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. 2020 Nov 18:11:581165.
doi: 10.3389/fimmu.2020.581165. eCollection 2020.

Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling

Jungmin Yang  1 , Inhwa Hwang  1 , Eunju Lee  1 , Sung Jae Shin  1 , Eun-Jin Lee  2 , Joon Haeng Rhee  3 , Je-Wook Yu  1
Affiliations

Bacterial Outer Membrane Vesicle-Mediated Cytosolic Delivery of Flagellin Triggers Host NLRC4 Canonical Inflammasome Signaling

Jungmin Yang et al. Front Immunol. .

Abstract

Bacteria-released components can modulate host innate immune response in the absence of direct host cell-bacteria interaction. In particular, bacteria-derived outer membrane vesicles (OMVs) were recently shown to activate host caspase-11-mediated non-canonical inflammasome pathway via deliverance of OMV-bound lipopolysaccharide. However, further precise understanding of innate immune-modulation by bacterial OMVs remains elusive. Here, we present evidence that flagellated bacteria-released OMVs can trigger NLRC4 canonical inflammasome activation via flagellin delivery to the cytoplasm of host cells. Salmonella typhimurium-derived OMVs caused a robust NLRC4-mediated caspase-1 activation and interleukin-1β secretion in macrophages in an endocytosis-dependent, but guanylate-binding protein-independent manner. Notably, OMV-associated flagellin is crucial for Salmonella OMV-induced inflammasome response. Flagellated Pseudomonas aeruginosa-released OMVs consistently promoted robust NLRC4 inflammasome activation, while non-flagellated Escherichia coli-released OMVs induced NLRC4-independent non-canonical inflammasome activation leading to NLRP3-mediated interleukin-1β secretion. Flagellin-deficient Salmonella OMVs caused a weak interleukin-1β production in a NLRP3-dependent manner. These findings indicate that Salmonella OMV triggers NLRC4 inflammasome activation via OMV-associated flagellin in addition to a mild induction of non-canonical inflammasome signaling via OMV-bound lipopolysaccharide. Intriguingly, flagellated Salmonella-derived OMVs induced more rapid inflammasome response than flagellin-deficient Salmonella OMV and non-flagellated Escherichia coli-derived OMVs. Supporting these in vitro results, Nlrc4-deficient mice showed significantly reduced interleukin-1β production after intraperitoneal challenge with Salmonella-released OMVs. Taken together, our results here propose that NLRC4 inflammasome machinery is a rapid sensor of bacterial OMV-bound flagellin as a host defense mechanism against bacterial pathogen infection.

Keywords: NLRC4; caspase-1; flagellin; host defense; inflammasome; interleukin-1; outer membrane vesicles.

