Neutrophils Directly Recognize Group B Streptococci and Contribute to Interleukin-1β Production during Infection

<div><p>Previous studies have shown that the pro-inflammatory cytokine IL-1β has a crucial role in host defenses against group B streptococcus (GBS), a frequent human pathogen, by recruiting neutrophils to infection sites. We examined here the cell types and mechanisms involved in IL-1β production during infection. Using a GBS-induced peritonitis model in mice, we first found that a large proportion of exudate cells contain intracellular IL-1β by immunofluorescence. Of the IL-1β positive cells, 82 and 7% were neutrophils and macrophages, respectively, suggesting that the former cell type might significantly contribute to IL-1β production. Accordingly, depletion of neutrophils with anti-Ly6G antibodies resulted in a significant reduction in the levels of IL-1β, but not of TNF-α or IL-6. We next found that neutrophils are capable of releasing mature IL-1β and TNF-α directly in response to <i>in vitro</i> stimulation with GBS. The production of pro-IL-1β and TNF-α in these cells required the Toll-like receptor (TLR) adaptor MyD88 and the chaperone protein UNC93B1, which is involved in mobilization of a subfamily of TLRs to the endosomes. Moreover, pro-IL-1β processing and IL-1β release was triggered by GBS hemolysin and required components of the canonical inflammasome, including caspase-1, ASC and NLRP3. Collectively our findings indicate that neutrophils make a significant contribution to IL-1β production during GBS infection, thereby amplifying their own recruitment. These cells directly recognize GBS by means of endosomal TLRs and cytosolic sensors, leading to activation of the caspase-1 inflammasome.</p></div

Neutrophils are the predominant cell type expressing IL-1β in response to challenge with GBS.

<p>Flow cytometry analysis showing cells positive for intracellular IL-1β staining in peritoneal lavage fluid samples from WT C57BL/6 mice challenged i.p. with HK-GBS. Neutrophils and macrophages were identified based on expression of Ly6G and F4/80, respectively. Data are from one representative experiment of three producing similar results.</p

IL-1β processing by neutrophils is mediated by caspase-1.

<p>Mouse neutrophils (5 x 10⁵/well) were incubated for 1 h in the presence of Z-VAD, YVAD-CHO, IETD, AEBSF, NE (all at a concentration of 10μM), or CGi (0.5μM). Live GBS (MOI 20) were added and secreted IL-1β (<b>A, C</b>) and TNF-α (<b>B, D</b>) were measured in culture supernatants after 24 h of incubation. Data are expressed as means + SD of three independent observations, each conducted with cells from a different animal. *, <i>p</i><0.05 versus untreated cells, as determined by one-way analysis of variance and the Student's–Keuls–Newman test.</p

The caspase 1 inflammasome is involved in pro-IL-1β processing and IL-1β release in GBS-infected neutrophils.

<p>Concentrations of IL-1β (<b>A</b> and <b>C</b>) or TNF-α (<b>B</b> and <b>D</b>) in culture supernatants of neutrophils lacking the indicated TLRs or inflammasome components. Supernatants were collected at 24 h after infection with live bacteria (MOI 20). Positive controls consisted of cells treated with LPS (0.1μg/ml) and then pulsed with ATP (5mM) for 30 min before collecting the supernatants. Data are expressed as means + SD of three independent observations, each conducted with cells from a different animal. *, <i>p</i><0.05 versus WT mice, as determined by one-way analysis of variance and the Student's-Keuls-Newman test. <b>E</b>, Western blot analysis, using anti-IL-1β antibodies, of lysates from neutrophils lacking the indicated inflammasome components. Neutrophils were infected with GBS (MOI 20) for 4h.</p

Effects of neutrophil depletion on cytokine production in response to GBS.

<p>WT C57BL/6 mice were pretreated with rat anti-Ly6G monoclonal antibody or isotype control Ig before i.p. challenge with HK-GBS. <b>A-C,</b> numbers of peritoneal cells positive for Ly6G (granulocytes), F4/80 (macrophages) and CD11c (dendritic cells) at the indicated times after HK-GBS challenge. <b>D-F</b>, Cytokine concentrations in peritoneal lavage fluid samples at the indicated times after HK-GBS challenge. Data are expressed as means±SD of three independent observations, each conducted on a different animal. *, <i>p</i><0.05, relative to isotype control-pretreated mice by one-way analysis of variance and the Student’s-Keuls-Newman test.</p

GBS β-hemolysin, but not CAMP factor, is involved IL-1β release.

<p>Concentrations of IL-1β in supernatants of WT neutrophil cultures collected at 24 h after infection with live GBS (MOI 20). WT strain NEM316 or its isogenic mutants deficient in β-hemolysin (ΔcylE), CAMP factor (Δcfb), or both (ΔcylE Δcfb) were used for stimulation. Data are expressed as means + SD of three independent observations, each conducted with cells from a different animal.</p

GBS-induced pro-IL-1β production is dependent on MyD88 and multiple endosomal TLRs.

<p>Western blot analysis of precipitated supernatants (<b>A</b>) or cell lysates (<b>B and C</b>) obtained from cultures of WT neutrophils treated with live (MOI 20) or HK-GBS (10 μg/ml). Immunoreactive bands were detected using anti-IL-1β. ns, non-stimulated samples. Concentrations of IL-1β (<b>D</b> and <b>F</b>) and TNF-α (<b>E</b> and <b>G</b>) in supernatants of neutrophils lacking the indicated gene products involved in TLR or cytokine signaling. 3d, neutrophils from 3d mutant mice lacking functional UNC93B1. Supernatants were collected at 24 h after infection with live GBS (MOI 20). The positive controls consisted of neutrophils treated with LPS (0.1μg/ml) and then pulsed with ATP (5mM) for 30 min before collecting supernatants. Data are expressed as means + SD of three independent observations, each conducted with cells from a different animal. *, <i>p</i><0.05 versus WT mice, as determined by one-way analysis of variance and the Student's–Keuls–Newman test.</p

Inhibition of the candidacidal activity of sera from animals immunized with plasmids containing selected peptide sequences (pT.TK1, left panel and pT.TK4, right panel) by laminarin, a soluble β-1,3-glucan, pustulan, a soluble β-1,6-D-glucan, and keyhole limpet hemocyanin (KLH)-conjugated peptides.

<p>Inhibition of the candidacidal activity of sera from animals immunized with plasmids containing selected peptide sequences (pT.TK1, left panel and pT.TK4, right panel) by laminarin, a soluble β-1,3-glucan, pustulan, a soluble β-1,6-D-glucan, and keyhole limpet hemocyanin (KLH)-conjugated peptides.</p

Plasmids used for immunization studies.

<p>*pT.neo is a previously described plasmid espression vector <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105727#pone.0105727-Beninati2" target="_blank">[26]</a> used for insertion of the selected peptide sequences (plasmids pT.TK1–4),</p><p>**pmIFN-γ is a previously described plasmid encoding for murine interferon-γ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0105727#pone.0105727-LoPasso1" target="_blank">[27]</a> used for co-immunization studies.</p><p>Letters in bold indicate the antigenic peptides selected from phage displayed libraries</p><p>Plasmids used for immunization studies.</p

Binding of mAb K20 to <i>Candida albicans</i> cell wall, particularly on germ tubes and budding cells.

<p>MAb K20 labeled with biotin was used in the immunofluorescence assay with streptavidin-fluorescein.</p