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A putative nitroreductase from the DosR regulon of Mycobacterium tuberculosis induces pro-inflammatory cytokine expression via TLR2 signaling pathway.

Peddireddy V, Doddam SN, Qureshi IA, Yerra P, Ahmed N - Sci Rep (2016)

Bottom Line: In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively.Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ.Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad 500046 India.

ABSTRACT
Tuberculosis caused by Mycobacterium tuberculosis is a global encumbrance and it is estimated that nearly one third population of the world acts as a reservoir for this pathogen without any symptoms. In this study, we attempted to characterise one of the genes of DosR regulon, Rv3131, a FMN binding nitroreductase domain containing protein, for its ability to alter cytokine profile, an essential feature of M. tuberculosis latency. Recombinant Rv3131 stimulated pro-inflammatory cytokines in THP-1 cells and human peripheral blood mononuclear cells in a time and dose dependent manner. In silico analyses using docking and simulations indicated that Rv3131 could strongly interact with TLR2 via a non-covalent bonding which was further confirmed using cell based colorimetric assay. In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively. Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ. Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway. Therefore, it can be surmised that cytokine secretion induced by Rv3131 might contribute to establishment of M. tuberculosis in the granulomas.

No MeSH data available.


Related in: MedlinePlus

Rv3131 stimulates the secretion of pro-inflammatory cytokines in PBMCs.Isolated PBMCs from human blood were treated with 10–2500 ng/ml of rRv3131 or LPS (100 ng/ml) or proteinase K digested Rv3131 protein for 24 h. The levels of secreted cytokines like IL-1β, IFN-γ, TNF-α and IL-8 were quantified by ELISA. Data represent the mean ± SEM of three experimental replicates.
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f1: Rv3131 stimulates the secretion of pro-inflammatory cytokines in PBMCs.Isolated PBMCs from human blood were treated with 10–2500 ng/ml of rRv3131 or LPS (100 ng/ml) or proteinase K digested Rv3131 protein for 24 h. The levels of secreted cytokines like IL-1β, IFN-γ, TNF-α and IL-8 were quantified by ELISA. Data represent the mean ± SEM of three experimental replicates.

Mentions: To determine whether recombinant Rv3131can induce pro-inflammatory responses, THP-1 cells and human PBMCs were treated with varying concentrations of this protein and the secreted cytokines (IL-1β, IFN-γ, TNF-α and IL-8) were measured in the supernatants. The positive control, LPS (100 ng) induced the production of these cytokines at similar conditions tested. Treatment of PBMCs with rRv3131 resulted in increased production of IFN-γ, TNF-α, IL-1 β and IL-8 in a dose dependent manner (Fig. 1). Similarly in the THP-1 cells rRv3131 was observed to induce TNF-α, IL-1 β and IL-8 production in a time and dose dependent manner (Fig. 2A). The negative control (rRv3131 digested with proteinase K) did not affect the cytokine production. In order to analyse whether the increased production of pro-inflammatory cytokines in THP-1 cells after rRv3131 treatment was due to modulation at the transcriptional level, real-time PCR was performed. Significant increase in the mRNA levels of IL-8 and TNF-α was observed (Fig. 2B).


A putative nitroreductase from the DosR regulon of Mycobacterium tuberculosis induces pro-inflammatory cytokine expression via TLR2 signaling pathway.

Peddireddy V, Doddam SN, Qureshi IA, Yerra P, Ahmed N - Sci Rep (2016)

Rv3131 stimulates the secretion of pro-inflammatory cytokines in PBMCs.Isolated PBMCs from human blood were treated with 10–2500 ng/ml of rRv3131 or LPS (100 ng/ml) or proteinase K digested Rv3131 protein for 24 h. The levels of secreted cytokines like IL-1β, IFN-γ, TNF-α and IL-8 were quantified by ELISA. Data represent the mean ± SEM of three experimental replicates.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837367&req=5

f1: Rv3131 stimulates the secretion of pro-inflammatory cytokines in PBMCs.Isolated PBMCs from human blood were treated with 10–2500 ng/ml of rRv3131 or LPS (100 ng/ml) or proteinase K digested Rv3131 protein for 24 h. The levels of secreted cytokines like IL-1β, IFN-γ, TNF-α and IL-8 were quantified by ELISA. Data represent the mean ± SEM of three experimental replicates.
Mentions: To determine whether recombinant Rv3131can induce pro-inflammatory responses, THP-1 cells and human PBMCs were treated with varying concentrations of this protein and the secreted cytokines (IL-1β, IFN-γ, TNF-α and IL-8) were measured in the supernatants. The positive control, LPS (100 ng) induced the production of these cytokines at similar conditions tested. Treatment of PBMCs with rRv3131 resulted in increased production of IFN-γ, TNF-α, IL-1 β and IL-8 in a dose dependent manner (Fig. 1). Similarly in the THP-1 cells rRv3131 was observed to induce TNF-α, IL-1 β and IL-8 production in a time and dose dependent manner (Fig. 2A). The negative control (rRv3131 digested with proteinase K) did not affect the cytokine production. In order to analyse whether the increased production of pro-inflammatory cytokines in THP-1 cells after rRv3131 treatment was due to modulation at the transcriptional level, real-time PCR was performed. Significant increase in the mRNA levels of IL-8 and TNF-α was observed (Fig. 2B).

Bottom Line: In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively.Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ.Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway.

View Article: PubMed Central - PubMed

Affiliation: Pathogen Biology Laboratory, Department of Biotechnology and Bioinformatics, University of Hyderabad, Hyderabad 500046 India.

ABSTRACT
Tuberculosis caused by Mycobacterium tuberculosis is a global encumbrance and it is estimated that nearly one third population of the world acts as a reservoir for this pathogen without any symptoms. In this study, we attempted to characterise one of the genes of DosR regulon, Rv3131, a FMN binding nitroreductase domain containing protein, for its ability to alter cytokine profile, an essential feature of M. tuberculosis latency. Recombinant Rv3131 stimulated pro-inflammatory cytokines in THP-1 cells and human peripheral blood mononuclear cells in a time and dose dependent manner. In silico analyses using docking and simulations indicated that Rv3131 could strongly interact with TLR2 via a non-covalent bonding which was further confirmed using cell based colorimetric assay. In THP-1 cells treated with Rv3131 protein, a significant upsurge in the surface expression, overall induction and expression of mRNA of TLR2 was observed when analysed by flow cytometry, western blotting and real time PCR, respectively. Activation of TLR2 by Rv3131 resulted in the phosphorylation of NF- κβ. Results of this study indicate a strong immunogenic capability of Rv3131 elicited via the activation of TLR2 signalling pathway. Therefore, it can be surmised that cytokine secretion induced by Rv3131 might contribute to establishment of M. tuberculosis in the granulomas.

No MeSH data available.


Related in: MedlinePlus