Novel role of PKR in inflammasome activation and HMGB1 release

Abstract

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1β, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.

Fig. 1. Role of PKR in pyroptosis-mediated HMGB1 release

a-b. Cells were stimulated with Poly I:C. (a) Macrophages from PKR^+/+ or PKR^-/- mice. (b) PKR^+/+ macrophages treated with indicated doses of the PKR inhibitor 2-AP. c. LPS-primed PKR^+/+ macrophages were stimulated or treated with or without potassium-substituted medium (KCl) as indicated. Cells were lysed at indicated time points and PKR activation was monitored by autophosphorylation. d-g. LPS-primed PKR^+/+ or PKR^-/- macrophages were stimulated or treated with 2-AP as indicated. HMGB1 levels in the supernatant were determined by Western blot. Cytotoxicity was determined by LDH assay. Data shown are means ± SD of 3 independent experiments. #, p<0.05 vs. wild-type stimulated groups. h. Mass-spectrometric analysis of acetylation status of nuclear location sequences (NLS) of HMGB1.

Fig. 2. PKR is important for inflammasome activation

a-b. LPS-primed PKR^+/+ or PKR^-/- macrophages were stimulated as indicated. c. PKR^+/+ macrophages were stimulated or treated with 2-AP or C₁₃H₈N₄OS (CNS) as indicated. d-e. PKR^+/+ or PKR^-/- mice (n=5) were injected with live E. coli. f-g. HEK293A cells were transfected as indicated. Caspase-1 activation and IL-1β cleavage were assessed by Western-blot. Data are representative of at least three independent experiments. Levels of IL-1β, IL-18, HMGB1, and IL-6, in the supernatant (b) or serum (d) were determined by ELISA. Peritoneal lavage fluid was collected and neutrophil content measured by flow cytometry (e). Data shown are means ± SD. #, p<0.05 vs. wild-type infected groups.

Fig. 3. PKR physically interacts with NLRP3 and facilitates inflammasome activation

a-b. Immunoprecipitation (IP) and Western-blot (WB) analysis of the physical interaction of PKR and NLRP3 in cell-free system using recombinant proteins (a) or LPS-primed macrophages stimulated with ATP or treated with of 2-AP or C₁₃H₈N₄OS (CNS) as indicated (b). c. The NLRP3 inflammasome was reconstituted using recombinant proteins and ATP/Poly I:C as indicated. Caspase-1 activity was measured by hydrolysis of WEHD-pNA. d. PKR^+/+ macrophages were stimulated or treated with 2-AP as indicated. Cell lysates were subjected to gel-filtration chromatograph and western-blot. Results are representative of three independent experiments.

Fig. 4. PKR regulates NLRP1, AIM2, NLRC4 inflammasome activation

a. HEK293A cells were transfected as indicated. The physical interaction of PKR and myc-tagged proteins was analyzed by Immunoprecipitation (IP) and Western-blot (WB) analysis. b-d. LPS-primed (b) or unprimed (c, d) PKR^+/+ or PKR^-/- macrophages were stimulated with Anthrax lethal toxin (LT), or transfected with Poly (dA:dT), or infected with S. Typhimurium (S.T). MOI, multiplicity of infection. e. PKR^+/+ macrophages were stimulated or treated with 2-AP as indicated. f. HEK293A cells were transfected as indicated. Caspase-1 activation, IL-1β cleavage, and HMGB1 secretion were assessed by Western-blot. Results are representative of at least three independent experiments.