LaQueta, M. space significantly inhibited ATP induced PKR activation (Fig. 1c). Inactivation of macrophage PKR by genetic deletion (Fig. 1d-e) or pharmacological inhibition (Fig. 1f, Supplementary Fig. 2) also significantly inhibited HMGB1 release. Open in a separate windows Fig. 1 Role of PKR in pyroptosis-mediated HMGB1 releaseCells 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. 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. 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 impartial experiments. #, p<0.05 vs. wild-type stimulated groups. Mass-spectrometric analysis of acetylation status of nuclear location sequences (NLS) of HMGB1. Pyroptosis, a form of programmed, inflammatory cell death, occurs with macrophage inflammasome activation, and we observed that deletion of PKR significantly inhibited LDH release (Fig. 1g). Analysis by tandem mass spectrometry of HMGB1 released in response to ATP, MSU, or ALU indicated that HMGB1 was highly acetylated in the nuclear location sequence (NLS) (Fig. 1h, Supplementary Fig. 3-6). In contrast, HMGB1 released from macrophages subjected to freeze/thaw cycles was not acetylated in the NLS (Fig. 1h). Together with evidence that inflammasome activation participates in the nuclear translocation of HMGB1 4, these results indicated that HMGB1 hyperacetylation and release, and inflammasome activation, are regulated by PKR. To address the role of PKR in activating the NLRP3 inflammasome, we measured caspase-1 activation and IL-1 cleavage in peritoneal macrophages from PKR+/+ and PKR-/- mice. Caspase-1 activation and IL-1 cleavage were significantly inhibited in PKR-/- macrophages stimulated by exposure to ATP, MSU and ALU (Fig. 2a). Comparable results were obtained in bone-marrow-derived dendritic cells (Supplementary Fig. 7) and macrophages (Supplementary Fig. 8). The expression of NLRP3 and pro-IL-1 did not differ significantly in PKR-/- macrophages as compared to PKR+/+ macrophages (Fig. 2a, Supplementary Fig. 9), but IL-1 secretion by macrophages exposed to live LPS-primed PKR+/+ or PKR-/- macrophages were stimulated as indicated. PKR+/+ macrophages were stimulated or treated with 2-AP or C13H8N4OS (CNS) as indicated. PKR+/+ or PKR-/- mice (n=5) were injected with live HEK293A cells were transfected as indicated. Caspase-1 activation and IL-1 cleavage were assessed by Western-blot. Data are representative of at least three impartial experiments. Levels of IL-1, 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 circulation cytometry (e). Data shown are means SD. #, p<0.05 vs. wild-type infected groups. Transfection with Poly I:C and RNA in bone-marrow derived dendritic cells significantly activated caspase-1 and stimulated IL-1 cleavage in PKR+/+, but not PKR-/- cells (Supplementary Fig. 11). Similar observations Molidustat were obtained in PKR-/- and PKR+/+macrophages stimulated by rotenone, which induces mitochondrial ROS production and PKR phosphorylation (Supplementary Fig. 12, 13). Pharmacological inhibition of PKR dose-dependently suppressed MSU-induced caspase-1 activation and IL-1 cleavage. The observed IC50s of 2-AP and C13H8N4OS were 0.5 mM and 0.25 M respectively, which agree closely with their known IC50 against PKR (Fig. 2c, Supplementary Fig. 14). PKR inhibition significantly reduced ATP- and ALU-induced inflammasome activation in murine macrophages (Supplementary Fig. 15, 16), and in human monocytic THP-1 cells (Supplementary Fig. 17). IL-18 release was significantly decreased in PKR-/- macrophages as compared with PKR+/+ macrophages stimulated with ATP, MSU or ALU, whereas TNF and IL-6 were not suppressed (Supplementary Fig. 18). Addition of 2-AP reduced MSU-induced IL-18 release, but not TNF and IL-6 (Supplementary Fig. 18). 