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Figures

Figure 1
Figure 1
Culture supernatants from Salmonella typhimurium promotes NLRC4-dependent inflammasome activation. (A) Illustration of experimental scheme to determine inflammasome activation in BMDMs by bacteria-secreted molecules using transwell plate. (B) Immunoblots from mouse BMDMs in the lower well (A) incubated with wild-type (WT) or ΔfliC-fljB S. typhimurium SL1344 in the upper well (A, MOI 30) for 7 h. (C) Immunoblots from mouse BMDMs untreated or treated with culture supernatant (CS, 1/33 or 1/10 volume of culture medium), derived from 6 h culture of WT or ΔfliC–fljB S. typhimurium, for 6 h. (D) Immunoblots from Nlrc4 +/+ or Nlrc4 −/− mice BMDMs treated with WT or ΔfliC–fljB S. typhimurium CS (1/10) for 6 h. (E) Immunoblots from Nlrp3 +/+ or Nlrp3 −/− mice BMDMs treated with WT or ΔfliC–fljB S. typhimurium CS (1/10) for 6 h, or primed with LPS (0.25 µg/ml, 3 h), followed by treatment with ATP (2.5 mM, 40 min). (F) Immunoblots from mouse BMDMs incubated with S. typhimurium CS (1/20) with or without heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min), for 6 h. (G, H) Quantification of IL-1β (G) or IL-6 (H) in the supernatant of mouse BMDMs treated with S. typhimurium CS (1/20) as same as in (F). (n = 3, one-way ANOVA) (I, J) Quantification of IL-1β (I) or IL-6 (J) in the culture supernatants of mouse BMDMs treated with WT, ΔfliC–fljB, or ΔfliC–fljB–prgJ S. typhimurium CS (1/100) for 6 h. (n = 4, one-way ANOVA). (K) Immunoblots from mouse BMDMs treated with culture supernatant (CS, 1/100 or 1/20), derived from 6 h culture of WT, ΔfliC–fljB, ΔfliC–fljB–prgJ S. typhimurium 14028s, for 6 h. (L) Immunoblots from mouse BMDMs treated with S. typhimurium flagellin (ST-FLA, 250 ng/ml) with or without premixing with DOTAP (DT) liposomal transfection reagent for 6 h or treated with LPS (0.25 µg/ml, 3 h), followed by ATP treatment (2.5 mM, 40 min). Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with S. typhimurium CS-treated group. (*P < 0.05, ***P < 0.001, n.s. not significant).
Figure 2
Figure 2
Outer membrane vesicles from Salmonella typhimurium activate inflammasome signaling via receptor-mediated endocytosis. (A) Immunoblots from the culture supernatants of mouse BMDMs treated with S. typhimurium CS, or supernatant (CS-Sup) or pellet (CS-Pel) from ultracentrifugation (150,000 × g, 18 h) of S. typhimurium CS. (B) Immunoblots from wild-type (WT) or ΔfliC–fljB S. typhimurium CS, filtrates from Centricon concentration ( Supplementary Figure S3A ) and the extract of OMVs. (C) Nanoparticle Tracking Analysis of OMVs isolated from WT or ΔfliC–fljB S. typhimurium. (D) Transmission electron microscopy of OMVs isolated from wild-type S. typhimurium. Scale bar, 100 nm. (E) Representative confocal microscopy images of BMDMs treated with Vybrant Dil-labeled PBS or S. typhimurium OMVs. Scale bar, 20 µm. (F) Immunoblots from mouse BMDMs treated with OMVs isolated from S. typhimurium SL1344 or 14028s (0.5 or 5 µg/ml) for 8 h or treated with Pam3CSK4 (1 µg/ml, 4 h), followed by the transfection of LPS (2 µg/ml, 5 h) using a DOTAP (DT). (G) Immunoblots from mouse BMDMs treated with WT or ΔfliC–fljB S. typhimurium (SL1344) OMV (1 or 5 µg/ml, 8 h). (H) Quantification of IL-1β in the culture supernatants of mouse BMDMs treated with WT, ΔfliC–fljB, or ΔfliC–fljB–prgJ S. typhimurium (SL1344) OMVs (5 µg/ml, 8 h) (n = 7, one-way ANOVA). (I) Immunoblots from mouse BMDMs treated with S. typhimurium OMV (5 µg/ml, 6 h) in the presence of cytochalasin D (5 µM) or Pitstop 2 (PS2, 10 µM), or treated with S. typhimurium CS (1/20) in the presence of PS2 (10 µM) pretreatment (10 min before Salmonella CS treatment). (J) Representative confocal microscopy images of BMDMs treated with Vybrant Dil-labeled S. typhimurium OMVs (5 µg/ml, 6 h) in the presence of Pitstop 2 treatment (10 µM, 30 min pretreat). Scale bar, 20 µm. (K) Relative Dil fluorescence intensity per DAPI signals of BMDMs as treated in (J). (n = 6, one-way ANOVA). (L) Quantification of IL-1β in the culture supernatants of mouse BMDMs treated as in (I, left panel). (n = 3, one-way ANOVA). Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with S. typhimurium CS-treated group. (*P < 0.05, ***P < 0.001).
