Fig 1.
Neutrophils are the predominant cell type expressing IL-1β in response to challenge with GBS.
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.
Fig 2.
Effects of neutrophil depletion on cytokine production in response to GBS.
WT C57BL/6 mice were pretreated with rat anti-Ly6G monoclonal antibody or isotype control Ig before i.p. challenge with HK-GBS. A-C, numbers of peritoneal cells positive for Ly6G (granulocytes), F4/80 (macrophages) and CD11c (dendritic cells) at the indicated times after HK-GBS challenge. D-F, 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. *, p<0.05, relative to isotype control-pretreated mice by one-way analysis of variance and the Student’s-Keuls-Newman test.
Fig 3.
Release of IL-1β and TNF-α in neutrophil cultures stimulated with GBS.
IL-1β and TNF-α concentrations in culture supernatants of neutrophils isolated from the bone marrow of WT C57BL/6 mice. Cytokines were measured at 24 h after treatment with increasing doses (1, 10 or 20 μg/ml) of HK-GBS (A and B) or increasing MOIs (5, 10, 20 or 40) of live GBS (C and D). Kinetics of IL-1β (E and G) and TNF-α (F and H) production in neutrophils stimulated with HK-GBS (10μg/ml) or with live GBS (MOI of 20). Neutrophils treated with LPS (0.1 μg/ml), and then pulsed with ATP (5mM) for 30 min before collecting supernatants, served as controls. Data are expressed as means+SD of three independent experiments.
Fig 4.
GBS-induced pro-IL-1β production is dependent on MyD88 and multiple endosomal TLRs.
Western blot analysis of precipitated supernatants (A) or cell lysates (B and C) 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 (D and F) and TNF-α (E and G) 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. *, p<0.05 versus WT mice, as determined by one-way analysis of variance and the Student's–Keuls–Newman test.
Fig 5.
The caspase 1 inflammasome is involved in pro-IL-1β processing and IL-1β release in GBS-infected neutrophils.
Concentrations of IL-1β (A and C) or TNF-α (B and D) 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. *, p<0.05 versus WT mice, as determined by one-way analysis of variance and the Student's-Keuls-Newman test. E, 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.
Fig 6.
IL-1β processing by neutrophils is mediated by caspase-1.
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 (A, C) and TNF-α (B, D) 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. *, p<0.05 versus untreated cells, as determined by one-way analysis of variance and the Student's–Keuls–Newman test.
Fig 7.
GBS β-hemolysin, but not CAMP factor, is involved IL-1β release.
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.