TLR4 Response to LPS Is Reinforced by Urokinase Receptor

Abstract

GPI-anchored uPAR is the receptor for the extracellular serine protease urokinase-type plasminogen activator (uPA). Though uPAR role in inflammatory processes is documented, underlying mechanisms are not fully understood. In this study we demonstrate that uPAR is a part of Toll-like receptor 4 (TLR4) interactome. Downregulation of uPAR expression resulted in diminished LPS-induced TLR4 signaling, less activation of NFκB, and decreased secretion of inflammatory mediators in myeloid and non-myeloid cells in vitro. In vivo uPAR-/- mice demonstrated better survival, strongly diminished inflammatory response and better organ functions in cecal ligation and puncture mouse polymicrobial sepsis model. Mechanistically, GPI-uPAR and soluble uPAR colocalized with TLR4 on the cell membrane and interacted with scavenger receptor CD36. Our data show that uPAR can interfere with innate immunity response via TLR4 and this mechanism represents a potentially important target in inflammation and sepsis therapy.

Keywords: CD36; LPS; TLR4; sepsis; urokinase receptor.

Figure 1

uPAR−/− leukocytes are less responsive to LPS stimulation. (A) EDTA whole blood was collected from healthy WT and uPAR−/− mice (n=6 mice per group) and stimulated ex vivo with 50 ng/ml LPS for 3 h. Non-stimulated blood samples from the same animals served as controls. Cytokines response after LPS stimulation is shown. Data are presented as mean ± SD, *P < 0.05; **P < 0.01; **P < 0.001 vs. WT mice. (B) Peritoneal macrophages were isolated by peritoneal lavage from healthy WT and uPAR−/− mice and stimulated or not with 50 ng/ml LPS for 3 h. Cytokines response after LPS stimulation was measured in conditioned medium. Data are presented as mean ± SD from two independent experiments performed in duplicates with pooled PM isolated from n=4 mice per group, *P < 0.05; **P < 0.01; ***P < 0.001 vs. WT mice.

Figure 2

(s)uPAR is a part of TLR4 interactome. (A) Primary peritoneal macrophages from uPAR−/− mice were stimulated with suPAR with or without LPS for 15 min. Then, cells were fixed and stained for TLR4 (Alexa 488, green) and uPAR (Alexa 647, red). DAPI was used as nuclear stain. Scale bar 10 μm. (B) Primary WT and uPAR−/− macrophages were stimulated with 50 ng/ml LPS and 1 μg/ml suPAR for 3 h. Data are presented as mean ± SD from two independent experiments performed in duplicates with pooled PM isolated from n=4 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001. (C) Raw 264.7 cells were stimulated with 100 ng/ml LPS, fixed and stained as in A. Scale bar 12.5 μm. (D) Raw 264.7 cells were stimulated with 1 μg/ml biotin-LPS for 30 min, then cell lysis was performed. Protein complexes were precipitated using streptavidin magnetic beads and analyzed by western blotting using anti-murine uPAR antibody. One of 3 independent experiments with similar results is shown.

Figure 3

uPAR is essential for the response of mesothelial epithelial cells to LPS. (A) Downregulation of uPAR expression in mouse mesothelial epithelial cells. (B) LPS response of SiCo and uPARsi transfected mouse mesothelial cells was assessed after stimulation with 100 ng/ml LPS for 3 h. Expression was analyzed by TaqMan RT-PCR. Data are presented as mean ± SD from three independent experiments, *P < 0.05; **P < 0.01; **P < 0.001. (C) LPS-induced protein tyrosine phosphorylation was assessed in SiCo and uPARsi mouse mesothelial cells by western blotting of the whole cell lysate with Ptyr antibody (upper panel) and P-p65 antibody (middle panel). GAPDH shows loading control (lower panel). (D) Quantification of tyrosine (left) and p65 phosphorylation (right) from three independent western blotting experiments.

