Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis

Abstract

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2(-/-) mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naïve WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein-coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2(-/-) mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.

Fig. 1.

TLR2 deficiency improves survival during septic peritonitis. (A) Survival rate after NS-CLP or S-CLP in WT or TLR2^−/− mice (n = 20). P < .05; log-rank test. (B–D) ELISA of TNF-α (B), IL-6 (C), and CXCL2 (D) in serum of WT mice (n = 10) and TLR2^−/− mice (n = 10) 6 h after CLP. (E) Neutrophil sequestration in lung measured 6 h after CLP. Data are mean ± SEM. *P < .05, **P < .01 relative to WT S-CLP mice. (F) Colony-forming units in peritoneal exudate and blood 6 h after CLP. Horizontal bars represent median values, and squares represent individual mice (n = 6–8 each).

Fig. 2.

TLR2 deficiency prevents impairment of neutrophil migration. (A) Neutrophils in peritoneal exudate of WT and TLR2^−/− mice 6 h after CLP (n = 15). (B) Adherent leukocytes in mesenteric venules 4 h after CLP as determined by intravital microscopy. (C) Flow cytometry of surface expression of CXCR2 on blood neutrophils of WT mice (Right) and TLR2^−/− mice (Left) 2 h after CLP. (D) Chemotaxis of neutrophils to CXCL2 isolated from whole blood 2 h after CLP. (E and F) Neutrophil number (E) and cfu (F) in peritoneal exudate of WT and TLR2^−/− mice treated with CXCR2 antagonist 6 h after NS-CLP (n = 10). Mice were injected i.v. with PBS or RTX (30 mg/kg) 30 min before CLP. Data are mean ± SEM. *P < .01 relative to WT S-CLP mice.

Fig. 3.

Systemic injection of TLR2 agonist suppresses neutrophil migration. (A) Flow cytometry of CXCR2 expression on blood neutrophils 2 h after i.v. injection of PBS or LTA (300 μg/mouse). (B) Chemotaxis of blood neutrophils to CXCL2 isolated 2 h after i.v. LTA injection. Data are mean ± SEM. *P < .001 relative to WT S-CLP mice. (C and D) Neutrophils in peritoneal exudate 4 h after i.p thioglycollate injection. (C) Mice were treated with PBS, PTX (4 μg/mouse), RPX (30 mg/kg), or LTA (100 and 300 μg/mouse) 30 min before thioglycollate injection. (D) WT and TLR2^−/− mice were injected with either PBS or LTA (300 μg/mouse, i.v.) 30 min before thioglycollate injection (n = 8). Data are mean ± SEM. *P < .001 relative to the PBS group plus thioglycollate. (E and F) Adoptive transfer of ex vivo LTA-activated neutrophils. BM neutrophils were treated with LTA or untreated (control), labeled with 0.5 or 5.0 μM CSFE, respectively, and infused i.v. into WT recipient mice in equal numbers. Then thioglycollate was injected i.p., and the peritoneal cells were harvested 4 h later. (E) Representative flow cytometry results of recovered CFSE⁺ cells from an individual mouse (n = 5). (F) Counts of recovered CFSE⁺ cells. Data are mean ± SEM. *P < .05.

Fig. 4.

Direct activation of TLR2 impairs chemotaxis in neutrophils. (A) Flow cytometry of CXCR2 expression on BM neutrophils treated with 1 μg/mL (Right) or 10 μg/mL (Left) of LTA or untreated (control) for 1 h. (B and C) Chemotaxis of BM neutrophils to CXCL2. (B) Neutrophils treated with anti-CXCR2 antibody, RPX (30 mg/kg), or LTA (0.1, 1, or 10 μg/mL) for 1 h. (C) Neutrophils treated with LTA (10 μg/mL) for different times, as indicated. Data are mean ± SEM. *P < .001 relative to the control group plus CXCL2. (D) F-actin polymerization of BM neutrophils to CXCL2 after LTA treatment. Representative images from 3 independent experiments are shown (Left). F-actin polymerization was quantified by mean fluorescence intensity (Right). *P < .001 relative to the control group plus CXCL2. (E) Chemotaxis of WT, TLR2^−/−, TLR6^−/−, and Myd88^−/− BM neutrophils to CXCL2 after LTA treatment. (F) Flow cytometry of CXCR2 expression on WT and TLR2^−/− BM neutrophils after LTA treatment.

Fig. 5.

TLR2 signaling up-regulates GRK2 expression in neutrophils. (A) CXCR2 and β-actin gene expression in LTA-treated neutrophils by RT-PCR. (B) Neutrophils chemotaxis to CXCL2 after treatment with CHX, a protein synthesis inhibitor, for 30 min and later with LTA (10 μg/mL) for 1 h or more. Data are mean ± SEM. *P < .001 relative to the WT control group plus CXCL2. (C) Representative fluorescence microscopy for GRK2 (red) in BM neutrophils after treatment with LTA (10 μg/mL) for 1 h. Nuclei were stained by DAPI (blue). (D) Representative immunoblot analysis of GRK2 and β-actin in neutrophil lysates after treatment with LTA (10 μg/mL) for 1 h. The graph shows data in arbitrary units of the density of GRK2 per β-actin band. *P < .01. (E) Representative fluorescence microscopy for GRK2 (red) in blood neutrophils isolated from septic WT and TLR2^−/− mice 2 h after CLP. Nuclei were stained by DAPI (blue).