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. 2012;2:786.
doi: 10.1038/srep00786. Epub 2012 Nov 8.

Formylpeptide Receptors Are Critical for Rapid Neutrophil Mobilization in Host Defense Against Listeria Monocytogenes

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Free PMC article

Formylpeptide Receptors Are Critical for Rapid Neutrophil Mobilization in Host Defense Against Listeria Monocytogenes

Mingyong Liu et al. Sci Rep. .
Free PMC article

Abstract

Listeria monocytogenes (Listeria) causes opportunistic infection in immunocompromised hosts with high mortality. Resistance to Listeria depends on immune responses and recruitment of neutrophils of the immune system into infected sites is an early and critical step. Mouse neutrophils express two G protein-coupled formylpeptide receptor subtypes Fpr1 and Fpr2 that recognize bacterial and host-derived chemotactic molecules including Listeria peptides for cell migration and activation. Here we report deficiency in Fprs exacerbated the severity of the infection and increased the mortality of infected mice. The mechanism involved impaired early neutrophil recruitment to the liver with Fpr1 and Fpr2 being sole receptors for neutrophils to sense Listeria chemoattractant signals and for production of bactericidal superoxide. Thus, Fprs are essential sentinels to guide the first wave of neutrophil infiltration in the liver of Listeria-infected mice for effective elimination of the invading pathogen.

