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Complement Receptor 3 and Toll-Like Receptor 4 Act Sequentially in Uptake and Intracellular Killing of Unopsonized Salmonella enterica Serovar Typhimurium by Human Neutrophils

Sanquin Research and Landsteiner Laboratory, Academic Medical Center, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands,¹ Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands,² Department of Infectious Diseases, Leiden University Medical Center, Albinusdreef 2, 2333 ZA Leiden, The Netherlands³

Received 15 July 2006/ Returned for modification 23 September 2006/ Accepted 24 February 2007

The uptake and subsequent killing of Salmonella enterica serovar Typhimurium by human neutrophils was studied. In particular, two pattern recognition receptors, complement receptor 3 (CR3) and Toll-like receptor 4 (TLR4), were found to be essential for the efficient uptake and activation, respectively, of the NADPH oxidase. The uptake of Salmonella was almost completely inhibited by various monoclonal antibodies against CR3, and neutrophils from a patient with leukocyte adhesion deficiency type 1, which lack CR3, showed almost no uptake of Salmonella. A lipopolysaccharide (LPS) mutant strain of Salmonella was used to show that the expression of full-length, wild-type, or so-called smooth LPS is important for the efficient killing of intracellular Salmonella. Infection with wild-type-LPS-expressing Salmonella resulted in the generation of reactive oxygen species (ROS) in TLR4-decorated, Salmonella-containing vacuoles, whereas ROS were not induced by an LPS mutant strain. In addition, the recognition of Salmonella by neutrophils, leading to ROS production, was shown to be intracellular, as determined by priming experiments with intact bacteria under conditions where the bacterium is not taken up. Finally, the generation of ROS in the wild-type-Salmonella-infected neutrophils was largely inhibited by the action of a TLR4-blocking, cell-permeable peptide, showing that signaling by this receptor from the Salmonella-containing vacuole is essential for the activation of the NADPH oxidase. In sum, our data identify the sequential recognition of unopsonized Salmonella strains by CR3 and TLR4 as essential events in the efficient uptake and killing of this intracellular pathogen.

Published ahead of print on 12 March 2007.

Editor: J. L. Flynn

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