRNA and IL4I1/IDO1 mRNA. ( B ) Maximum values observed in each experiment with the indicated PS ASOs for BJAB cells (top) and THP1-TLR9 cells (bottom). ( C ) Kinetics of IL4I1 and CCL22 qRT-PCR levels in BJAB cells, and ( D ) THP1-TLR9 cells after dosing with the indicated PS ASOs. All data are presented as a percentage of UTC control (mRNA expression/Ribogreen/UTC) and expressed as mean ± S.E.M. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Activation Assay, Quantitative RT-PCR, Control, Expressing

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Immunogenic PS ASOs stimulate Kyn production via the tryptophan catabolism pathway, and genetic manipulation of tryptophan catabolizing enzymes alters TLR9-mediated PS ASO activation. Total extracellular ( A ) tryptophan and ( B ) kynurenine metabolite levels from THP1-TLR9 cells 24- or 48-h post-treatment with 0.32, 1.6, or 8 μM of indicated PS ASOs. ( C ) Relative CCL22 and IDO1 mRNA expression levels in IDO1 siRNA-treated THP1-TLR9 stimulated with CpG ASO. ( D ) Validation of IDO1 siRNA knockdown and representative western blot measuring IDO1 protein expression. THP1-TLR9 cells were electroporated with 1 μM of siRNA 24 h prior to treatment with 1.6 μM CpG ASO for 2 h. Protein lysates were collected 24 h following treatment. ( E ) Relative CCL22 and IL4I1 mRNA expression levels in IL4I1 overexpression plasmid-treated BJAB cells stimulated with CpG ASO. All bar graph data are expressed as mean ± S.E.M.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Activation Assay, Expressing, Biomarker Discovery, Knockdown, Western Blot, Over Expression, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Directed metabolomics of tryptophan metabolites after PS ASO treatment in THP1-TLR9 cells. Cells were treated with 8 μM PS-ASOs for 2 h, recovered for 48 h, and the collected cellular supernatant was subjected to directed metabolomic analysis of tryptophan metabolites to quantify specific changes. Biological replicates ( n = 3) were submitted for metabolomic analysis. Significant differences in means of metabolite quantities (ng/mL) were determined via one-way ANOVA with Dunnett post-hoc tests (statistical significance is denoted at P < 0.05*, P < 0.005**, and P < 0.0005***).

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Metabolomic

Journal: Nucleic Acids Research

Article Title: Phosphorothioate antisense oligonucleotide induced innate immune activation is attenuated by tryptophan oxidation products

doi: 10.1093/nar/gkag311

Figure Lengend Snippet: Exogenous kynurenine and indole metabolites attenuate PS-ASO-induced innate immune responses. ( A ) Relative CCL22 mRNA expression levels in BJAB and THP1-TLR9 cells and ( B ) Relative IRF reporter activity and NF-kB reporter activity in THP1-TLR9 cells following treatment with 100 µM of the indicated metabolite and 0.8 µM CpG ASO. Relative TLR9 activation of ( C ) THP1-TLR9 cells and ( D ) BJAB cells pretreated with various doses (1.5 mM–0.08 µM) of kynurenine, kynurenic acid, 3-hydroxy anthranilic acid, and indole-3-pyruvate for 2 h, and subsequently treated with 1.6 µM of ASO-20 or ASO-95 for 24 h. TLR9 activation was measured as CCL22 mRNA expression. Metabolite IC50s derived from the top five doses are shown for each ASO, calculated using linear regression with normalized response and variable slope. All experiments were reproduced in at least three independent biological experiments, with multiple replicates per experiment.

Article Snippet: THP1-Dual hTLR9 cells (InvivoGen) overexpress the human TLR9 gene and are engineered with two secreted reporters.

Techniques: Expressing, Activity Assay, Activation Assay, Derivative Assay