Protective Role of Peroxiredoxin I in Heat-Killed Staphylococcus Aureus-infected Mice

Abstract

Background/aim: Staphylococcus aureus (S. aureus) is a major gram-positive pathogen, which can cause toxic and immunogenic injuries both in nosocomial and community-acquired infections. Peroxiredoxin (Prx) I plays crucial roles in cellular apoptosis, proliferation, and signal transduction as well as in immunoregulation. The present study aimed to investigate whether Prx I protects mice from death caused by the heat-killed Staphylococcus aureus.

Materials and methods: In the present study, we challenged the wild-type and Prx I-deficient mice with heat-killed S. aureus (HKSA). The effects of Prx I were evaluated by a series of in vitro and in vivo experiments including western blot, Haematoxylin and Eosin staining, splenocyte analysis and cytokines analysis.

Results: Intra-peritoneal (ip) inoculation of HKSA resulted in increased mortality of Prx I-knockout (KO) mice with severe liver damage and highly populated spleens with lymphocytes. Furthermore, HKSA infections also bursted the production of both pro-inflammatory and anti-inflammatory serum cytokines in Prx I KO compared to wild-type mice.

Conclusion: Enhanced mortality of S. aureus-infected mice with Prx I deficiency suggested that Prx I may protect against the infection-associated lethality of mice.

Keywords: Peroxiredoxin I; Staphylococcus aureus; inflammatory; intra-peritoneal; knockout.

Figure 1. Prx I deficiency increases the HKSA induced mortality in mice. (A) (Upper Panel) PCR analysis of genomic DNA isolated from wild-type (lane 1) and Prx I homozygous knockout mice (lane 2). The 700 bp (Neo) and 250 bp (Prx I) arrows indicate the PCR products of Prx I KO and wild-type mice, respectively. (Lower Panel) Western blot analysis of Prx I expression in the liver from wild and Prx I KO mice. (B) The wild (n=12) and Prx I KO (n=12) mice were ip injected with (approximately 2.85×1010 CFU/mouse) HKSA, and mortality was observed at the indicated times. No additional mice died 96 h following the injection. HSKA: Heat-killed Staphylococcus aureus; PRX I KO: peroxiredoxin I knockout; h: hours.

Figure 2. Impaired production of cytokines and immune cells in HKSA-infected Prx I KO mice. (A) Wild type (n=10) and Prx I KO mice (n=10) were challenged with HKSA (approximately 2.85×1010 CFU/mouse) at indicated times. The serum TNF-α, (B) IL-6 and (C) IL-10 were detected using ELISA. (D) The wild-type and Prx I KO mice were challenged with HKSA (approximately 2.85×1010 CFU/mouse) at indicated times and the splenocytes were collected for analysis of immune cell distribution. The number of CD8-positive T cells, CD4/CD25 positive, (E) Treg, (F) CD11b positive monocytes and (G) NK1.1 positive NK cells were analyzed using flow cytometry. n=6, *p< 0.05, **p<0.01. HSKA: Heat-killed Staphylococcus aureus; PRX I KO: peroxiredoxin I knockout; h: hours.

Figure 3. Severe liver inflammation and increased tissue cytokines in Prx I KO mice in response to HKSA infection. (A, B) The H&E-stained sections from wild type and Prx I KO mice at the indicated times (0, 36, 48 and 60 h) following the HKSA infection, microscope images at 400× show signs of immune cell infiltration in the liver (A) (yellow arrow, immune cell infiltration) and (B) lungs. n=6 (C-F) The cytokine levels IL-6 and IL-10 were analysed in the liver and lung of wild type and Prx I KO mice at the indicated times following the HKSA infection. The produced levels of cytokines are represented as pg/mg of total protein, (n=6 for each experiment) *p< 0.05, **p< 0.01.

Figure 4. Increased apoptosis in the liver of Prx I KO mice in response to HKSA infection. The protein expression levels of cleaved caspase-3 and Bcl-2 in both liver (A) and lungs (B) of wild type and Prx I KO mice following HKSA infection. The protein expression levels are presented as means±SD (C-F). (n=6 for each experiment) *p< 0.05 and **p< 0.01.