Inflammatory macrophages exploited by oral streptococcus increase IL-1B release via NLRP6 inflammasome

Abstract

Chronic inflammatory periodontal disease develops in part from the infiltration of a large number of classically activated inflammatory macrophages that release inflammatory cytokines important for disease progression, including inflammasome-dependent interleukin (IL)-1β. Streptococcus gordonii is a normally commensal oral microorganism; while not causative, recent evidence indicates that commensal oral microbes are required for the full development of periodontal disease. We have recently reported that inflammatory macrophages counterintuitively allow for the increased survival of phagocytosed S. gordonii over nonactivated or alternatively activated macrophages. This survival is dependent on increased reactive oxygen species production within the phagosome of the inflammatory macrophages, and resistance by the bacterium and can result in S. gordonii damaging the phagolysosomes. Here, we show that activated macrophages infected with live S. gordonii release more IL-1β than non-activated macrophages infected with either live or dead S. gordonii, and that the survival of oral Streptococci are more dependent on macrophage activation than other Gram positive microbes, both classical pathogens and commensals. We also find that S. gordonii-dependent inflammatory macrophage inflammasome activation requires the cytoplasmic NLRP6. Overall, our results suggest S. gordonii is capable of evading immune destruction, increasing inflammatory mediators, and increasing inflammatory macrophage response, and that this ability is increased under conditions of inflammation. This work reveals additional mechanisms by which normally commensal oral streptococci-macrophage interactions can change, resulting in increased release of mature IL-1β, potentially contributing to an environment that perpetuates inflammation.

Keywords: commensal; inflammasome; macrophages; streptococcus gordonii.

Fig. 1.

Oral viridans streptococci have a unique ability to survive within M1-activated macrophages over unstimulated macrophages. (A–D) Relative survival of S. gordonii DL1 within macrophages from different sources comparing unstimulated and IFNγ/LPS-activated macrophages: (A) RAW264.7 mouse macrophages, (B) WT iBMDMs, (C) THP-1 human monocytic cell line–differentiated macrophages, (D) human primary peripheral blood monocyte–derived macrophages. Shown are mean ± SEM of 3 or more independent experiments. P values calculated by Student's t test. (E) Survival of Gram-positive bacteria within RAW264.7 macrophages. Shown are mean ± SEM of 3 independent experiments. (F) Survival of various S. gordonii strains within RAW264.7 macrophages. S. gordonii SK12 and SK9 are strains known to not cause endocarditis in animal models, and SK12 has reduced reactive oxygen resistance compared with S. gordonii DL1. S. gordonii DL1 and 38 are known to be more pathogenic in animal models of endocarditis.P values calculated by ordinary 1-way analysis of variance followed by Sidak's multiple comparisons test. Sa = Staphylococcus aureus; Se = Staphylococcus epidermidis; Sg = Streptococcus gordonii; Sm = Streptococcus mutans.

Fig. 2.

Live S. gordonii stimulates significantly increased IL-1β release from IFNγ-activated macrophages. Inflammatory cytokine release measured by enzyme-linked immunosorbent assay (ELISA) from PMA differentiated the THP-1 human monocytic cell line. Cytokines measured were IL-1β (A, C) and TNFα (B, D). (A, B) THP-1 cells were polarized with IFNγ or left unstimulated prior to incubation with S. gordonii DL1 for 6 h (B) or 24 h (A). (C, D) THP-1 cells were polarized with IFNγ and incubated with live or heat-killed (HK) S. gordonii DL1 for 6 h (D) or 24 h (C). In each case, control indicated the THP-1 cells alone. Shown are mean ± SEM of 4 or more independent experiments. P values were calculated by ordinary 1-way analysis of variance followed by Tukey's multiple comparisons test where appropriate. NS indicates that the analysis of variance was not significant with α = 0.05. Concentrations (pg/mL) are per 1 × 10⁵ cells/mL. Sg = Streptococcus gordonii.

Fig. 3.

Live S. gordonii activates inflammasomes in IFNγ-stimulated macrophages. (A, B) Percent ASC speckle–positive cells after incubation (A: 6 h; B: 12 h) with live or heat-killed (HK) S. gordonii in PMA-differentiated THP-1 Asc-GFP cells polarized with IFNγ. Positive control: 4 h 1 µg/mL LPS + 30 min 20 µM nigericin. (C) Representative image of ASC speckle–positive cells. (D) Representative image of ASC speckle–positive cell containing S. gordonii. Shown are mean ± SEM of at least 4 independent experiments in which at least 1,000 cells per condition were analyzed. P values were calculated by ordinary 1-way analysis of variance followed by Tukey's multiple comparisons test. Sg = Streptococcus gordonii.

Fig. 4.

Live S. gordonii activates caspase-1 in macrophages. (A) Representative immunoblot showing caspase-1 and IL-1β cleavage. THP-1 cells were incubated with live or heat-killed (HK) S. gordonii DL1 for 24 h. Actin was used as a loading control. (B) Fold change in caspase-1 activation relative to THP-1 cells without bacteria added measured by YVAD-AFC fluorescence. (C) Propidium iodide (PI) acquisition by THP-1 cells after 24 h incubation. THP-1 cells were polarized with IFNγ or left unstimulated prior to incubation with live or HK S. gordonii. (D) LDH cytotoxicity after 2, 6, and 12 h incubation with live or HK S. gordonii with IFNγ-stimulated THP-1 cells. Transfected LTA was used as a positive control for pyroptosis. Percent release was determined compared with maximum LDH release by cells incubated with lysis buffer. Shown are mean ± SEM of 3 independent experiments. P value was calculated by unpaired t test (B) or ordinary 2-way analysis of variance followed by Dunnett's multiple comparisons in which each group was compared with control (+ indicates control vs LTA, * indicates control vs S. gordonii DL1) (C). Ctrl = control; Sg = Streptococcus gordonii.

Fig. 5.

Determining inflammasome pathway activation by S. gordonii in macrophages. (A) IL-β release from PMA-differentiated, IFNγ-activated THP-1 macrophages with indicated inflammasome inhibitor added during incubation with S. gordonii for 6 h. MCC950 (MCC) indicates NLRP3 inhibitor, Ac-LEVD-CHO (LEVD) indicate scaspase-4/5 inhibitor, and Ac-YVAD-CHO (YVAD) indicates caspase-1 inhibitor. (B) IL-1β release as determined by enzyme-linked immunosorbent assay (ELISA) from IFNγ-stimulated immortalized BMDMs derived from WT, NLRP3 knockout, or NLRP6 knockout mice incubated with like or heat-killed (HK) S. gordonii for 24 h. (C) IL-1β release as determined by ELISA from BMDM derived from WT or NLRP6 knockout mice incubated with live or HK S. gordonii for 24 h. Transfected LTA (28 μg) was used as a positive control for NLRP6 activation. (D) Representative immunoblot showing caspase-1 and IL-1β cleavage. WT or NLRP6 knockout primary BMDMs were incubated with live or HK S. gordonii for 24 h. Actin was used as a loading control. (E) IL-1β release from caspase-11 knockout or WT iBMDMs incubated with live or HK S. gordonii for 24 h. (F) Representative immunoblot showing caspase-1 and IL-1β cleavage. WT or caspase-4 knockout THP-1 cells were incubated with live or HK S. gordonii for 6 h. Again, actin was used as a loading control. Shown are mean± SEM of 3 independent experiments. P values were calculated by 1-way analysis of variance followed by Dunnett's multiple comparisons (A, C, E) or 2-way analysis of variance followed by Sidak's multiple comparisons test (B). Ctrl = control; Sg = Streptococcus gordonii.