Effect of the synthetic Toll-like receptor ligands LPS, Pam3CSK4, HKLM and FSL-1 in the function of bovine polymorphonuclear neutrophils

Iván Conejeros,Amanda J Gibson,Dirk Werling,Tamara Muñoz-Caro,Carlos Hermosilla,Anja Taubert,Rafael A Burgos

doi:10.1016/j.dci.2015.05.012

Abstract

Toll-like receptors (TLR) are a family of pattern recognition receptors that sense microbial associated molecular patterns (MAMP) such as microbial membrane components and nucleic acids of bacterial origin. Polymorphonuclear neutrophils (PMN) are the first cell of the innate immune system to arrive at the site of infection or injury and elicit oxidative and non-oxidative microbicidal mechanisms. Observations in human and mouse suggest that TLR ligands can induce direct responses in PMN. So far, there is no information of the effect of synthetic TLR ligands on the response of bovine PMN. The objective of this study was to evaluate the functional response of bovine PMN incubated with four synthetic TLR ligands: ultrapure LPS (TLR4), Pam3CSK4 (TLR2/1), HKLM (TLR2) and FSL-1 (TLR2/6). The results show that all the ligands increment cells size as identified by changes in the FSC-SSC as part of the flow cytometric analysis. Interestingly, only Pam3CSK4 consistently induced a calcium influx, increased ROS production and secretion of gelatinase granules, whereas no response was seen using other ligands. Furthermore, exposure of bovine PMN to ultrapure LPS, Pam3CSK4, HKLM or FSL-1 for 24 hours did not impact on apoptosis of these cells. Our data provide evidence for a selective response of bovine PMNs to TLR ligands.

Highlights

Polymorphonuclear neutrophils (PMN) are the first line of cellular defense against bacterial and fungal agents (Yu and Czuprynski, 1996), and rapidly arrive at the site of injury or infection, recognizing and attempting to resolve the infection through various antimicrobial mechanisms (Segal, 2005)
Given changes in PMN morphology upon stimulation with synthetic Toll-like receptors (TLR) ligands and lack of CD11b- or CD62L-shedding we examined the expression of TLR2 and TLR4 cell surface protein levels
As the initial observations supported that TLR ligands induces the rapid activation of isolated bovine PMN, we investigated whether TLR-ligand interaction increased intracellular calcium concentration in bovine PMN

Summary

Introduction

Polymorphonuclear neutrophils (PMN) are the first line of cellular defense against bacterial and fungal agents (Yu and Czuprynski, 1996), and rapidly arrive at the site of injury or infection, recognizing and attempting to resolve the infection through various antimicrobial mechanisms (Segal, 2005). The complex process of mounting these steps of the inflammatory response must be tightly regulated in order to avoid subsequent damages to host cells by overshooting responses (Nathan, 2006) In this context, the detection and sensing of the molecules produced in the first steps of infection or injury (i.e. chemoattractants) and microbial associated molecular patterns (MAMPs) are relevant. Toll like receptors (TLR) are a family of pattern recognition receptors (PRR) which sensing different MAMPs that includes lipoproteins, lipopolysaccharide, flagellin and nucleic acids from bacterial origin These receptors bind endogenous ligands such as heat shock proteins (HSP) and structural molecules such as fibrinogen, heparan sulfate and soluble hyaluronan. These molecules are constituents of the extracellular matrix and are termed as danger-associated molecular pattern (DAMP), as their recognition by TLRs is associated with inflammatory response during tissue damage and tissue repair

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