Figure 3
Figure 3
Salmonella-released outer membrane vesicles promotes NLRC4-dependent inflammasome activation. (A, B) Immunoblots from Nlrp3 +/+ or Nlrp3 −/− (A) or Nlrc4 +/+ or Nlrc4 −/− (B) mice BMDMs treated with S. typhimurium OMVs (3 or 10 µg/ml, 6 h, A; 5 or 12.5 µg/ml, 6 h, B), or treated with Pam3CSK4 (1 µg/ml, 4 h), followed by the transfection of LPS (1 µg/ml, 6 h) using a DOTAP (DT). (C) Immunoblots from Asc +/+ or Asc −/− immortalized BMDMs treated with S. typhimurium OMVs (5 or 12.5 µg/ml, 6 h), or primed with LPS (1 µg/ml, 6 h), followed by the treatment with nigericin (5 µM, 40 min). (D) Immunoblots from Nlrp3 +/+ or Nlrp3 −/− mice BMDMs treated with ΔfliC–fljB S. typhimurium OMVs (10 µg/ml) for 6 h. (E, F) Quantification of IL-1β in the culture supernatants of Nlrc4 +/+ or Nlrc4 −/− (E) or Nlrp3 +/+ or Nlrp3 −/− (F) mice BMDMs treated with S. typhimurium OMVs (1 or 7 µg/ml, E; 10 µg/ml, F) for 8 h, or treated with Pam3CSK4 (1 µg/ml, 3 h), followed by the transfection of LPS (2 µg/ml, E; 1 µg/ml, F) for 6 h. (n = 3) (G, H) Quantification of IL-1β in the culture supernatants of Nlrc4 +/+ or Nlrc4 −/− (G) or Nlrp3 +/+ or Nlrp3 −/− (H) mice BMDMs treated with as same as in (E, F). (n = 3) Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with the group in the Nlrp3 +/+ cells. (*P < 0.05, ***P < 0.001).
Figure 4
Figure 4
Flagellated bacteria-released outer membrane vesicles trigger NLRC4-dependent inflammasome activation. (A) Immunoblots from mouse BMDMs treated with OMV (0.5 or 3 µg/ml) isolated from Pseudomonas aeruginosa PAO1 for 8 h. (B) Immunoblots from Nlrc4 +/+ or Nlrc4 −/− mice BMDMs treated with P. aeruginosa OMV (1 or 7 µg/ml) for 8 h. (C) Immunoblots from Nlrp3 +/+ or Nlrp3 −/− mice BMDMs treated with P. aeruginosa OMV (5 µg/ml) for 8 h. (D, E) Quantification of IL-1β in the culture supernatants of Nlrc4 +/+ or Nlrc4 −/− (D) or Nlrp3 +/+ or Nlrp3 −/− (E) mice BMDMs treated with P. aeruginosa OMV (1 or 7 µg/ml, D; 5 µg/ml, E) for 8 h, or treated with Pam3CSK4 (1 µg/ml, 3 h), followed by the transfection of LPS (2 µg/ml, D; 1 µg/ml, E) for 6 h. (n = 3) (F) Immunoblots from Nlrp3 +/+ or Nlrp3 −/− mice BMDMs treated with E. coli BL21 or DH5α OMVs (5 µg/ml) for 8 h. (G) Immunoblots from Nlrc4 +/+ or Nlrc4 −/− mice BMDMs treated with E. coli BL21 OMVs (1 or 5 µg/ml) for 8 h. (H, I) Quantification of IL-1β in the culture supernatants of Nlrp3 +/+ or Nlrp3 −/− (H) or Nlrc4 +/+ or Nlrc4 −/− (I) mice BMDMs treated with E. coli BL21-derived OMVs (5 µg/ml, H; 1 or 5 µg/ml, I) or DH5α-derived OMVs (5 µg/ml, H) for 8 h, or treated with Pam3CSK4 (1 µg/ml, 3 h), followed by the transfection of LPS (1 µg/ml, H; 2 µg/ml, I) for 6 h. (n = 3) Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences compared with the group in the Nlrp3 +/+ cells. (*P < 0.05, ***P < 0.001, n.s. not significant).
Figure 5
Figure 5
