Fig 1.
Pam3CSK4 pretreatment protects mice from MRSA infection.
KM mice were anesthetized (i.p.) with 100 μl 10% chloralhydrate (CHO), then inhaled Pam3CSK4 (0, 25, 50, 100 μg /mice) in 70 μl normal saline through nasal cavity. 24 h later, mice inhaled MRSA. Survival curve of mice pretreated with Pam3CSK4 versus normal saline after intranasal infection with 1.5x109 cfu MRSA(n = 13) (A). Bacterial burden in lung (B) and bronchus (C) was calculated 12 h after pulmonary infection with MRSA (2×108 cfu/mice, n = 6). Note: *P<0.05; **P<0.01 versus normal saline-treated mice.
Fig 2.
Pam3CSK4-pretreated mice have less neutrophil recruitment in MRSA-induced pneumonia.
Infiltrating neutrophils were digested from lung tissues or washed out from the bronchus 6 and 12 h post infection. Cells were incubated with PE/CY5.5-anti-Gr-1, PE-anti-CD11b and analyzed by flow cytometry. Numbers of neutrophils in lung and bronchial lavage (BAL) in naïve and MRSA-challenged mice at 6 h (A) and 12 h (B) post infection were detected. The absolute numbers of neutrophils in the lungs and bronchus from mice 12 h post infection were calculated (C). Sections from MRSA-infected mice and naïve mice were stained by H&E (D). (The representative percentages of neutrophils in lung or bronchus came from one mice per group (n = 5)), *P<0.05; **P<0.01 versus normal saline-treated mice.
Fig 3.
Pretreatment with Pam3CSK4 decreases inflammatory cytokine production.
Lung tissues were collected and homogenized at 6, 12 and 24 h after MRSA infection. Organ extracts were centrifuged and supernatants were collected. Cytokine concentrations were measured by ELISA kits for TNF-α, IL-6, IL-1β, IL-10 and TGF-β according to the manufacturer’s instructions (A). Kinetics of cytokine mRNA levels of TNF-a, IL-6, IL-1β, IL-10 and TGF-β were tested in mouse lung at indicated times by qPCR (B). The mRNA levels of TNF-a, IL-6 and IL-10 in mouse bronchus were detected using qPCR (C). These data are representative of three independent experiments in duplicate wells (each group, n = 3–5), and the results are shown as means ± SEM. Note: *P<0.05; **P<0.01; ***P<0.001 versus normal saline-treated mice.
Fig 4.
Pam3CSK4 pretreatment reduces expression of neutrophil-inducing chemokines.
Lung tissues were collected and homogenized at indicated times after MRSA infection. Kinetics of cytokine mRNA levels of CXCL-1 and CXCL-2 were tested in mouse lung at 6 h and 12 h using qPCR. Data are expressed as the mean ± SEM of 3–5 mice/time from three independent experiments. Note: *P<0.05 or **p<0.01 versus saline-treated mice.
Fig 5.
Pam3CSK4 enhances mRNA expression of Fcγ receptor (FcγR) and complement receptor (CR).
KM mice were treated with Pam3CSK4 for 24 h prior to MRSA infection. After 6 h (A)and 12 h(B), the mRNA expression of FcγR(Ⅰ,Ⅲ) and CR(1,3) in lungs were performed by quantitative PCR. Data are expressed as the mean ± SEM of 3–5 mice/time from three independent experiments. Note: *P<0.05; **P<0.01 and ***P<0.001 versus normal saline-treated mice.
Table 1.
Primers used in this study.
Fig 6.
Pretreatment with Pam3CSK4 increases antimicrobial activity of neutrophils.
Neutrophils were purified according to the instructions (Miltenyi,Gemany). Neutrophils were purified and measured by flow cytometry (A), and PE/CY5.5-Rat IgG2b(κ), PE-anti Rat IgG 2b(κ) as isotype controls(left panel in Fig 6A). The bacterial cfu of neutrophils was calculated (B), the left panel (the medium contol) indicated the well including MRSA and medium (RPMI1640) but Pam3CSK4 and neutrophils. FITC-positive neutrophils were quantified by flow cytometry(C). Survival of mice injected with neutrophils co-cultured with Pam3CSK4, and previously inoculated with MRSA 1 hour earlier (D). Generation of ROS was measured by flow cytometry (E) and lactoferrin release was tested using mouse Lactoferrin ELISA Kit (F). Note: *P<0.05; **P<0.01 versus normal saline-treated mice.