2-AP failed to inhibit MSU-induced caspase-1 activation and IL-1 cleavage in PKR-/- macrophages (Supplementary Fig.19). Collectively, these findings establish a critical role for PKR in activating the NLRP3 inflammasome. PKR+/+ and PKR-/- mice were then exposed to live in order to activate the NLRP3 inflammasome 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). Molidustat 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. 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. Exposure of PKR+/+ macrophages to ATP significantly enhanced formation of NLRP3 and PKR complexes (Fig. 3b). Pharmacological inhibition of PKR phosphorylation by 2-AP and C13H8N4OS significantly suppressed.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 (PKR+/+ macrophages were stimulated or treated with 2-AP as indicated. 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. 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. Mass-spectrometric analysis of acetylation status of nuclear location sequences (NLS) of HMGB1. Pyroptosis, a form of programmed, inflammatory cell death, occurs with macrophage inflammasome activation, and we observed that deletion of PKR significantly inhibited LDH release (Fig. 1g). Analysis by tandem mass spectrometry of HMGB1 released in response to ATP, MSU, or ALU indicated that HMGB1 was highly acetylated in the nuclear location sequence (NLS) (Fig. 1h, Supplementary Fig. 3-6). In contrast, HMGB1 released from macrophages subjected to freeze/thaw cycles was not acetylated in the NLS (Fig. 1h). Together with evidence that inflammasome activation participates in the nuclear translocation of HMGB1 4, these results indicated that HMGB1 hyperacetylation and release, and inflammasome activation, are regulated by PKR. To address the role of PKR in activating the NLRP3 inflammasome, we measured caspase-1 activation and IL-1 cleavage in peritoneal macrophages from PKR+/+ and PKR-/- mice. Caspase-1 activation and IL-1 cleavage were significantly inhibited in PKR-/- macrophages stimulated by exposure to ATP, MSU and ALU (Fig. 2a). Similar results were obtained in bone-marrow-derived dendritic cells (Supplementary Fig. 7) and macrophages (Supplementary Fig. 8). The expression of NLRP3 and pro-IL-1 did not differ significantly in PKR-/- macrophages as compared to PKR+/+ macrophages (Fig. 2a, Supplementary Fig. 9), but IL-1 secretion by macrophages exposed to live LPS-primed PKR+/+ or PKR-/- macrophages were stimulated as indicated. PKR+/+ macrophages were stimulated or treated with 2-AP or C13H8N4OS (CNS) as indicated. PKR+/+ or PKR-/- mice (n=5) were injected with live HEK293A cells were transfected as indicated. Caspase-1 activation and IL-1 cleavage were assessed by Western-blot. Data are representative of at least three independent experiments. Levels of IL-1, 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. Transfection with Poly I:C and RNA in bone-marrow derived dendritic cells significantly activated caspase-1 and stimulated IL-1 cleavage in PKR+/+, but not PKR-/- cells (Supplementary Fig. 11). Similar observations were obtained in PKR-/- and PKR+/+macrophages stimulated by rotenone, which induces mitochondrial ROS production and PKR phosphorylation (Supplementary Fig. 12, 13). Pharmacological inhibition of PKR dose-dependently suppressed MSU-induced caspase-1 activation and IL-1 cleavage. The observed IC50s of 2-AP and C13H8N4OS were 0.5 mM and 0.25 M respectively, which agree closely with their known IC50 against PKR (Fig. 2c, Supplementary Fig. 14). PKR inhibition significantly reduced ATP- and ALU-induced inflammasome activation in murine macrophages (Supplementary Fig. 15, 16), and in human monocytic THP-1 cells (Supplementary Fig. 17). IL-18 release was significantly decreased in PKR-/- macrophages as compared with PKR+/+ macrophages stimulated with ATP, MSU or ALU, whereas TNF and IL-6 were not suppressed (Supplementary Fig. 18). Addition of 2-AP reduced MSU-induced IL-18 launch, but not TNF and IL-6 (Supplementary Fig. 18). 