Figures

Figure 1
Figure 1. Increased susceptibility and Listeria load in Fpr-deficient mice.
(a) The survival of mice post Listeria infection. Mice were i.v. injected with 2 × 104 Listeria in 100 µl DPBS and observed for up to 10 days. Results shown are the means of three experiments. * significantly reduced survival of Fpr-deficient mice compared with WT littermates, p = 0.031. n = 8 mice for each group in each experiment. (b) Listeria load in the liver. Mouse livers were harvested 3 d after infection and homogenized in DPBS. The tissue suspension was diluted, inoculated and incubated in agar plates at 37°C for 24 h. The bacterial colony forming units (CFUs) were counted. n = 3−4 mice per group in each experiment. * significantly increased Listeria CFUs formed by liver lysates from Fpr-deficient mice compared with WT mice (p = 0.008). Data are the mean ± SD from a representative experiment out of three performed. (c) Neutrophils in the liver of Listeria-infected mice. Mice were i.v. injected with 2 × 104 Listeria in 100 µl DPBS. Neutrophils in the liver were purified and analyzed with flow cytometry at different time points. n = 5 mice per group in each experiment. * significantly decreased neutrophils in the liver of Fpr-deficient mice at all measurement time points as compared with WT mice (p = 0.006). (d) Immunofluorescence staining of infiltrating neutrophils. The livers of mice were cryosectioned and stained with Ly6G (Green) and DAPI (Blue) 6 h and 48 h after Listeria infection (400 ×). (e) Abscess formation in the liver of Listeria-infected mice. Mice were injected with 2 × 104 Listeria and the livers were harvested at 48 h. Paraffin liver sections (5 μm) were stained with H&E. Marked areas delineate the edges of abscesses (200 ×).
Figure 2
Figure 2. Competitive repopulation of neutrophils and chemokine production in the liver of Listeria-infected mice.
(a) Competitive repopulation of WT and Fpr-deficient mouse neutrophils in Listeria-infected Fpr1/2−/− mice. Bone marrow cells (1 × 107) from WT (Red) and Fpr1/2−/− mice (Green) were labeled and mixed at ratio 1: 1 then were i.v. injected into Fpr1/2−/− mice immediately after Listeria infection. The livers were harvested and analyzed by immunofluorescence microscopy at 4, 24 and 48 h. n = 10 (400 ×). (b) Quantification of infiltrating cells in the Fpr1/2−/− mouse liver at 4 h. Fluorescence stained cells in 3 high powered fields (400 ×) in liver sections were counted with Image J. * significantly decreased repopulation of Fpr1/2−/− mouse cells (green) as compared with WT mouse cells (red) (p = 0.0004). (c) Survival rate of Fpr-deficient mice receiving transfer of WT mouse bone marrow. Fpr2−/− and Fpr1/2−/− mice were irradiated and transferred with 1 × 107 WT mouse bone marrow cells. All recipient mice were then i.v. injected with 2 × 104 Listeria. * significantly increased survival rate of Fpr-deficient mice receiving WT mouse bone marrow (Fpr2−/−_WT-BM, and Fpr1/2−/−_WT-BM) as compared with mice without WT bone marrow transfer (Fpr2−/− and Fpr1/2−/−) (p = 0.001). n = 10 mice per group in each experiment. (d) Restoration of Ly6G+ cell infiltration in the liver Fpr1/2−/− mice after transfer of WT mouse bone marrow cells. Myeloid cells purified from infected mice and labeled with CD45, CD11b and Ly6G. The percentage of Ly6G+ cells in CD45+CD11b+ cells was analyzed. Grey areas: isotype control; Black line: cells from infected WT mouse liver; Blue: cells from the liver of infected Fpr1/2−/− mice receiving bone marrow cells from WT mice; Red line: cells from the liver of infected Fpr1/2−/− mice receiving bone marrow cells from Fpr1/2−/− mice. (e) CXCL1 and CXCL2 production in the liver of Listeria-infected mice. The livers of Listeria-infected mice were homogenized. CXCL1 and CXCL2 in the supernatant were measured with ELISA. n = 5 mice per group in each experiment.
Figure 3
Figure 3. The neutrophil chemotactic and activating effects of Listeria products.
(a) Chemotactic activity of Listeria-derived peptide fMIVIL for mouse neutrophils. * significantly increased cell migration in response to the peptide as compared with medium control (0) (p = 0.0007). (b) Chemotactic activity of Listeria lysate for HEK293 cells transfected with Fprs. * significantly increased cell chemotaxis in response to Listeria lysate as compared with medium control (0) (p = 0.004). (c) Migration of mouse neutrophils to Listeria lysate. * significantly increased chemotaxis response of neutrophils to lysates as compared with medium control (0) (p = 0.003). (d) Listeria lysate-induced phosphorylation of Erk1/2 in WT mouse neutrophils in the presence or absence of Fpr antagonists and a TLR2 neutralizing antibody. (e) Semiquantitative analysis of phosphorylated Erk1/2. * significantly decreased Erk1/2 phosphorylation compared with cells stimulated with Listeria lysate at 1: 10 dilution in the absence of Fpr antagonists or TLR2 antibody. Results are from 1 experiment out of 3 performed. (f) Induction of Erk1/2 phosphorylation in WT and Fpr1/2−/− mouse neutrophils by Listeria lysate (at 1:10 dilution) in the presence or absence of an Fpr antagonist Boc-2. (g) Semiquantitative analysis of phosphorylated Erk1/2. # significantly reduced Erk1/2 phosphorylation in Fpr1/2−/− mouse neutrophils as compared with WT mouse neutrophils. * significantly decreased Erk1/2 phosphorylation in WT mouse neutrophils stimulated with Listeria lysate in the presence of Fpr antagonist Boc-2 as compared with cells stimulated with Listeria lysate alone (p = 0.001).
Figure 4
Figure 4. Listeria phagocytosis, killing and Listeria-induced H2O2 production by neutrophils.
(a) Listeria phagocytosis by mouse neutrophils. Neutrophils were incubated with 100-fold heat-inactivated Listeria for 1 h at 37°C then were stained with anti-Ly6G and anti-Listeria (ANTIBODY-ONLINE.com, Cat#ABIN576776) antibodies. The cells were then analyzed for % positivity and mean fluorescence index (MFI) with flow cytometry. Cells with Listeria on ice were used as control (Black areas: neutrophils with Listeria on ice; Red lines: neutrophils with phagocytosed Listeria). (b) Immunofluorescence of Listeria phagocytosed by mouse neutrophils. Neutrophils were attached to chamber slides. Listeria were pre-treated with anti-Listeria antibody for 30 min and then incubated with neutrophils for 1 h followed by analysis with immunofluorescence confocal microscopy. Neutrophil nuclei were counterstained with DAPI (Green: Listeria; Blue: DAPI). (c) Listeria killing capacity of neutrophils. Mouse neutrophils were incubated with 100-fold live Listeria for 1 h at 37°C and non-phagocytosed bacteria were removed by washing. Permeabilized neutrophils were inoculated on agar plates and Listeria CFUs released by neutrophils were counted after 24 h. * significantly increased CFUs released by Fpr-deficient mouse neutrophils as compared with WT mouse cells (p = 0.006). (d) Listeria-induced H2O2 production by WT mouse neutrophils. Neutrophils (5 ×106 cells) from WT mice were primed with 1 ng/ml GM-CSF for 60 minutes then were incubated with an Fpr1-specific antagonist Boc-MLF (1 µM, 10 min), an Fpr2 specific antagonist WRW4 (2 µM, 10 min) or a TLR2 neutralizing antibody (100 ng/ml, 30 min) followed by stimulation with 100-fold heat-inactivated Listeria for 30 min at 37°C. * significantly decreased H2O2 production by neutrophils pretreated with Fpr antagonists or anti-TLR2 antibody as compared with neutrophils treated with inactivated Listeria only (p = 0.001). (e) Neutrophil production of H2O2 in response to heat-inactivated Listeria. Neutrophils (5 × 106) primed with 1 ng/ml GM-CSF for 60 minutes were incubated with 100-fold heat-inactivated Listeria. H2O2 production was measured at indicated time points. * significantly decreased H2O2 production by Fpr-deficient mouse neutrophils as compared with WT mouse neutrophils (p = 0.007, n = 3).

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