The Urokinase Receptor (uPAR) Facilitates Clearance of Borrelia burgdorferi
Figure 2
The urokinase receptor (uPAR) is involved in clearance of B. burgdorferi.
(A) Urokinase receptor knock-out C57BL/6 mice display higher systemic B. burgdorferi numbers. WT and uPAR −/− mice were inoculated with B. burgdorferi and sacrificed two and four weeks post infection. DNA was extracted from the indicated tissues and subjected to quantitative Borrelia flab and mouse β-actin PCR. In sham inoculated mice (2 to 3 per group) we did not detect B. burgdorferi DNA. Six to eight mice per group were used and bars represent the mean±SEM. (B and C) Urokinase receptor knock-out C57BL/6 mice develop more rigorous IgG responses. Sera from C57BL/6 WT and uPAR knock-out mice, 2 and 4 weeks post B. burgdorferi (B burg) or sham inoculation (SHAM) was used for whole cell B. burgdorferi ELISA. Thus, we determined total IgG directed against B. burgdorferi (B) and IgG subclasses, of which only IgG1 (C) is shown. (D) WT and uPAR −/− macrophages produce similar levels of pro-inflammatory cytokines when exposed to viable B. burgdorferi in vitro. Peritoneal macrophages were stimulated with control medium (medium) or B. burgdorferi (B burg) for 16 hours. The supernatant was analyzed for cytokine production using a mouse inflammation cytometric bead array. (E and F) Urokinase receptor deficient granulocytes and macrophages are incapable of adequately phagocytosing B. burgdorferi. Whole blood or peritoneal macrophages were incubated with CFSE-labeled viable or heat-killed FITC-labeled B. burgdorferi at 37°C or at 4°C as a control. Phagocytosis was stopped by transferring the tubes to ice and extracellular bacteria were quenched by addition of a quenching dye containing Trypan blue. When whole blood was used erythrocytes were lysed before cells were DAPI stained and subjected to fluorescent microscopy (E) or stained for Gr-1 (granulocytes) and subjected to FACS analysis (F; left panel). Peritoneal macrophages were directly subjected to FACS analysis (F; right panel). Phagocytosis was depicted as the phagocytosis index [64],[65]: mean fluorescence intensity (MFI)×percentage (%) positive cells) at 37°C minus (MFI×% positive cells at 4°C). Six to eight mice per group were used, graphs represent the mean±SEM and are representative of three independent experiments. (G) B. burgdorferi binds equally well to WT and uPAR −/− macrophages. A similar experiment as described in (F) was performed, albeit at 4°C and without the addition of quenching dye to determine binding of B. burgdorferi to peritoneal macrophages. Binding is expressed as the binding index: % CFSE positive cells×MFI. Four to six mice per group were used and bars represent the mean±SEM. The experiment was repeated twice. A p-value<0.05 was considered statistically significant. * indicating p<0,05; ** p<0,01 and *** p<0,001.