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A role for syntaxin 3 in the secretion of IL-6 from dendritic cells following activation of toll-like receptors.

Collins LE, DeCourcey J, Rochfort KD, Kristek M, Loscher CE - Front Immunol (2015)

Bottom Line: This correlated with secretion of IL-6 and MIP-1α.Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels.Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators.

View Article: PubMed Central - PubMed

Affiliation: Immunomodulation Research Group, School of Biotechnology, Dublin City University , Dublin , Ireland.

ABSTRACT
The role of dendritic cells (DCs) in directing the immune response is due in part to their capacity to produce a range of cytokines. Importantly, DCs are a source of cytokines, which can promote T cell survival and T helper cell differentiation. While it has become evident that soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors (SNAREs) are involved in membrane fusion and ultimately cytokine release, little is known about which members of this family facilitate the secretion of specific cytokines from DCs. We profiled mRNA of 18 SNARE proteins in DCs in response to activation with a panel of three Toll-like receptors (TLR) ligands and show differential expression of SNAREs in response to their stimulus and subsequent secretion patterns. Of interest, STX3 mRNA was up-regulated in response to TLR4 and TLR7 activation but not TLR2 activation. This correlated with secretion of IL-6 and MIP-1α. Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels. Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators. Given the role of IL-6 in Th17 differentiation, these findings suggest the potential of STX3 as therapeutic target in inflammatory disease.

No MeSH data available.


Related in: MedlinePlus

Effect of TLR ligand stimulation on Qb SNARES (Vti1a and Vti1b), Qbc SNARE (SNAP23), Qc SNARE (STX6) mRNA expression in JAWS II DCs. JAWS II DCs were plated 1 × 106/ml and stimulated with 100 ng/ml LPS, 1 mM Loxoribine, or 5 μg/ml PGN at 1, 4, and 12 h. The amount of specific SNARE protein was quantified by reverse transcription followed by RT-qPCR and normalized with S18 levels. Fold differences were calculated relative to SNARE levels at time zero (assigned value of 1). Results are mean ± SEM of quadruplicate assays. A two sample, two tailed student’s t-test comparing ΔCts of control and 1, 4, or 12 h stimulated sample *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
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Figure 2: Effect of TLR ligand stimulation on Qb SNARES (Vti1a and Vti1b), Qbc SNARE (SNAP23), Qc SNARE (STX6) mRNA expression in JAWS II DCs. JAWS II DCs were plated 1 × 106/ml and stimulated with 100 ng/ml LPS, 1 mM Loxoribine, or 5 μg/ml PGN at 1, 4, and 12 h. The amount of specific SNARE protein was quantified by reverse transcription followed by RT-qPCR and normalized with S18 levels. Fold differences were calculated relative to SNARE levels at time zero (assigned value of 1). Results are mean ± SEM of quadruplicate assays. A two sample, two tailed student’s t-test comparing ΔCts of control and 1, 4, or 12 h stimulated sample *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.

Mentions: Using RT-quantitative polymerase chain reaction (qPCR) we analyzed the expression of Qa SNAREs (STX2, STX3, STX4, STX5, STX7, STX11, STX12, and STX16) (Figure 1), Qb SNARES (Vti1a and Vti1b), Qbc SNARE (SNAP23), Qc SNARE (STX6)(Figure 2), and R SNAREs (VAMP1, VAMP2, VAMP3, VAMP4, VAMP7, and VAMP8)(Figure 3) in JAWS II DCs following stimulation with 100 ng/ml LPS, 1 mM Loxoribine, or 5 μg/ml PGN. RT-qPCR was normalized with S18 levels. This housekeeping gene showed smallest standard deviation compared to β-actin and GAPDH between the technical replicates and LPS, Loxoribine and PGN stimulation. Thus was used for all subsequent experiments (Figure S1 in Supplementary Material).


A role for syntaxin 3 in the secretion of IL-6 from dendritic cells following activation of toll-like receptors.

