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SERPINB1-mediated checkpoint of inflammatory caspase activation

Nature Immunologyvolume 20pages276287 (2019) | Download Citation

Abstract

Inflammatory caspases (caspase-1, caspase-4, caspase-5 and caspase-11 (caspase-1/-4/-5/-11)) mediate host defense against microbial infections, processing pro-inflammatory cytokines and triggering pyroptosis. However, precise checkpoints are required to prevent their unsolicited activation. Here we report that serpin family B member 1 (SERPINB1) limited the activity of those caspases by suppressing their caspase-recruitment domain (CARD) oligomerization and enzymatic activation. While the reactive center loop of SERPINB1 inhibits neutrophil serine proteases, its carboxy-terminal CARD-binding motif restrained the activation of pro-caspase-1/-4/-5/-11. Consequently, knockdown or deletion of SERPINB1 prompted spontaneous activation of caspase-1/-4/-5/-11, release of the cytokine IL-1β and pyroptosis, inducing elevated inflammation after non-hygienic co-housing with pet-store mice and enhanced sensitivity to lipopolysaccharide- or Acinetobacter baumannii–induced endotoxemia. Our results reveal that SERPINB1 acts as a vital gatekeeper of inflammation by restraining neutrophil serine proteases and inflammatory caspases in a genetically and functionally separable manner.

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The data that support the findings of this study are available from the corresponding author upon request.

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Change history

  • 07 March 2019

    In the version of this article initially published, the label (CASP4-C285A-HA) above the second and fifth lanes in the right blot in Fig. 1e is incorrect; the correct label is CASP4-C258A-HA. Also, the two labels at right above the plot in Fig. 6c were switched; the far right label should be ‘Co-housed Serpinb1a–/–’ (in red font) and the label just to its left (above the fourth column) should be ‘Co-housed WT’ (in black font). Finally, the bottom two symbols in the key to Fig. 7d were switched; the red circle should identify 1CARD-SUMO (TEV) and the blue triangle should identify 1CARD-SUMO + SERPINB1 (TEV). The errors have been corrected in the HTML and PDF versions of the article.

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Acknowledgements

We thank E. Remold-O’Donnell (Boston Children’s Hospital) for Serpinb1a–/– mice, J. Yuan (Harvard Medical School) for Casp11–/– mice, T.S. Xiao (Case Western Reserve University), H.D. Beer (University of Zurich) and K.L. Rock (University of Massachusetts Medical School) for reagents, M.J. Kwak and B.H. Oh for protein purification, and J.W. Bowman and R. Amatya for manuscript preparation. This work was partly supported by grant nos. CA180779, CA200422, AI073099, AI116585, AI129496, AI140718, AI140705, DE023926, DE027888 and DE028521 (J.U.J.), NRF-2016R1D1A1B03931761 (H.-R.L.), Al124491 and HD087988 (H.W.), and AI081719, AI117211, AI127954 and AI106375 (B.S.).

Author information

Affiliations

  1. Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    • Youn Jung Choi
    • , Stephanie Kim
    • , Younho Choi
    • , Travis B. Nielsen
    • , Jun Yan
    • , Brad Spellberg
    •  & Jae U. Jung
  2. Department of Pharmacology and Pharmaceutical Sciences, School of Pharmacy, University of Southern California, Los Angeles, CA, USA

    • Youn Jung Choi
    •  & Jae U. Jung
  3. Department of Medicine, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    • Travis B. Nielsen
    • , Jun Yan
    •  & Brad Spellberg
  4. Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA

    • Alvin Lu
    • , Jianbin Ruan
    •  & Hao Wu
  5. Program in Cellular and Molecular Medicine, Boston Children’s Hospital, Boston, MA, USA

    • Alvin Lu
    • , Jianbin Ruan
    •  & Hao Wu
  6. Department of Biotechnology and Bioinformatics, Collage of Science and Technology, Korea University, Sejong, South Korea

    • Hye-Ra Lee

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Contributions

Y.J.C. performed and analyzed all experiments, prepared the figures and wrote the first draft of the manuscript. S.K., Y.C., T.B.N., J.Y., A.L., J.R., H.-R.L., H.W. and B.S. collaborated on the experimental design, execution and interpretation. Y.J.C. and J.U.J. jointly conceived the experimental design, interpreted the results and wrote subsequent drafts of the manuscript.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Jae U. Jung.

