Immune Netw.  2016 Dec;16(6):373-380. 10.4110/in.2016.16.6.373.

Peroxiredoxin-3 Is Involved in Bactericidal Activity through the Regulation of Mitochondrial Reactive Oxygen Species

• Lee S
• Wi SM
• Min Y
• Lee KY

• KMID: 2362802
• DOI: http://doi.org/10.4110/in.2016.16.6.373

Abstract

Peroxiredoxin-3 (Prdx3) is a mitochondrial protein of the thioredoxin family of antioxidant peroxidases and is the principal peroxidase responsible for metabolizing mitochondrial hydrogen peroxide. Recent reports have shown that mitochondrial reactive oxygen species (mROS) contribute to macrophage-mediated bactericidal activity in response to Toll-like receptors. Herein, we investigated the functional effect of Prdx3 in bactericidal activity. The mitochondrial localization of Prdx3 in HEK293T cells was confirmed by cell fractionation and confocal microscopy analyses. To investigate the functional role of Prdx3 in bactericidal activity, Prdx3-knockdown (Prdx3(KD)) THP-1 cells were generated. The mROS levels in Prdx3(KD) THP-1 cells were significantly higher than those in control THP-1 cells. Moreover, the mROS levels were markedly increased in response to lipopolysaccharide. Notably, the Salmonella enterica serovar Typhimurium infection assay revealed that the Prdx3(KD) THP-1 cells were significantly resistant to S. Typhimurium infection, as compared with control THP-1 cells. Taken together, these results indicate that Prdx3 is functionally important in bactericidal activity through the regulation of mROS.

Keyword

Peroxiredoxin-3; Mitochondrial reactive oxygen species; Bactericidal activity; Lipopolysaccharides; Toll like receptors

MeSH Terms

Cell Fractionation
Humans
Hydrogen Peroxide
Lipopolysaccharides
Microscopy, Confocal
Mitochondrial Proteins
Peroxidase
Peroxidases
Reactive Oxygen Species*
Salmonella enterica
Serogroup
Thioredoxins
Toll-Like Receptors
Hydrogen Peroxide
Lipopolysaccharides
Mitochondrial Proteins
Peroxidase
Peroxidases
Reactive Oxygen Species
Thioredoxins
Toll-Like Receptors

Figure

• 

  Figure 1 Peroxiredoxin-3 (Prdx3) is implicated in the generation of mitochondrial reactive oxygen species. (A) HEK293T cells were fractionated and protein extracts were analyzed by western-blotting with antibodies for Prdx3, IκB-α for cytoplasmic marker or GRIM19 for mitochondrial marker. The western blotting shown is representative of three independent experiments. (B) HEK293T cells were fixed and immunostained with MitoTracker (green) and an anti-Prdx3 antibody (red), and counterstained with 4’,6-diamidino-2-phenylindole (blue). Data are representative of three independent experiments. (C) THP-1 cells were infected with lentivirus containing shRNA targeted to human Prdx3, or control lentivirus, according to the manufacture's protocol. Control THP-1 (Ctrl) and Prdx3-knockdown THP-1 (Prdx3KD THP-1) cells were cultured in puromycin-containing medium (4 µg/mL) for 2 weeks to select stable clones. Immunoblotting with anti-Prdx3 or anti-GAPDH antibodies was performed to evaluate the knockdown efficacy. The western blotting shown is representative of three independent experiments. (D) Ctrl and Prdx3KD THP-1 cells were cultured without or with 500 ng/mLlipopolysaccharide (LPS) for 60 min, stained with MitoSOX, and analyzed by immunofluorescence microscopy. Data are representative of three independent replicates. (E) Ctrl and Prdx3KD THP-1 cells were cultured without or with 500 ng/mL LPS for 60 min, stained with MitoSOX, and analyzed by flow cytometry. Data are presented as the mean fluorescence intensity (M.F.I)±SEM from triplicate samples. *p<0.05,**p<0.01.

• 

  Figure 2 Prdx3 may not interrupt formation of the TRAF6-ECSIT complex. (A) HEK293T cells were transiently transfected with mock, Flag-TRAF6, Myc-ECSIT, or Myc-Prdx3 as indicated. After 38 h, an immunoprecipitation (IP) assay with an anti-Myc antibody was performed, followed by immune blotting (IB) with anti-Myc or anti-Flag antibodies. The western blotting shown is representative of three independent experiments. (B) HEK293T cells were transfected with mock, Myc-Prdx3, Flag-TRAF6 wild type (wt), Flag-TRAF6 110-522, Flag-TRAF6 260-522, or Flag-TRAF6 349-522, as indicated. At 38 h after transfection, cells were extracted, immunoprecipitated with an anti-Flag antibody, and an immune blotting (IB) assay performed with anti-Flag or anti-Myc antibodies. The western blotting shown is representative of three independent experiments. (C) A schematic model for how TRAF6 interacts with Prdx3 and ECSIT.

• 

  Figure 3 Salmonella survival assay in peroxiredoxin-3 (Prdx3)-knockdown THP-1 cells. (A) Control (Ctrl) and Prdx3-knockdown (Prdx3KD) THP-1 cells were infected with wild type Salmonella enterica serovar Typhimurium (14028s strain) at a multiplicity of infection of 10 bacteria/cell as described in Methods. Cells were lysed by 0.5% deoxycholate in Dulbecco's phosphate-buffered saline, and the bacteria were diluted (50:1) and plated on Luria-Bertani agar. (B) The number of colonies was counted. Data are presented as means±SEM of three independent experiments.*p<0.05,**p<0.01.

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