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. 2019 Aug 15;203(4):795-800.
doi: 10.4049/jimmunol.1800720. Epub 2019 Jul 10.

Cutting Edge: Protein Arginine Deiminase 2 and 4 Regulate NLRP3 Inflammasome-Dependent IL-1β Maturation and ASC Speck Formation in Macrophages

Neha Mishra  1 , Lidja Schwerdtner  1 , Kelly Sams  2 , Santanu Mondal  3 , Fareed Ahmad  4 , Reinhold E Schmidt  5 , Scott A Coonrod  2 , Paul R Thompson  3 , Markus M Lerch  6 , Lukas Bossaller  7
Affiliations

Cutting Edge: Protein Arginine Deiminase 2 and 4 Regulate NLRP3 Inflammasome-Dependent IL-1β Maturation and ASC Speck Formation in Macrophages

Neha Mishra et al. J Immunol. .

Abstract

Protein arginine deiminase (PAD) enzymes catalyze the conversion of protein-bound arginine into citrulline, an irreversible posttranslational modification with loss of a positive charge that can influence protein-protein interactions and protein structure. Protein arginine deiminase activity depends on high intracellular calcium concentrations occurring in dying cells. In this study, we demonstrate that protein citrullination is common during pyroptotic cell death in macrophages and that inhibition of PAD enzyme activity by Cl-amidine, a pan-PAD inhibitor, blocks NLRP3 inflammasome assembly and proinflammatory IL-1β release in macrophages. Genetic deficiency of either PAD2 or PAD4 alone in murine macrophages does not impair IL-1β release; however, pharmacological inhibition or small interfering RNA knockdown of PAD2 within PAD4-/- macrophages does. Our results suggest that PAD2 and 4 activity in macrophages is required for optimal inflammasome assembly and IL-1β release, a finding of importance for autoimmune diseases and inflammation.

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Conflict of interest statement

Disclosures: P.R.T. is a consultant for Celgene and Disarm Therapeutics, founded Padlock Therapeutics, and has received fees from Bristol Myers Squibb. The other authors have no financial conflicts of interest.

Figures

Figure 1.
Figure 1.. Inflammasome activation induces protein citrullination in murine macrophages.
(A) Confocal images of BMDMs stained with anti-ASC Ab (red), F95 (green), and DAPI (nuclei, blue) either left untreated (left panel) or primed with LPS for 2h (right panel). (B) BMDMs were stimulated with LPS and nigericin for 15 min and prepared for confocal imaging as in (A). Citrullinated and non-citrullinated ASC specks are indicated by an arrowhead or triangle, respectively, and nonactivated cells with evenly distributed cytoplasmic ASC are marked by stars. (C) Confocal images of BMDMs stained with anti-vimentin Ab (red) and F95 (green) stimulated for 6h (Ca2+) and 60 min (nigericin) as indicated. (D) Anti-modified citrulline (AMC) immunoblot of cell lysates from LPS-primed and nigericin-stimulated BMDMs at the indicated time points (one representative immunoblot of three independent experiments and quantification by band densitometry; mean ± SEM). Images were analyzed by Image J. Scale bar, 20 μm.
Figure 2.
Figure 2.. PAD enzyme inhibition blocks IL-1β release in NLRP3-activated macrophages.
ELISA for IL-1β (A) and TNF-α (B) of supernatants (SNs) from NLRP3-activated BMDMs stimulated as indicated and treated with increasing concentrations of the pan-PAD inhibitor Cl-amidine (n=4, mean ± SEM for duplicate samples). (C) Immunoblot for IL-1β, caspase-1 and β-actin of SNs and cell lysate (CL) from BMDMs stimulated as in (A) (one representative out of four independent experiments is shown). (D) AMC immunoblot of CL from BMDMs treated with LPS plus ATP or (E) LPS plus nigericin and increasing concentrations of Cl-amindine (one representative of two independent experiments). (F) Immunoblot analysis of DSS cross-linked ASC of LPS- and ATP-stimulated BMDMs treated with increasing concentrations of Cl-amidine (one representative of two independent experiments and quantification by band densiometry). dA:dT, poly (dA:dT).
Figure 3.
Figure 3.. PAD2 and PAD4 are required for NLRP3-dependent IL-1β release.
(A) IL-1β ELISA of supernatant (SN) from WT compared to PAD4-/- BMDMs. BMDMs were primed with LPS and stimulated with Ca2+, ATP, nigericin and poly(dA:dT) (WT n=3; PAD4-/- , n=3; mean ± SEM for duplicate samples). (B) Immunoblot for IL-1β and caspase-1 from SN and cell lysates (CL) of WT and PAD4−/− BMDMs stimulated as indicated. (C) Production of IL-1β from WT BMDMs stimulated with LPS and nigericin and treated with AFM 30a (n=3), CI-amidine (n=4), GSK484 (n=4), or BB-CI-amidine (n=4), as measured by ELISA. Cytokine levels are normalized to uninhibited control cells. Nonlinear regression analysis was performed, and log (M) of the indicated inhibitors versus the normalized response (variable slope) is presented. (D) IL-1β ELISA of SN from WT and PAD4−/− BMDMs primed with LPS and stimulated with ATP, or nigericin (E) and treated with increasing concentrations of AFM 30a (n=3, mean ± SEM for duplicate samples). *p<0.05; **p<0.01; ***p<0.001, ****p<0.0001 by two-way ANOVA. dA:dT, poly(dA:dT).
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