Integrated supplementary information

  1. Supplementary Figure 1 Caspase-4 CARD region responsible for SERPINB1 binding.

    a, Schematic diagram of caspase-4-CARD consisting of six α-helices (α1-α6). The full-length and truncated forms of caspase-4-CARD were co-transformed with the SERPINB1 carboxy-terminal region (aa 330–379) to yeast for the growth on two or four dropout (DO) plates. b, I-TASSER prediction of α-helical bundles of caspase-4-CARD. Model 1, among top five models generated by I-TASSER, with a C-score of 0.38, estimated TM-score of 0.76 ± 0.10, and estimated RMSD of 2.9 ± 2.1 Å. Three residues in α-helices 2 and 3 regions (D27/V30/E40) marked with red color are predicted as a ligand binding site. Structure was prepared using PyMOL. c, The loss of SERPINB1-binding activity of caspase-4-CARD mutant. HA-tagged full-length enzymatically inactive caspase-4 containing wild-type CARD or mutant CARD (D27A/V30A/E40A), and Flag-tagged SERPINB1 were transfected to 293T cells, and whole-cell extracts (WCEs) were subjected to co-immunoprecipitation (IP) with anti-Flag, followed by immunoblotting (IB) with anti-HA or anti-Flag antibody. Data in a,c are representative of two independent experiments.

  2. Supplementary Figure 2 The CARD-binding motif of SERPINB1 targets caspase-1 and caspase-4.

    a, Schematic diagram of SERPINB1 deletion mutant constructs. GST-pulldown (GST-PD) assay of SERPINB1 deletion mutant (Δ7 and Δ13) binding to caspase-1-CARD. Flag-tagged SERPINB1 wild-type, Δ7, or Δ13 and GST-caspase-1-CARD were transfected to 293T cells, and WCEs were subjected to GST-pulldown, followed by immunoblotting (IB) using anti-Flag or anti-GST antibody. b, Co-immunoprecipitation (IP) assay of SERPINB1 wild-type or RCL mutant (F343A/C344A) binding to proteinase-3 (PRTN3) or caspase-4. Flag-SERPINB1 wild-type or F343A/C344A and PRTN3-HA or caspase-4-HA were transfected to 293T cells, and WCEs were subjected to co-immunoprecipitation with anti-Flag, followed by immunoblotting using anti-HA or anti-Flag antibody. % indicates acrylamide percentage. c, Neutrophil elastase (NE) enzymatic assay; NE was incubated with SERPINB1 and the reactions were diluted into assay buffer containing chromogenic substrate (MeO-SucAAPV-pNA). Free p-nitroanilide (pNA) after cleavage from the substrate was measured at 400 nm. d, Caspase-1 or caspase-4 enzymatic assay; caspase-1 or caspase-4 was incubated with SERPINB1 and the reactions were diluted into assay buffer containing substrate (caspase-1; Ac-YVAD-pNA, caspase-4; Ac-LEVD-pNA). The reaction mixture was further incubated at 37 °C for hydrolysis of substrate and residual protease activity was quantified at the indicated time points. Data in a,b,c,d are representative of two independent experiments.

  3. Supplementary Figure 3 SERPINB1 depletion-induced IL-1β secretion and cytotoxicity.

    a, Validation of silencing efficiency of three shRNAs targeting SERPINB1 in THP1 and U937. Cells transiently transduced with scramble or shSERPINB1 lentivirus for 48 h and WCEs were immunoblotted with anti-SERPINB1 antibody. b, IL-1β, TNF and IL-6 cytokine secretion upon SERPINB1 depletion. c, Effects of caspase inhibitors on IL-1β secretion upon SERPINB1 depletion. d, Generation of caspase-1 or caspase-4 knockout cells. Location of sgRNA used for CRISPR/Cas9-mediated editing of the caspase-1 and caspase-4 locus. The sgRNA target and PAM sequences are shown in red and blue, respectively. e,f, Validation of silencing efficiency of shRNAs targeting neutrophil elastase (NE), proteinase-3 (PRTN3), and/or cathepsin G (CG), ASC or NLRP3 in THP1. Whole-cell lysates (WCL) were immunoblotted with indicated antibodies. g, IL-1β release upon inflammasome stimulus in SERPINB1-depleted cells. h,i, Lactate dehydrogenase (LDH) release-based cytotoxicity of THP1 and U937 cells after SERPINB1 depletion. Cytotoxicity was measured at 3 days post-transduction. j,k, ATP-based cell viability of THP1 and U937 cells upon SERPINB1 depletion. Cells were transduced by scramble or shSERPINB1 lentivirus for 48 h. Viability was determined at the indicated time point after lentiviral transduction. Data in a,d,e,f are representative of two independent experiments. Data are presented as mean ± s.e.m. from n=3 independent experiments in b,c,g,h,i and from n=4 pooled from two independent experiments in j,k. P values were determined by two-way analysis of variance (ANOVA) with Bonferroni’s comparison relative to scramble in b,h,i, and by one-way ANOVA with Dunnett’s comparison relative to shSERPINB1-4 LPS in c. NS, not significant.