Collins LE, DeCourcey J, Rochfort KD, Kristek M, Loscher CE - Front Immunol (2015)

Effect of TLR ligand stimulation on Qb SNARES (Vti1a and Vti1b), Qbc SNARE (SNAP23), Qc SNARE (STX6) mRNA expression in JAWS II DCs. JAWS II DCs were plated 1 × 106/ml and stimulated with 100 ng/ml LPS, 1 mM Loxoribine, or 5 μg/ml PGN at 1, 4, and 12 h. The amount of specific SNARE protein was quantified by reverse transcription followed by RT-qPCR and normalized with S18 levels. Fold differences were calculated relative to SNARE levels at time zero (assigned value of 1). Results are mean ± SEM of quadruplicate assays. A two sample, two tailed student’s t-test comparing ΔCts of control and 1, 4, or 12 h stimulated sample *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4306318&req=5

Figure 2: Effect of TLR ligand stimulation on Qb SNARES (Vti1a and Vti1b), Qbc SNARE (SNAP23), Qc SNARE (STX6) mRNA expression in JAWS II DCs. JAWS II DCs were plated 1 × 106/ml and stimulated with 100 ng/ml LPS, 1 mM Loxoribine, or 5 μg/ml PGN at 1, 4, and 12 h. The amount of specific SNARE protein was quantified by reverse transcription followed by RT-qPCR and normalized with S18 levels. Fold differences were calculated relative to SNARE levels at time zero (assigned value of 1). Results are mean ± SEM of quadruplicate assays. A two sample, two tailed student’s t-test comparing ΔCts of control and 1, 4, or 12 h stimulated sample *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.
Mentions: Using RT-quantitative polymerase chain reaction (qPCR) we analyzed the expression of Qa SNAREs (STX2, STX3, STX4, STX5, STX7, STX11, STX12, and STX16) (Figure 1), Qb SNARES (Vti1a and Vti1b), Qbc SNARE (SNAP23), Qc SNARE (STX6)(Figure 2), and R SNAREs (VAMP1, VAMP2, VAMP3, VAMP4, VAMP7, and VAMP8)(Figure 3) in JAWS II DCs following stimulation with 100 ng/ml LPS, 1 mM Loxoribine, or 5 μg/ml PGN. RT-qPCR was normalized with S18 levels. This housekeeping gene showed smallest standard deviation compared to β-actin and GAPDH between the technical replicates and LPS, Loxoribine and PGN stimulation. Thus was used for all subsequent experiments (Figure S1 in Supplementary Material).

Bottom Line: This correlated with secretion of IL-6 and MIP-1α.Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels.Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators.

View Article: PubMed Central - PubMed

Affiliation: Immunomodulation Research Group, School of Biotechnology, Dublin City University , Dublin , Ireland.

ABSTRACT
The role of dendritic cells (DCs) in directing the immune response is due in part to their capacity to produce a range of cytokines. Importantly, DCs are a source of cytokines, which can promote T cell survival and T helper cell differentiation. While it has become evident that soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptors (SNAREs) are involved in membrane fusion and ultimately cytokine release, little is known about which members of this family facilitate the secretion of specific cytokines from DCs. We profiled mRNA of 18 SNARE proteins in DCs in response to activation with a panel of three Toll-like receptors (TLR) ligands and show differential expression of SNAREs in response to their stimulus and subsequent secretion patterns. Of interest, STX3 mRNA was up-regulated in response to TLR4 and TLR7 activation but not TLR2 activation. This correlated with secretion of IL-6 and MIP-1α. Abolishment of STX3 from DCs by RNAi resulted in the attenuation of IL-6 levels and to some extent MIP-1α levels. Analysis of subcellular location of STX3 by confocal microscopy showed translocation of STX3 to the cell membrane only in DCs secreting IL-6 or MIP-1α, indicating a role for STX3 in trafficking of these immune mediators. Given the role of IL-6 in Th17 differentiation, these findings suggest the potential of STX3 as therapeutic target in inflammatory disease.

No MeSH data available.


Related in: MedlinePlus