Mouse peptidoglycan recognition protein PGLYRP-1 plays a role in the host innate immune response against Listeria monocytogenes infection

Abstract

The role of mouse peptidoglycan recognition protein PGLYRP-1 in innate immunity against Listeria monocytogenes infection was studied. The recombinant mouse PGLYRP-1 and a polyclonal antibody specific to PGLYRP-1 were prepared. The mouse PGLYRP-1 showed antibacterial activities against L. monocytogenes and other Gram-positive bacteria. PGLYRP-1 mRNA expression was induced in the spleens and livers of mice infected with L. monocytogenes. The viable bacterial number increased, and the production of cytokines such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) was reduced in mice when mice had been injected with anti-PGLYRP-1 antibody before infection. The levels of IFN-γ and TNF-α titers in the organs were higher and the viable bacterial number was reduced in mice injected with recombinant mouse PGLYRP-1 (rmPGLYRP-1) before infection. PGLYRP-1 could directly induce these cytokines in spleen cell cultures. The elimination of intracellular bacteria was upregulated in NMuLi hepatocyte cells overexpressing PGLYRP-1. The enhancement of the elimination of L. monocytogenes from the organs was observed in IFN-γ(-/-) mice by rmPGLYRP-1 administration but not in TNF-α(-/-) mice. These results suggest that PGLYRP-1 plays a role in innate immunity against L. monocytogenes infection by inducing TNF-α.

FIG. 1.

Expression of PGLYRP-1-coding tag7 gene and PGLYRP-1 protein after L. monocytogenes infection. (A) Mice were infected with 5 × 10⁵ CFU of L. monocytogenes, the spleens and livers were obtained 0, 0.5, 1, 2, 4, 6, and 12 h after infection, and RNA was extracted from the organs. The expression of tag7 and gapdh was estimated by RT-PCR. Representative data are shown for three independent experiments. (B) Mice were infected with 5 × 10⁵ CFU of L. monocytogenes, and the sera were obtained 0, 3, 6, and 12 h after infection. The protein concentration was adjusted to 20 μg/well and applied to Western blotting. Representative data are shown for three independent experiments.

FIG. 2.

Inhibition of elimination of L. monocytogenes and endogenous cytokine production by administration of anti-mPGLYRP-1 antibody in mice. C57BL/6 mice were injected with 1 mg of anti-PGLYRP-1 antibody or normal rabbit globulin (NRG) 24 h before infection with 5 × 10⁵ CFU of L. monocytogenes, and viable bacterial numbers in the spleens (A) and livers (B) from mice were determined 1, 2, 3, and 5 days after infection. Similarly, titers of IFN-γ (C) and TNF-α (D) in the sera and spleens 24 h after infection were determined in an anti-PGLYRP-1 antibody-treated group and NRG-treated group. The threshold of detection is indicated by <12.5. Each group consists of six mice. Bars show SD. An asterisk indicates a significant difference from the NRG-treated group at P < 0.05.

FIG. 3.

Enhancement of host resistance against L. monocytogenes infection and cytokine production by administration of rmPGLYRP-1. C57BL/6 mice were injected with 100 μg of rmPGLYRP-1, heat-inactivated rmPGLYRP-1, or PBS, and titers of IFN-γ (A) and TNF-α (B) in the sera were determined 24 h after administration. Similarly, C57BL/6 mice were injected with 100 μg of rmPGLYRP-1, heat-inactivated rmPGLYRP-1, or PBS 6 h before infection with 5 × 10⁶ CFU of L. monocytogenes, and viable bacterial numbers in the spleens (C) and livers (D) from mice were determined 1, 2, 3, and 5 days after infection. Titers of IFN-γ (E) and TNF-α (F) in the sera and spleens were determined for the rmPGLYRP-1-treated group, heat-inactivated rmPGLYRP-1-treated group, and PBS-treated group 24 h after infection. The threshold of detection is indicated by <12.5. Each group consists of six mice. Bars show SD. An asterisk indicates a significant difference from the PBS-treated group and heat-inactivated rmPGLYRP-1-treated group at P < 0.05.

FIG. 4.