  4. Supplementary Figure 4 Mouse SERPINB1 isoforms for inhibition of mouse caspase-1 and caspase-11.

    a, Interaction between murine SERPINB1a,b,c and caspase-1/-11. HA-tagged caspase-1-C254A or caspase-1 and Flag-tagged SERPINB1a,b,c were transfected to 293T cells, and WCEs were subjected to co-immunoprecipitation (IP) with anti-Flag, followed by immunoblotting (IB) using anti-HA or anti-Flag antibody. b, Relative mRNA quantification of murine Serpinb1a,b,c in myeloid cell lines. Gapdh was used as a normalization control. c, Detection of caspase-1 activation upon Serpinb1a,b,c depletion in DC 2.4 cells. d, Verification of Serpinb1-targeting shRNAs silencing efficiency by quantitative PCR with reverse transcription (qRT-PCR) in BMDMs. mRNA expression was normalized to 18S and fold change was calculated relative to scramble. Data in a are representative of two independent experiments. Data are presented as mean ± s.e.m. from n=3 independent experiments in b,c,d. P values were determined by one-way ANOVA with Dunnett’s comparison relative to scramble in c,d.

  5. Supplementary Figure 5 Spleen histopathology during A. baumannii infection.

    Hematoxylin-eosin-stained spleen sections from wild-type (WT) and Serpinb1a–/– mice at 6 hpi. Images are representative of two analyzed mice for each condition. Bar, 100 µm.

  6. Supplementary Figure 6 Gene expression of Serpinb1a–/– mice upon co-housing.

    a,b, Peripheral leukocytes and lung Il6 mRNAs of wild-type (WT), Serpinb1a–/–, Casp1–/–Casp11–/– and Serpinb1a–/–Casp1–/–Casp11–/– mice either housed in SPF facility or co-housed with pet-store mice (n=8 for wild-type or Serpinb1a–/– mice, n=4 for Casp1–/–Casp11–/– or Serpinb1a–/–Casp1–/–Casp11–/–). mRNA expression was normalized to 18S and fold change was calculated relative to the average of SPF-housed wild-type mice. Data are presented as floating bars (min to max) with line at mean. P values were determined by two-way ANOVA with Bonferroni’s comparison relative to co-housed wild-type mice.

  7. Supplementary Figure 7 Protein purification and Sortase A-mediated FITC labeling.

    a, Schematic diagram of a sandwich-tagged caspase-1-CARD protein oligomerization assay. b, SDS-PAGE of size-exclusion chromatography fractions was either stained with Coomassie Blue (left) or immunoblotted with anti-SMT3 antibody (right). c, Schematic diagram of Sortase A-mediated fluorescein isothiocyanate- (FITC)-conjugation of the sandwich-tagged caspase-1-CARD protein. d, Sortase A-mediated FITC labeling. MBP-1CARD-SUMO, Sortase A and GGG-FITC peptide were incubated at the indicated ratio, followed by size-exclusion chromatography. The indicated FITC labeled fractions were visualized (bottom). The corresponding SDS-PAGE Coomassie Blue gel is shown (top). e, The SUMO cleavage from His-SUMO-SERPINB1 by ULP1 protease to exclude the potential SUMO-SUMO interaction. After ULP1 treatment, protein was either stained with Coomassie Blue (left) or immunoblotted with anti-His antibody (right). f, Illustration of SERPINB1 function as a protease inhibitor with dual specificity. SERPINB1 binds to neutrophil serine protease through the RCL domain and interacts with inflammatory caspase through the CBM region. Human native SERPINB1 structure; PDB 4GA7. The CBM of SERPINB1 consists of β-strands (s1C, s4B and s5B) depicted in red color. The surrounding α-helices (αH, αG and αA) are colored in light cyan and β-strands (s3B, s2B, s1B and s6B) are shown in dark gray. The RCL of SERPINB1 is shown in yellow dashed lines indicate missing residues of the RCL in the structure. Structure was prepared using PyMOL. Data in b,d,e are representative of two independent experiments.

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