The diacylated lipopeptide FSL-1 enhances phagocytosis of bacteria by macrophages through a Toll-like receptor 2-mediated signalling pathway

Abstract

A significant amount of evidence has been accumulated to show that Toll-like receptors (TLRs) function as sensors for microbial invasion. However, little is known about how signalling triggered by TLRs leads to the phagocytosis of pathogens. This study was designed to determine whether stimulation of TLR2 mainly with the lipopeptide FSL-1 plays a role in the phagocytosis of pathogens by macrophages. FSL-1 enhanced the phagocytosis of Escherichia coli to a markedly greater extent than it did that of Staphylococcus aureus, but did not enhance the phagocytosis of latex beads. FSL-1 stimulation resulted in enhanced phagocytosis of bacteria by macrophages from TLR2(+/+) mice but not by those from TLR2(-/-) mice. Chinese hamster ovary cells stably expressing TLR2 failed to phagocytose these bacteria, but the cells expressing CD14 did. FSL-1 induced upregulation of the expression of phagocytic receptors, including MSR1, CD36, DC-SIGN and Dectin-1 in THP-1 cells. Human embryonic kidney 293 cells transfected with DC-SIGN and MSR1 phagocytosed these bacteria. These results suggest that the FSL-1-induced enhancement of phagocytosis of bacteria by macrophages may be explained partly by the upregulation of scavenger receptors and the C-type lectins through TLR2-mediated signalling pathways, and that TLR2 by itself does not function as a phagocytic receptor.

A role of the Ca 2+ binding site of DC-SIGN in the phagocytosis of E. coli

“…This is not in agreement with our previous finding that the p38 inhibitor SB203580 reduced the bacterial phagocytosis by a monocytic cell line, THP-1 cells [7]. This disagreement suggests that the bacterial phagocytosis by professional phagocytes like THP-1 cells [7] is different from that by nonphagocytic cells transfected with the phagocytic receptor DC-SIGN and/or is not mediated by DC-SIGN alone. This speculation is supported by the findings that the PI3K inhibitor wortmannin did not inhibit the DC-SIGN-mediated phagocytosis (Fig.…”

“…This speculation is supported by the findings that the PI3K inhibitor wortmannin did not inhibit the DC-SIGN-mediated phagocytosis (Fig. 2C), but did the phagocytosis by macrophages [7].…”

“…The phagocytosis was performed by the method described previously [6,7]. 293/DC-SIGN stable and transient transfectants (2.5 x 10 5 ) were seeded in each well of a 24-well plate and then incubated at 37°C on the day before the assay.…”

“…293/DC-SIGN stable and transient transfectants (2.5 x 10 5 ) were seeded in each well of a 24-well plate and then incubated at 37°C on the day before the assay. After the cells had been washed three times with base medium warmed at 37°C, they were incubated for 1 h or 2 h with indicated doses of Alexa Fluor ® 488-conjugated E. coli by confocal laser scanning microscopy (CLSM) was previously described [6,7].…”

“…Immunoblotting was performed as described using either an anti-DC-SIGN mAb (MAB161) or an anti--actin mAb as a primary Ab and an HRP-conjugated anti-mouse IgG (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) as a secondary Ab [6,7]. Immunoreactive proteins were detected by using ECL™ detection reagents (GE Healthcare, Piscataway, NJ).…”