Figure 4

uPAR is essential for the response of kidney proximal tubular epithelial cells to LPS. (A, B) LPS- induced expression of IL-6 and IL-8 by human renal proximal tubule epithelial cell (HK-2) was assessed by TaqMan RT-PCR (A) and ELISA (B). (C) HMGB1-dependent IL-6 and IL-8 expression by HK-2 cells was assessed by TaqMan RT-PCR. (D) Human renal epithelial HK-2 cells were lentivirus-infected to express Gaussia luciferase under control of NFκB and GAPDH promoters. Enzyme activity was measured in cell conditioned media 10 h after stimulation with LPS. Data are presented as mean ± SD from three independent experiments, *P < 0.05; **P < 0.01; **P < 0.001.

Figure 5

uPAR is a part of TLR4 interactome. (A) Biotin-LPS binding was assessed in SiCo and uPARsi HK-2 cells as described in Methods. (B) Duolink proximity ligation assay to assess uPAR/TLR4 and uPAR/CD36 interaction was performed on HK-2 cells stimulated with 100 ng/ml LPS for 15 min as described in Methods. (C) Duolink images were quantified using Particles analysis tool of ImageJ. (D) HK-2 cells were stimulated with 100 ng/ml LPS for 3 h after cell pre-treatment with 10 µM of CD36 inhibitor SSO. Expression of IL-6 and IL-8 was assessed by TaqMan RT-PCR. Data are presented as mean ± SD from three independent experiments, *P < 0.05; **P < 0.01; **P < 0.001; n.s. not significant.

Figure 6

Effect of uPAR deficiency on systemic inflammatory response in polymicrobial sepsis model. (A) Peritoneum of sham and LPS-injected WT mice was fixed and stained for uPAR (red) and TLR4 (green). DAPI used as nuclear stain. Scale bar 100 μm. (B) High-grad sepsis was induced in WT and uPAR−/− mice by CLP surgery with ligation of 75% of the cecum length (n=10 mice per group). Sham operated mice served as controls (n=6 mice per group). Blood sample were obtained at 20 h after surgery and inflammatory response was evaluated by cytokine measurements in plasma. (C) IL-6/IL-10 ratio in plasma of WT and uPAR−/− septic mice. Data in are presented as mean ± SD, *P < 0.05; **P < 0.01; **P < 0.001 vs. WT mice.

Figure 7

Effect of uPAR deficiency on local inflammatory response in polymicrobial sepsis model. High-grad sepsis was induced in WT and uPAR−/− mice as described above (n=10 mice per group). Sham operated mice served as controls (n=6 mice per group). Peritoneal lavage was performed at 20 h after surgery and inflammatory response was evaluated by cytokine measurement and inflammatory cell population analysis. (A) CLP-induced cytokine release in PL. (B) White blood cell (WBC) counts and differential cell counts from PL samples at 20 h after CLP or sham operation (n=6 to 10 mice per group).. *P < 0.05; **P < 0.01; **P < 0.001. PMN, polymorphonuclear neutrophils. (C) Bacterial load in PL of WT and uPAR−/− mice was assessed by plating serial dilutions of PL fluid on Columbia blood-agar base plates. (D) Phagocytic capacity of WT and uPAR−/− naïve peritoneal macrophages and cells treated overnight with PL obtained from CLP-WT mice was assessed using FITC-labelled E. Coli. Data in are presented as mean ± SD, *P < 0.05; **P < 0.01; **P < 0.001 vs. WT mice.

Figure 8

Effect of uPAR deficiency on multiorgan failure and survival in polymicrobial sepsis model. High-grad sepsis was induced in WT and uPAR−/− mice by CLP surgery as described in above (n= 10 mice per group). Sham operated mice served as controls (n=6 mice per group). (A) Kidney function was estimated by the measurements of serum creatinine and blood urea nitrogen (BUN) levels at 20 h after CLP or sham surgery. (B) Liver function was estimated by the measurements of AST and ALT levels. (C) Serum lactate dehydrogenase (LDH) levels were determined 20 h after sham or CLP surgery. Data are presented as mean ± SD, *P < 0.05; **P < 0.01; **P < 0.001 vs. WT mice. (D) Mid-grade sepsis was induced in WT and uPAR−/− mice by CLP surgery with ligation of 50% of the cecum length (n= 10 mice per group). Survival was monitored daily for 21 days, Kaplan-Mayer curves were generated and Gehan-Breslow-Wilcoxon test was performed for statistical analysis.