Fig. 7: Lpp expression leads to an imbalanced coagulation/fibrinolysis hemostasis and fibrinogen depletion abrogates this Lpp-induced effect.
The levels of (A) tissue factor (TF) and (B) plasminogen activator inhibitor-1 (PAI-1) in the supernatant of healthy NMRI skin biopsy homogenates (n = 5), or in the skin biopsy homogenates on day 3 after subcutaneous (s.c.) skin injection with 20 μl of purified Staphylococcus aureus lipoprotein, denoted as Lpl1(+sp) (2.5 or 10 μg; n = 7/group) or unlipidated Lpl1 protein, denoted as Lpl1(−sp) (10 μg; n = 3) in NMRI mice. The levels of C TF and D PAI-1 in the supernatant of skin biopsy homogenates from NMRI mice 3 days after s.c. skin coinjection with 20 μl of SA113Δlgt mutant strain (2.4 × 106 colony-forming units [CFU]/site) with either phosphate-buffered saline (PBS) or Lpl1(+sp) (5 µg/site; n = 15/group). The levels of E TF and F PAI-1 in the supernatant of skin biopsy homogenates from NMRI mice 3 days after s.c. skin coinjection with 20 μl of SA113Δlgt mutant strain (2.4 × 106 CFU/site) with either PBS or Lpl1(−sp) (5 µg/site; n = 5/group). G PAI-1 levels in the supernatants collected from C57BL/6 wildtype (WT) and TLR2 deficient (TLR2−/−) (n = 4/group) mouse peritoneal macrophage cell cultures (5 × 105 cells/mL) after stimulation for 24 h with Lpl1(+sp) (0.02 or 0.2 μg/mL), Pam3CSK4 (20 ng/mL), or culture medium. NMRI mice depleted of fibrinogen using Ancrod or treated with PBS as control and s.c. infected with 20 μl of Staphylococcus aureus Newman parental strain or NewmanΔlgt mutant strain (2 × 106 CFU/site) to assess H the skin lesion size (mm2) up to 3 days postinfection (n = 9–11/group), and I the bacterial counts in the supernatant of skin biopsy homogenates on day 3 postinfection (n = 8–10/group). Representative photomicrographs (J) of mouse skin lesions in NMRI mice s.c. infected with Newman parental strain or NewmanΔlgt mutant strain treated with either PBS (left upper panel and right upper panel, respectively) or Ancrod (left lower panel and right lower panel, respectively) on day 3 postinfection, stained with hematoxylin and eosin. Original magnification ×2.5. The data were pooled from 2 to 3 independent experiments. Statistical evaluations were performed using the Mann–Whitney U test, with data presented as scatterplot with line indicating median value (A–F, I), or the mean ± standard error of the mean (G-H). 1,2,3 = Comparisons of corresponding stimuli between WT and TLR2−/− mice. *P < 0.05; **P < 0.01; ns = not significant.