Salmonella-released outer membrane vesicles exhibit heat-resistant and GBP2-independent inflammasome activation potential. (A) Immunoblots of OptiPrep density gradient fractions of S. typhimurium OMVs. (B) Immunoblots of the extracts of OMVs in the presence of heat treatment (97°C, 30 min) or proteinase K treatment (10 µg/ml, 30 min) by anti-ST-flagellin antibody. (C) Immunoblots from mouse BMDMs primed with LPS (0.25 µg/ml, 3 h), followed by the treatment with intact or heat-treated S. typhimurium flagellin (250 ng/ml) for 6 h. (D) Immunoblots of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs (5 µg/ml) for 6 h. (E) Quantification of IL-1β in the culture supernatants of mouse BMDMs treated with intact or heat-treated S. typhimurium OMVs for 6 h. (n = 5). (F) Immunoblots from Gbp2 +/+ or Gbp2 −/− mice BMDMs treated with E. coli BL21 OMVs (5 µg/ml) or S. typhimurium (14028s) OMVs (5 µg/ml) or for 8 h. (G, H) Quantification of IL-1β in the culture supernatants of Gbp2 +/+ or Gbp2 −/− mice BMDMs treated as same as (F) (n = 3). (I, J) Immunoblots of mouse BMDMs treated with intact or proteinase-treated S. typhimurium OMVs (10 µg/ml) in the presence of MCC 950 (100 nM) as indicated for 8 h. After proteinase treatment, PMSF was added to eliminate proteinase K activity. Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences. (**P < 0.01, ***P < 0.001, n.s. not significant).
Figure 6
Figure 6
Salmonella-released outer membrane vesicles trigger in vivo NLRC4-dependent interleukin-1β secretion. (A) Immunoblots from mouse BMDMs treated with wild-type (WT) or ΔfliC–fljB S. typhimurium (SL1344) OMV (5 µg/ml) for 2, 4, 8 h. (B) Quantification of IL-1β in the culture supernatants of mouse BMDMs treated with WT or ΔfliC–fljB S. typhimurium (SL1344) OMVs (5 µg/ml) for 2, 8, 16 h. (n = 3) (C) Quantification of LDH release into culture supernatants of mouse BMDMs treated with WT or ΔfliC–fljB S. typhimurium (SL1344) OMVs (5 µg/ml) for 2, 8, 16 h. (n = 3) (D) Immunoblots from mouse BMDMs treated with S. typhimurium (SL1344) or E. coli (BL21) OMVs (5 µg/ml) for 2, 4, 8 h as indicated. GSDMD, gasdermin D (E) Quantification of LDH release into culture supernatants of mouse BMDMs treated with S. typhimurium (SL1344) or E. coli (BL21) OMVs (5 µg/ml) for 2, 4, 8 h as indicated. (n = 3). (F, G) Quantification of IL-1β in the peritoneal lavage fluid of Nlrc4 +/+ or Nlrc4 −/− (F), or Nlrp3 +/+ or Nlrp3 −/− (G) mice 6 h after intraperitoneal injection of PBS or S. typhimurium OMVs (50 µg/mice). (n = 2, PBS; n = 3, OMV) (H) Quantification of IL-1β in the peritoneal lavage fluid of Nlrp3 +/+ or Nlrp3 −/− mice 6 h after intraperitoneal injection of ΔfliC–fljB S. typhimurium OMVs (50 µg/mice). (n = 5, Nlrp3 +/+; n = 6, Nlrp3 −/−) (I, J) Quantification of IL-6 in the peritoneal lavage fluid of Nlrc4 +/+ or Nlrc4 −/− (I), or Nlrp3 +/+ or Nlrp3 −/− (J) mice 6 h after intraperitoneal injection of PBS or S. typhimurium OMVs (50 µg/mice). (n = 2, PBS; n = 3, OMV) Culture supernatants (Sup) or cellular lysates (Lys) were immunoblotted with the indicated antibodies. Data were expressed as the mean ± SEM. Asterisks indicate significant differences. (*P < 0.05, ***P < 0.001, n.s. not significant).
Figure 7
Figure 7
Proposed mechanism of host inflammasome activation by flagellated or non-flagellated bacteria-released outer membrane vesicles. Flagellated bacteria such as S. typhimurium or P. aeruginosa facilitate the delivery of bacterial flagellin and LPS into the cytoplasm of host cells via OMV cargo. In the initial phase of OMV exposure, OMV-delivered flagellin causes the assembly of NLRC4 inflammasome leading to caspase-1 activation and the subsequent IL-1β secretion. Besides flagellin, intracellular OMV-associated LPS can activate caspase-11 non-canonical inflammasome signaling, which includes GSDMD-dependent pyroptosis and NLRP3-mediated caspase-1 activation and IL-1β secretion. On the contrary, OMVs derived from non-flagellated bacteria such as E. coli mediates the cytosolic delivery of bacterial LPS. Non-flagellated bacterial -delivered LPS mainly causes the activation of caspase-11 and GSDMD-mediated pyroptosis, contributing to potential endotoxic shock. NAIP5, NLR family, apoptosis inhibitory protein 5; NLRC4, NLR family, CARD domain-containing protein 4; NLRP3, NLR family, pyrin domain-containing protein 3; GSDMD, gasdermin D.
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