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Intracellular immune sensing promotes inflammation via gasdermin D–driven release of a lectin alarmin

Abstract

Inflammatory caspase sensing of cytosolic lipopolysaccharide (LPS) triggers pyroptosis and the concurrent release of damage-associated molecular patterns (DAMPs). Collectively, DAMPs are key determinants that shape the aftermath of inflammatory cell death. However, the identity and function of the individual DAMPs released are poorly defined. Our proteomics study revealed that cytosolic LPS sensing triggered the release of galectin-1, a β-galactoside-binding lectin. Galectin-1 release is a common feature of inflammatory cell death, including necroptosis. In vivo studies using galectin-1-deficient mice, recombinant galectin-1 and galectin-1-neutralizing antibody showed that galectin-1 promotes inflammation and plays a detrimental role in LPS-induced lethality. Mechanistically, galectin-1 inhibition of CD45 (Ptprc) underlies its unfavorable role in endotoxin shock. Finally, we found increased galectin-1 in sera from human patients with sepsis. Overall, we uncovered galectin-1 as a bona fide DAMP released as a consequence of cytosolic LPS sensing, identifying a new outcome of inflammatory cell death.

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Fig. 1: Proteomic identification of intracellular LPS-elicited release of galectin-1.
Fig. 2: Cytosolic LPS-induced galectin-1 release in vitro is dependent on caspase-11/4 and GSDMD.
Fig. 3: Cytosolic LPS-induced release of galectin-1 is dependent on caspase-11 and GSDMD in vivo.
Fig. 4: Necroptosis triggers galectin-1 release.
Fig. 5: Galectin-1 plays a detrimental role during LPS septic shock.
Fig. 6: Galectin-1 amplifies systemic inflammatory responses during LPS shock.
Fig. 7: Galectin-1 inhibition of CD45 underlies its detrimental role in endotoxin shock.

Data availability

The RNA-seq data have been deposited with the Gene Expression Omnibus (accession number GSE140892). Source data and uncropped immunoblot images are included in the paper as supplementary information. All other data supporting the findings of the paper are available from the corresponding author upon reasonable request.

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Acknowledgements

We thank J. Conejo-Garcia for the Lgals1−/− mice and V. Dixit (Genentech) and K. Fitzgerald (University of Massachusetts Medical School) for the Casp11−/− and Gsdmd−/− mice, S. Yeung and K. Khanna for help with the multiplex analysis and G. Fong for the MS1 cell line (University of Connecticut Health). This work was supported by the National Institutes of Health (grant nos. AI119015 and AI135528 to V.A.R.); Agencia Nacional de Promoción Científica y Tecnológica, Fundación Sales and Fundación Bunge y Born to G.A.R.; the Federal Ministry of Education and Research, Germany (grant no. 01EO1502) and Deutsche Forschungsgemeinschaft (German Research Foundation) under Germany’s Excellence Strategy-EXC 2051, project ID 390713860 (to M.B.).

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Authors

Contributions

V.A.R. and A.J.R. conceived the study. V.A.R., G.A.R. and A.J.R. designed the experiments and wrote the manuscript. A.J.R., S.O.V., A.M., S.P.M.H., P.K., J.M.P.S., C.W., K.C., S.K.V., J.R., G.A.R. and A.T.V. performed the experiments, analyzed the data or provided technical or conceptual help. S.D., S.D.D., M.B. and C.S. performed the human sample analysis. M.M.F., R.L., A.W. and G.A.H. provided the recombinant galectin-1 and GFP. B.Z. and C.L. performed the RNA-seq analysis.

Corresponding author

Correspondence to Vijay A. Rathinam.

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The authors declare no competing interests.

Additional information

Peer review information Nature Immunology thanks Derek Abbott and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Zoltan Fehervari was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Galectin-1 is released as a consequence of inflammasome activation.

a, Experimental design for profiling caspase-11-mediated release of DAMPs by PF2D-based proteomics. WT and Casp11–/– BMDMs were infected with EHEC (MOI=50) for 16 h and supernatants were harvested and concentrated and processed on the Beckman Coulter ProteomeLab PF2D platform. b, Galectin-1 secretion by Pam3CSK4-primed WT and Lgals1–/– BMDMs stimulated with EHEC (MOI=50) or poly(dA:dT) transfection for 16 h or 10 µM nigericin for 1 h. c, Immunoblot of galectin-1 (14.5 kDa) in the supernatant and lysates of BMDMs stimulated as in (b). d, IL-1α secretion by Pam3CSK4-primed WT, Casp11–/–, and Casp1–/– BMDMs stimulated with 10 μM nigericin for 1 h. e, Immunoblot of caspase-11 and β-actin in lysates of MS1 endothelial cells transduced with LentiCRISPR V2 plasmid expressing control (GFP) sgRNA or caspase-11 sgRNA. f, Immunoblot of caspase-4 and β-actin in lysates of HeLa cells transduced with LentiCRISPR V2 plasmid expressing control (GFP) sgRNA or caspase-4 sgRNA. g, Immunoblot of GSDMD in the lysates of Pam3CSK4-primed WT BMDMs infected with EHEC or S. flexneri (MOI=50) for 16 h or 10 µM nigericin for 1 h in the presence or absence of 50 mM glycine. h, Galectin-1 release from liposomes packaged with galectin-1 and incubated with either 1 µg recombinant active caspase-11, 2 µg recombinant gasdermin D, both caspase-11 and gasdermin D, or 0.1% Triton X-100. Data are presented as mean ± SEM of one experiment representative of two (b,h; h is the replicate for the Fig. 2i). Combined data from two independent experiments (d) are shown as mean ± SEM. Immunoblots (c,eg) are representative of two independent experiments.

