Linkage of inflammation and oxidative stress via release of glutathionylated peroxiredoxin-2, which acts as a danger signal

Abstract

The mechanism by which oxidative stress induces inflammation and vice versa is unclear but is of great importance, being apparently linked to many chronic inflammatory diseases. We show here that inflammatory stimuli induce release of oxidized peroxiredoxin-2 (PRDX2), a ubiquitous redox-active intracellular enzyme. Once released, the extracellular PRDX2 acts as a redox-dependent inflammatory mediator, triggering macrophages to produce and release TNF-α. The oxidative coupling of glutathione (GSH) to PRDX2 cysteine residues (i.e., protein glutathionylation) occurs before or during PRDX2 release, a process central to the regulation of immunity. We identified PRDX2 among the glutathionylated proteins released in vitro by LPS-stimulated macrophages using mass spectrometry proteomic methods. Consistent with being part of an inflammatory cascade, we find that PRDX2 then induces TNF-α release. Unlike classical inflammatory cytokines, PRDX2 release does not reflect LPS-mediated induction of mRNA or protein synthesis; instead, PRDX2 is constitutively present in macrophages, mainly in the reduced form, and is released in the oxidized form on LPS stimulation. Release of PRDX2 is also observed in human embryonic kidney cells treated with TNF-α. Importantly, the PRDX2 substrate thioredoxin (TRX) is also released along with PRDX2, enabling an oxidative cascade that can alter the -SH status of surface proteins and thereby facilitate activation via cytokine and Toll-like receptors. Thus, our findings suggest a model in which the release of PRDX2 and TRX from macrophages can modify the redox status of cell surface receptors and enable induction of inflammatory responses. This pathway warrants further exploration as a potential novel therapeutic target for chronic inflammatory diseases.

Keywords: cysteine oxidation; redox proteomics; thiol oxidation.

Fig. 1.

Glutathionylated proteins are released by LPS-stimulated RAW264 cells. Supernatants from BioGEE-preloaded cells exposed to 100 ng/mL LPS for 24 h were analyzed by Western blot analysis with streptavidin peroxidase. Left lane, nonreduced sample; right lane, sample reduced with 10 mM DTT before loading on the gel.

Fig. 2.

LPS induces PRDX2 release. (A) Western blot of supernatants from RAW264.7 cells treated for 24 h with and without 100 ng/mL LPS. PRDX2 was measured in triplicate samples each derived from an independent well. (Upper) Western blot with anti-PRDX2. (Lower) Ponceau red staining. (B) Intracellular PRDX2 in the cell lysates of the experiment in A. (Upper) Western blot with anti-PRDX2. (Lower) Western blot with anti-GAPDH. (C) PRDX2 in supernatant from RAW264 cells treated for 24 h with different concentrations of LPS. (D) PRDX2 in the corresponding lysates. Percentages of viable cells, as mean ± SD of quadruplicate samples, were 98 ± 2% with 0.01 ng/mL LPS, 95 ± 7% with 0.1 ng/mL LPS, 92 ± 4% with 1 ng/mL LPS, 91 ± 3% with 10 ng/mL LPS, and 90 ± 2% with 100 ng/mL LPS. (E) Time course of PRDX2 release in supernatants from cells treated with 100 ng/mL LPS for 8, 16, or 24 h.

Fig. 3.

PRDX2 release in different experimental conditions. (A) PRDX2 mRNA in RAW264 cells at 4 h or 24 h after stimulation with 100 ng/mL LPS. PRDX2 mRNA expressed in arbitrary units (normalized to GAPDH as described in Materials and Methods), representing the fold change relative to one of the control samples at 4 h. Results are the mean ± SD of triplicate samples analyzed in duplicate. (B) Release of PRDX2 by human macrophages (Left) or PBMCs stimulated with 100 ng/mL LPS for 24 h. PRDX2 was measured by ELISA in triplicate samples of macrophages from four donors or PBMCs from five donors. Mean ± SD values for macrophages were control, 3.22 ± 1.92 ng/mL and LPS, 4.97 ± 2.69 ng/mL. Mean ± SD values for PBMCs were control, 2.90 ± 2.54 ng/mL and LPS, 8.10 ± 6.31 ng/mL. Increases were statistically significant for both macrophages and PBMCs (P < 0.05, t test for paired data). (C) Release of PRDX2 by PBMCs at physiological oxygen concentration. PBMCs were stimulated with 100 ng/mL LPS for 24 h under physiological (5%; Left) or atmospheric (20%; Right) oxygen levels. PRDX2 was measured by ELISA in triplicate PBMC samples from three donors. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective control (without LPS), Student t test. (D) PRDX2 released by HEK 293T cells stimulated with 50 ng/mL TNF-α for 24 h. Each lane represents an independent biological sample.

Fig. 4.

PRDX2 in supernatants from LPS-treated cells is glutathionylated. The sample was immunoprecipitated with anti-PRDX2. Lane 1, immunoprecipitate; lane 2, total supernatants before immunoprecipitation. (Left) Western blot with anti-GSH. (Right) Western blot with anti-PRDX2.

Fig. 5.

hPRDX2 induces TNF-α production. (A) hrPRDX2 was added at 10 µg/mL to RAW264 cells, and TNF-α production was measured 24 h later by ELISA in triplicate samples. Solid bars represent PRDX2 preincubated with polymixin B, as described in the text; open bars represent PRDX2 preincubated without polymyxin B. (B) TNF-α induction by hrPRDX2 in RAW cells pretreated with 100 U/mL IFN-γ or not pretreated. *P < 0.01 vs. control (A) or vs. PRDX2 alone (B). (C) hrPRDX2 was added to human primary macrophage cultures from four donors, and TNF-α production was measured by ELISA 24 h later in triplicate.

Fig. 6.

LPS induces the release of TRX1 by PBMCs. Cells were stimulated with 100 ng/mL LPS for 24 h under physiological (5%; Left) or atmospheric (20%; Right) oxygen levels. TRX1 was measured by ELISA in triplicate PBMC samples from three donors. *P < 0.05; **P < 0.01; ***P < 0.001 vs. respective control (without LPS), Student t test.

Fig. 7.

Metabolic pathways of redox signaling by released PRDX2 and TRX1.