The NLRP6 Inflammasome Recognizes Lipoteichoic Acid and Regulates Gram-Positive Pathogen Infection

Abstract

The activator and composition of the NLRP6 inflammasome remain poorly understood. We find that lipoteichoic acid (LTA), a molecule produced by Gram-positive bacteria, binds and activates NLRP6. In response to cytosolic LTA or infection with Listeria monocytogenes, NLRP6 recruited caspase-11 and caspase-1 via the adaptor ASC. NLRP6 activation by LTA induced processing of caspase-11, which promoted caspase-1 activation and interleukin-1β (IL-1β)/IL-18 maturation in macrophages. Nlrp6^-/- and Casp11^-/- mice were less susceptible to L. monocytogenes infection, which was associated with reduced pathogen loads and impaired IL-18 production. Administration of IL-18 to Nlrp6^-/- or Casp11^-/- mice restored the susceptibility of mutant mice to L. monocytogenes infection. These results reveal a previously unrecognized innate immunity pathway triggered by cytosolic LTA that is sensed by NLRP6 and exacerbates systemic Gram-positive pathogen infection via the production of IL-18.

Keywords: IL-18; Listeria monocytogenes; NLRP6; caspase-1; caspase-11; gram-positive bacteria; inflammasome; lipoteichoic acid.

Figure 1.. Caspase-11 is processed in macrophages infected with Listeria through NLRP6 and ASC to promote caspase-1 activation.

Primary BMDMs were left uninfected or infected with Listeria at MOI = 10 or indicated MOI for 12 h or indicated times. (A, B) The supernatants were subjected to ELISA. (C−E, G, H, J) The cell lysates (Lysate) and supernatants (Sup) were subjected to immunoblotting, or (F, I, K) the lysates and (F) supernatants were subjected to caspase substrate cleavage assay. Blots of caspase-11 were cropped to reveal protein bands at different exposures. Results are representative of at least three independent experiments, and error bars denote s.d. of triplicate wells. ND, not detected. **P < 0.01, ***P < 0.001, ****P < 0.0001. See also Figures S1 and S2.

Figure 2.. Cytosolic LTA triggers caspase-11 cleavage.

Primary BMDMs were primed with poly(I:C) for 4 h, then transfected with (A and L) Listeria extracts, (B−K, M, N) indicated ligands for 4 h. (A−D, F, H, I, K−N) The cell lysates (Lysate) and supernatants (Sup) were subjected to immunoblotting, or (E and J) the lysates were subjected to caspase substrate cleavage assay. Blots of caspase-11 were cropped to reveal protein bands at 36 different exposures. (G) The supernatants were subjected to LDH assay. Results are representative of at least (A−M) three or (N) two independent experiments, and error bars denote s.d. of triplicate wells. PDE, phosphodiesterase; sLTA, synthetic LTA; GPR, glycerophosphate repeat; lmo0927 + lmo0927, lmo0927 strain reconstituted with lmo0927-expressing plasmid. ****P < 0.0001. See also Figures S2 and S3.

Figure 3.. LTA binds to the LRR domain of NLRP6.

(A, C−E) Indicated tagged NLRP6, NLRP3 or caspase-11 constructs were expressed in HEK293T cells, lysed and incubated with LTA or LPS. The lysates were immunoprecipitated (IP) and analyzed by immunoblotting (IB). (B) Full Western blots of LTA and LPS are shown as a reference. (F−H) BLI analysis of the interaction between biosensor-immobilized purified S-NLRP6 PYD and LTA, sLTA GPR or LPS. (I) Immortalized Nlrp6^−/− BMDMs reconstituted with WT NLRP6 or chimeric NLRP6 with NLRP3 PYD were infected with Listeria for 9 h. The cell lysates (Lysate) were subjected to immunoblotting. Blots of caspase-11 were cropped to reveal protein bands at different exposures. Results are representative of at least (A−D, F−I) three or (E) two independent experiments. LRR, leucine-rich repeat; PYD, pyrin domain; K_D, the equilibrium dissociation constant; sLTA, synthetic LTA; GPR, glycerophosphate repeat. See also Figure S4.

Figure 4.. Caspase-11 and caspase-1 are recruited to the NLRP6 inflammasome through ASC.

(A−E) Immortalized BMDMs were left uninfected or infected with Listeria for 12 h or indicated times. (F) Poly(I:C)-primed immortalized BMDMs were incubated with LTA in Opti-MEM supplemented with 0.005% saponin for 4 h. (A, D−F) The cells were lysed, immunoprecipitated (IP) and analyzed by immunoblotting (IB). (D−F) Cells were treated with DSP and DTBP before IP. Whole cell lysates are shown as the input. Asterisk, IgG. Blots of caspase-11 were cropped to reveal protein bands at different exposures. (B and C) The cells were fixed, immunostained and ASC specks (arrowheads) were counted. ASC, green; caspase-11 (Flag), red; and nuclei, blue. Scale bars, 10 µm. ND, not detected. Results are representative of at least (A, D−F) three or (B and C) two independent experiments, and error bars denote s.d. of each group. See also Figure S5.

Figure 5.. Processed caspase-11 promotes caspase-1-mediated IL-18 secretion.

(A) Schematic representation of predictive cleavage sites in pro-caspase-11. (B−E) Immortalized Casp11^−/− BMDMs reconstituted with WT or mutants of caspase-11 were infected with Listeria for 12 h. (B and C) The cell lysates (Lysate) were subjected to immunoblotting, or (D and E) the supernatants were subjected to ELISA. Blots of caspase-11 were cropped to reveal protein bands at different exposures. Results are representative of at least three independent experiments, and error bars denote s.d. of triplicate wells. **P < 0.01, ****P < 0.0001. See also Figure S5.

Figure 6.. Type I IFN signaling is required for NLRP6 and caspase-11 expression.

(A, B, E, G, H) Primary BMDMs were left uninfected or infected with Listeria for 12 h or indicated times. (C) Immortalized or (D) primary BMDMs were stimulated with indicated ligands for 22 h, immunoprecipitated (IP), and analyzed by immunoblotting. (F) Primary WT BMDMs were primed with indicated ligands for 4 h, then transfected with LTA for 4 h. The cell lysates (Lysate) and supernatants (Sup) were subjected to (A, C−H) immunoblotting or (B) caspase substrate cleavage assay. Blots of caspase-11 were cropped to reveal protein bands at different exposures. Results are representative of at least (A−C, F−H) three or (D, E) two independent experiments, and error bars denote s.d. of triplicate wells. **P < 0.01.

Figure 7.. NLRP6-caspase-11 axis exacerbates Listeria infection.

Mice were infected with (A, C−H) 10⁴ or (B) 10⁵ cfu of Listeria intravenously. (A, F−H) The organs were removed on day 4 for cfu counting. (B) Mouse survival was monitored for 10 days. (C and The spleen homogenates were immunoblotted. (E) The sera were collected on day 4, and cytokine levels were determined by ELISA. (G) Recombinant IL-18 (rIL-18) was administrated on day 2 post-infection (P.I.). Blots of caspase-11 were cropped to reveal protein bands at different exposures. Results are (A, B, F, G) pooled from two independent experiments or (C−E, H) representative of three independent experiments, and error bars denote s.d. of each group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See also Figures S6 and S7.