Regulation of neutrophil chemotaxis by toll-like receptor 9 is important for sepsis survival

Introduction

Successful clearance of bacterial infection depends on efficient neutrophil migration to infected tissues [1]. Chemotaxis is a crucial event for neutrophil migration to local infection and is controlled mainly through activation of G-protein-coupled receptors. Furthermore, the functionality of these receptors is regulated by G-protein-coupled receptor kinases (GRKs) [1]. Impaired chemotactic responses in sepsis was correlated with dysregulated neutrophil toll-like receptor (TLR) signaling, TLR2 and TLR4, while TLR9 inhibition in dendritic cells was associated with reduction of mortality in polymicrobial sepsis [2]. Despite the TLR9 expression, the role of this receptor in neutrophil chemotaxis has not been studied. Thus, the aim of the present study was to verify the importance of TLR9 activation on neutrophil migration during sepsis.

Methods

C57BL/6 wildtype (WT) and TLR9^-/- mice were submitted to the cecal ligation and puncture (CLP) sepsis model and the survival rate was evaluated over 7 days. Also, neutrophil migration to the peritoneal cavity was measured 6 hours after CLP. Chemotaxis of blood neutrophils to CXCL2 in the Boyden camera, CXCR2 expression, and GRK2 induction on blood neutrophils, measured by flow cytometry and immunofluorescence, respectively, were performed 2 hours after CLP. All experiments were developed in accordance with the ethical guidelines of the School of Medicine of Ribeirão Preto, University of São Paulo (protocol number 150/2009).

Results

TLR9^-/- mice submitted to CLP had an enhanced survival rate when compared with WT mice (P = 0.096), and these knockout mice had increased the neutrophil migration to infectious focus (P = 0.0445). Investigating the mechanism by which the deficiency of TLR9 could recover neutrophil migration, it was observed that neutrophil derived from TLR9^-/- CLP-treated mice had restored the ability to migrate in vitro (chemotact assay) toward MIP-2, CXCR2 ligand (P = 0.0133). Moreover, the recovery in neutrophil chemotaxis was associated with an enhancement in CXCR2 expression on the neutrophil surface and a reduction in GRK2 induction.

Conclusions

In sepsis, TLR9 activation, similar to that previously observed with TLR2 and TLR4, can also be harmful to control bacterial growth, because it impairs neutrophils from reaching the infection focus.