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

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

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

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

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

Enrichment of NHBA fragments after affinity-selection of NHBA libraries with the 31E10/E7 mAb.

<p>Panels A, C and D show the NHBA fragments that were mostly enriched by affinity selection, ranked by frequency. A and C, fragments identified by next-generation sequencing of lambda (A) or M13 filamentous phage (C) libraries; B, cumulative occurrence, per single amino acid, of all fragments identified in the affinity-selected M13 filamentous phage library. D, fragments identified by Sanger sequencing of immunoreactive clones after immunoblotting in the lambda library.</p

Epitope Mapping of a Monoclonal Antibody Directed against Neisserial Heparin Binding Antigen Using Next Generation Sequencing of Antigen-Specific Libraries

<div><p>We explore here the potential of a newly described technology, which is named PROFILER and is based on next generation sequencing of gene-specific lambda phage-displayed libraries, to rapidly and accurately map monoclonal antibody (mAb) epitopes. For this purpose, we used a novel mAb (designated 31E10/E7) directed against Neisserial Heparin-Binding Antigen (NHBA), a component of the anti-group B meningococcus Bexsero^® vaccine. An NHBA phage-displayed library was affinity-selected with mAb 31E10/E7, followed by massive sequencing of the inserts present in antibody-selected phage pools. Insert analysis identified an amino acid stretch (D91-A128) in the N-terminal domain, which was shared by all of the mAb-enriched fragments. Moreover, a recombinant fragment encompassing this sequence could recapitulate the immunoreactivity of the entire NHBA molecule against mAb 31E10/E7. These results were confirmed using a panel of overlapping recombinant fragments derived from the NHBA vaccine variant and a set of chemically synthetized peptides covering the 10 most frequent antigenic variants. Furthermore, hydrogen-deuterium exchange mass-spectrometry analysis of the NHBA-mAb 31E10/E7 complex was also compatible with mapping of the epitope to the D91-A128 region. Collectively, these results indicate that the PROFILER technology can reliably identify epitope-containing antigenic fragments and requires considerably less work, time and reagents than other epitope mapping methods.</p></div

Enrichment of phage clones predicted to display authentic NadA fragments on their surface after selection with a serum pool from volunteers immunized with the Bexero vaccine.

<p>Frequency values reported in the vertical axis in panels A–C refer to the occurrence, per single amino acid position, of sequences predicted to express authentic NadA fragments, relative to those predicted to express irrelevant or no polypeptides. The inset in figure A reports the same data with a higher y-axis magnification. The horizontal axis reports the amino acid positions of the translated NadA sequence. A, unselected library; B and C, library outputs after one and two rounds of selection, respectively. D, Cumulative enrichment factors for each amino acid position derived from NadA fragments obtained after one (blue line) and two (red line) rounds of selection; colored bars in the horizontal axis refer to NadA domains; the area between the dashed vertical lines correspond to the cell binding region of NadA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114159#pone.0114159-Tavano1" target="_blank">[18]</a>. E and F, enrichment factors of NadA fragments after one and two rounds of selection, respectively. Only the fragments laying in the upper quartile of enrichment factors values are shown.</p