Antibacterial activities of rmPGLYRP-1 against Gram-positive and Gram-negative bacteria. (A to C) An assay mixture for antibacterial activity contained 1 × 10⁶ CFU of S. aureus (A), S. epidermidis (B), and L. monocytogenes (C) rmPGLYRP-1 or bovine serum albumin (BSA) as a control in 1 ml of the assay buffer with or without anti-mPGLYRP-1 antibody (1 mg/ml). After 0.5, 1, 3, and 6 h of incubation, 100 μl of the mixture was taken and plated on tryptic soy agar plates after serial dilution. Each result was obtained from six independent assays. An asterisk indicates that a value is significantly different from the value obtained from BSA controls at P < 0.01. (D, E) An assay mixture for antibacterial activity contained 1 × 10⁶ CFU of S. aureus (D) and L. monocytogenes (E) rmPGLYRP-1 or BSA as a control in 1 ml of the assay buffer with or without 1 mM zinc chloride or 1 mM EDTA. Each result was obtained from six independent assays. An asterisk indicates that a value is significantly different from the value obtained from BSA controls at P < 0.01.

FIG. 5.

Cytokine production by stimulation with rmPGLYRP-1 in vitro. (A) Naïve mouse spleen cells were stimulated with 0.4, 2, or 10 μg of rmPGLYRP-1 or 10 μg of heat-inactivated rmPGLYRP-1 for 48 h, and IFN-γ in culture supernatant fluids was assayed. (B) Splenic macrophages were stimulated with 0.4, 2, or 10 μg of rmPGLYRP-1 or 10 μg of heat-inactivated rmPGLYRP-1 for 48 h, and TNF-α in culture supernatant fluids was assayed. h10 means 10 μg of heat-inactivated rmPGLYRP-1. Each group consists of six wells of the plates. Bars show SD. An asterisk indicates that the value is significantly different from that of the unstimulated culture and heat-inactivated rmPGLYRP-1 at P < 0.01.

FIG. 6.

Enhancement of cytokine production in spleen cells and macrophages by systemic administration of rmPGLYRP-1. (A) Spleen cells were obtained from C57BL/6 mice 6 h after the injection of 100 μg of rmPGLYRP-1 or PBS in vivo and then were stimulated with PBS or 1 × 10⁷ CFU of viable L. monocytogenes cells for 12 h in vitro. IFN-γ titers in culture supernatant fluids were assayed. (B) Splenic macrophages from C57BL/6 mice pretreated with rmPGLYRP-1 or PBS were stimulated with PBS or viable L. monocytogenes cells for 12 h in vitro. TNF-α titers in culture supernatant fluids were assayed. The threshold of detection is indicated by <12.5. Each group consists of six wells of the plates. Bars show SD. An asterisk indicates that the value is significantly different from that obtained from PBS-pretreated groups at P < 0.01.

FIG. 7.

Increased elimination of L. monocytogenes in PGLYRP-1-overexpressing hepatocytes. NMuLi hepatocyte cells seeded at a concentration of 2 × 10⁵ cells/well were transfected with pEGFP-C2/PGLYRP-1 or pEGFP-C2 control vector. Both transfected cells were infected with L. monocytogenes for 30 min, and cell cultivation was continued with the elimination of extracellular bacteria by gentamicin. After 6 h of cultivation, L. monocytogenes cells were labeled with rhodamine, and rhodamine-labeled bacterial numbers in GFP-labeled cells were counted and the ratio of the bacterial number to the cell number was calculated. An asterisk indicates a significant difference from the pEGFP-C2-transfected group at P < 0.05.

FIG. 8.

Effect of rmPGLYRP-1 on host resistance against L. monocytogenes infection in IFN-γ^−/− mice and TNF-α^−/− mice. IFN-γ^−/− mice and TNF-α^−/− mice were injected with 100 μg of rmPGLYRP-1 or PBS 6 h before infection with 1 × 10⁵ CFU of L. monocytogenes. The number of viable L. monocytogenes cells in organs from IFN-γ^−/− mice (A) and TNF-α^−/− mice (B) was determined on day 3 after infection. Each group consists of six mice. An asterisk indicates the significant difference from the PBS-treated group at P < 0.01.