Neutrophils amplify their own recruitment to group B streptococcus infection sites by means of chemokine production

Neutrophil recruitment to sites of infection is a critical first step in the innate immune response to bacterial pathogens. The mechanisms underlying this process are incompletely understood. Here we used a model of peritonitis induced by Streptococcus agalactiae (Group B streptococcus, GBS), a frequent gram-positive pathogen, to investigate in vivo neutrophil recruitment. We found that cell influx occurred concomitantly with the release of Cxcl1 (chemokine (C-X-C motif) ligand 1) and Cxcl2. GBS-induced peritoneal exudates had higher numbers of neutrophils and higher concentrations of Cxcl2, but not Cxcl1, than those induced by classical pro-inflammatory stimuli, such as lipopolysaccharide (LPS) and zymosan. Moreover, GBS-induced neutrophil recruitment was significantly reduced by the administration of either anti-Cxcl1 or anti-Cxcl2 neutralizing antibodies. Cell depletion experiments demonstrated that Cxcl1 production was predominantly dependent on macrophages, while both neutrophils and macrophages were required for optimal Cxcl2 release. In vitro studies indicated that bone-marrow derived macrophages could secrete high levels of Cxcl1/2 when stimulated with either GBS or LPS. Bone marrow-derived neutrophils, in contrast, produced low levels of either chemokine in response to LPS while releasing high amounts of Cxcl2 when stimulated with GBS. This first set of data suggested that inflammatory responses induced by live bacteria are characterized by the release of high levels of Cxcl2, which is at least in part produced by neutrophils and mediates further neutrophil influx in concert with Cxcl1. To study the mechanisms underlying high-level Cxcl2 production induced by live GBS in neutrophils, we examined the role of Toll-like receptors (TLRs), which were previously shown to mediate pro-inflammatory cytokine responses to these bacteria. In both neutrophils and macrophages, synthesis of Cxcl1/2 occurred de novo after GBS stimulation and was totally dependent on the Toll-like receptor (TLR) adaptor MyD88. Moreover, chemokine production was significantly reduced in the absence of functional UNC93B1, a chaperone protein involved in the recruitment of nucleic acid sensing TLRs to the endosomes. The phenotype of UNC93B1-defective cells could be recapitulated by the simultaneous absence of TLR 7, 9 and 13, but not by the absence of individual TLRs, indicating that optimal chemokine production requires the simultaneous activation of multiple endosomal TLRs. We observed that high-level Cxcl2 release by neutrophils in response to live, but not heat-killed, bacteria requires, in addition, signaling by formylated peptide receptors (FPRs), as suggested by significant inhibition of chemokine production in the presence of Boc-2, an antagonist of both FPR1 and FPR2. Conversely, an FPR1 agonist, as well as bacterial culture supernatants, was capable of strongly synergizing with heat-killed GBS in the induction of Cxcl2 production. Collectively our data show that both macrophages and neutrophils participate in inflammatory cell recruitment through de novo chemokine production after recognition of gram positive bacteria by means of multiple endosomal TLRs and FPRs. Moreover, neutrophils amplify their own recruitment through high-level Cxcl2 production in response to live, but not killed, bacteria. Finally, these cells are apparently capable of discriminating between live and killed bacteria by detecting extracellular FPR ligands. These data may be useful to devise alternative strategies to treat bacterial infections by potentiating the recruitment and the functional activity of polymorphonuclear leukocytes.