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. 2015 Dec 1;6:1340.
doi: 10.3389/fmicb.2015.01340. eCollection 2015.

Interferon-γ From Brain Leukocytes Enhances Meningitis by Type 4 Streptococcus Pneumoniae

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Free PMC article

Interferon-γ From Brain Leukocytes Enhances Meningitis by Type 4 Streptococcus Pneumoniae

Elena Pettini et al. Front Microbiol. .
Free PMC article

Abstract

Streptococcus pneumoniae is the leading cause of bacterial meningitis. Pneumococcal meningitis is a life-threatening disease with high rates of mortality and neurological sequelae. Immune targeting of S. pneumoniae is essential for clearance of infection; however, within the brain, the induced inflammatory response contributes to pathogenesis. In this study we investigate the local inflammatory response and the role of IFN-γ in a murine model of pneumococcal meningitis induced by intracranial injection of type 4 S. pneumoniae. Lymphoid and myeloid cell populations involved in meningitis, as well as cytokine gene expression, were investigated after infection. Animals were treated with a monoclonal antibody specific for murine IFN-γ to evaluate its role in animal survival. Intracranial inoculation of 3 × 10(4) colony-forming units of type 4 strain TIGR4 caused 75% of mice to develop meningitis within 4 days. The amount of lymphocytes, NK cells, neutrophils, monocytes and macrophages in the brain increased 48 h post infection. IFN-γ mRNA levels were about 240-fold higher in brains of infected mice compared to controls. Pro-inflammatory cytokines such as IL-1β and TNF-α, and TLR2 were also upregulated. In vivo treatment with anti-IFN-γ antibody increased survival of infected mice. This study shows that IFN-γ produced during meningitis by type 4 S. pneumoniae enhances bacterial pathogenesis exerting a negative effect on the disease outcome.

Keywords: IFN-γ; Streptococcus pneumoniae; TIGR4; intracranial infection; meningitis; murine model.

Figures

Figure 1
Figure 1
Murine model of meningitis by S. pneumoniae. C57BL/6J mice were infected by the intracranial route with 3 × 104 CFU of S. pneumoniae strain TIGR4 (filled circles) or injected with bacterial growth medium (control, open circles). Data are from two independent experiments with 8 mice/group, and animals were monitored up to 8 days post infection. (A) Kaplan-Meier curve of mouse survival. Results were expressed as percentage of survival over time, and statistical differences were evaluated between infected mice and control group using Log-Rank test. Asterisks indicate statistical significance (***P < 0.0001). (B) Clinical score analysis was evaluated using a scale for severity of disease, from 0 (normal) to 5 (moribund), and values were reported as mean value ± SEM.
Figure 2
Figure 2
Cytokine and TLR gene expression in the mouse brain 48 h post infection. The relative IFN-γ, TNF-α, IL-1β, IL-10, TLR2, and TLR4 gene expression was determined by RT-PCR in brains of C57BL/6J mice infected by the intracranial route with 3 × 104 CFU of S. pneumoniae TIGR4 (filled circles). As control, a group of mice was injected with bacteria growth medium (open circles). All animals were sacrificed 48 h post infection. Symbols represent individual mice and bars represent the geometric mean of each group. Data are from two independent experiments with 4–6 mice/group. Statistical analysis was performed on log-transformed data between infected and control group using the two tailed Mann-Whitney test. Asterisks indicate statistical significance (*P0.05; ***P0.001).
Figure 3
Figure 3
Leukocytes in the brain 48 h post infection. The recruitment of leukocytes was quantified by multiparametric flow cytometric analysis in the brains of C57BL/6J mice collected 48 h post intracranial infection with 3 × 104 CFU of S. pneumoniae TIGR4. As control, a group of mice was injected with bacterial growth medium. (A) Gating strategy adopted for the identification of leukocyte populations in brain of mice. Lymphocytes (CD45hiCD3+), NKs cells (CD45hiCD3NKp46+), neutrophils (CD45hiCD11bhiLy6G+), monocytes (CD45hiCD11bhiLy6GLy6C+), and macrophages (CD45hiCD11bhiF4/80+) were detected. (B) Percentages (%) of cell populations detected in brain of mice infected with S. pneumoniae TIGR4 or injected with bacterial growth medium. Infection with TIGR4 induced the entry in the brain of about 4% lymphocytes, 5% NK cells, 27% neutrophils, 21% monocytes, and 19% macrophages, all evaluated on CD45+ cells. In control group all cell populations detected were ≤ 0.7% of CD45+ cells. Data are from four independent experiments with 3–6 animals/group. Bars represent mean ± SEM. Two tailed Mann-Whitney test was used for comparison between infected and control group and asterisks indicate statistical significance (***P < 0.001).
Figure 4
Figure 4
Cellular source of IFN-γ in the infected brain. Lymphocytes (CD45hiCD3+), NKs (CD45hiCD3NKp46+), neutrophils (CD45hiCD11bhiLy6G+), monocytes (CD45hiCD11bhi Ly6GLy6C+), and macrophages (CD45hiCD11bhiF4/80+) were analyzed for IFN-γ intracellular staining. Flow cytometric analysis was performed in brain of mice 48 h post intracranial infection with 3 × 104 CFU of S. pneumoniae TIGR4. (A) Representative flow cytometry dot plot graphs showing the percentage of IFN-γ+ cells among different cell populations in the brain. The gate for IFN-γ+ cells was set using an appropriate APC-conjugated isotype control. (B) Symbols represent individual mice and bars represent the mean. Mean values for each cell population were 0.6 ± 0.1% for lymphocytes, 7 ± 2.4% for NK cells, 1.8 ± 0.4% for neutrophils, 3.4 ± 0.8% for monocytes, and 3 ± 0.9% for macrophages.
Figure 5
Figure 5
IFN-γ specific monoclonal antibody treatment. Mice were infected by the intracranial route with 3 × 104 CFU of S. pneumoniae TIGR4 and administered with a neutralizing anti-IFN-γ antibody (black) or with an isotype control antibody (white) at the time of the bacteria inoculum. To analyze the role of IFN-γ in pneumococcal meningitis, animals were monitored up to 4 days post infection. Data are from two independent experiments with 4 mice/group. (A) Kaplan-Meier curve of mouse survival. Results were expressed as percentage of survival over time. (B) Clinical score was evaluated using a scale for severity of disease, from 0 (normal) to 5 (moribund), and reported as mean value ± SEM. (C) Bacterial load estimation in brain of mice 48 h post infection. Mean values ± SEM are reported.

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