Source data

Extended Data Fig. 2 Galectin-1 contributes to lethality during sepsis.

a, Plasma amounts of ALT and LDH in WT, Lgals1–/–, Casp11–/–, and Gsdmd–/– mice injected i.p. with 5 mg/kg LPS for 18 h. b, Survival of WT (n=9), Lgals1–/– (n=9), and Casp11–/– mice (n=4) injected i.p. with E. coli (5x108 CFU). c, Survival of Lgals1–/– mice injected i.p. with LPS (3 mg/kg) followed by i.p. injection of PBS (n=4), 100 µg of recombinant GFP (n=4), or 100 µg rGal-1 (n=4) 1 h later. d, Survival of Lgals1–/– mice injected i.p. with LPS (3 mg/kg) followed by i.p. injection of 100 µg of rGal-1 (n=6) or heat inactivated rGal-1 (HI rGal-1) (n=6) 1 h later. e, TNF and IL-6 secretion by WT BMDMs stimulated with 5 µM rGal-1 or 1 µg LPS for 16 h. f, Survival of WT and Casp11–/– mice injected i.p. with PBS (n=3) or 100 µg of rGal-1 (n=3). Combined data from two independent experiments are shown (b,d, and e). In (e) data are presented as mean ± SEM. In (a), each circle represents a mouse and the horizontal lines represent mean. ns, not significant (one-way ANOVA). *p < 0.05, one-way ANOVA (a); Mantel-Cox test (d).

Source data

Extended Data Fig. 3 Galectin-1 amplifies systemic inflammatory responses during LPS shock.

a,b, Cytokine and chemokine levels in the lung (a) and spleen (b) homogenates of WT and Lgals1–/– mice injected i.p. with 5 mg/kg LPS for 20 h. Combined data from two independent experiments are shown. Each circle represents a mouse and the horizontal lines represent mean. *p < 0.05; unpaired two-tailed t test (ab); ns, not significant.

Source data

Extended Data Fig. 4 Galectin-1 amplifies systemic inflammatory responses during endotoxemia.

a, Indicated cytokine and chemokine levels in the plasma of WT (n=5), Lgals1–/– (n=5), Casp11–/– (n=5), and Gsdmd–/– (n=5) mice injected i.p. with 5 mg/kg LPS for 20 h. Each circle represents a mouse and the horizontal lines represent mean. *p < 0.05; one-way ANOVA followed by Sidak’s post-test.

Source data

Extended Data Fig. 5 Galectin-1 functions in a glycan-dependent manner during endotoxemia.

a, Survival of WT, Mgat5–/–, and C2gnt1–/– mice injected i.p. with 5 mg/kg LPS followed by i.p. injection of PBS or 100 µg of rGal-1 1 h later (WT LPS PBS n=6, WT LPS rGal-1 n=7, Mgat5–/– LPS PBS n=5, Mgat5–/– LPS rGal-1 n=5, C2gnt1–/– LPS PBS n=9 and C2gnt1–/– LPS rGal-1 n=11). b, Plasma amounts of ALT and LDH in WT (n=6), Mgat5–/– (n=5), and C2gnt1–/– (n=9) mice injected i.p. with 5 mg/kg LPS for 18 h. c, Galectin-1 levels in the plasma of WT (n=6), Mgat5–/– (n=5), and C2gnt1–/– (n=9) mice injected i.p. with 5 mg/kg LPS for 18 h. d, Immunoblot of CD45 that was immunoprecipitated with anti-CD45 antibody or IgG control antibody from the spleen homogenates of WT mice. Each circle represents a mouse and the horizontal lines represent mean (b,c). ns, not significant; one-way ANOVA followed by the Sidak’s post-test. Immunoblots (d) are representative of two independent experiments (d).

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Russo, A.J., Vasudevan, S.O., Méndez-Huergo, S.P. et al. Intracellular immune sensing promotes inflammation via gasdermin D–driven release of a lectin alarmin. Nat Immunol 22, 154–165 (2021). https://doi.org/10.1038/s41590-020-00844-7

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