2-AP failed to inhibit MSU-induced caspase-1 activation and IL-1 cleavage in PKR-/- macrophages (Supplementary Fig.19). Collectively, these findings establish a essential part for PKR in activating the NLRP3 inflammasome. PKR+/+ and PKR-/- mice were then exposed to live in order to activate the NLRP3 inflammasome Immunoprecipitation (IP) and Western-blot (WB) analysis of the physical connection 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). 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. PKR+/+ macrophages were stimulated or treated with 2-AP as indicated. Cell lysates were subjected to gel-filtration chromatograph and western-blot. Results.2a, Supplementary Fig. Addition of potassium to the extracellular space significantly inhibited ATP induced PKR activation (Fig. 1c). Inactivation of macrophage PKR by genetic deletion (Fig. 1d-e) or pharmacological inhibition (Fig. 1f, Supplementary Fig. 2) also significantly inhibited HMGB1 launch. Open in a separate windowpane Fig. 1 Part of PKR in pyroptosis-mediated HMGB1 releaseCells 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. 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. 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 demonstrated are means SD of 3 self-employed experiments. #, p<0.05 vs. wild-type stimulated groups. Mass-spectrometric analysis of acetylation status of nuclear location sequences (NLS) of HMGB1. Pyroptosis, a form of programmed, inflammatory cell death, happens with macrophage inflammasome activation, and we observed that deletion of PKR significantly inhibited LDH launch (Fig. 1g). Analysis by tandem mass spectrometry of HMGB1 released in response to ATP, MSU, or ALU indicated that HMGB1 was highly acetylated in the nuclear location sequence (NLS) (Fig. 1h, Supplementary Fig. 3-6). In contrast, HMGB1 released from macrophages subjected to freeze/thaw cycles was not acetylated in the NLS (Fig. 1h). Together with evidence that inflammasome activation participates in the nuclear translocation of HMGB1 4, these results indicated that HMGB1 hyperacetylation and launch, and inflammasome activation, are controlled by Molidustat PKR. To address the part of PKR in activating the NLRP3 inflammasome, we measured caspase-1 activation and IL-1 cleavage in peritoneal macrophages from PKR+/+ and PKR-/- mice. Caspase-1 activation and IL-1 cleavage were significantly inhibited in PKR-/- macrophages stimulated by exposure to ATP, MSU and ALU (Fig. 2a). Related results were acquired in bone-marrow-derived dendritic cells (Supplementary Fig. 7) and macrophages (Supplementary Fig. 8). The manifestation of NLRP3 and pro-IL-1 did not differ significantly in PKR-/- macrophages as compared to PKR+/+ macrophages (Fig. 2a, Supplementary Fig. 9), but IL-1 secretion by macrophages exposed to live LPS-primed PKR+/+ or PKR-/- macrophages were stimulated as indicated. PKR+/+ macrophages were stimulated or treated with 2-AP or C13H8N4OS (CNS) as indicated. PKR+/+ or PKR-/- mice (n=5) were injected with live HEK293A cells were transfected as indicated. Caspase-1 activation and IL-1 cleavage were assessed by Western-blot. Data are representative of at least three self-employed experiments. Levels of IL-1, 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 material measured by circulation cytometry (e). Data demonstrated are means SD. #, p<0.05 vs. wild-type infected organizations. Transfection with Poly I:C and RNA in bone-marrow derived dendritic cells significantly triggered caspase-1 and stimulated IL-1 cleavage in PKR+/+, but not PKR-/- cells (Supplementary Fig. 11). Related observations were acquired in PKR-/- and PKR+/+macrophages stimulated by rotenone, which induces mitochondrial ROS production and PKR phosphorylation (Supplementary Fig. 12, 13). Pharmacological inhibition of PKR dose-dependently suppressed MSU-induced caspase-1 activation and IL-1 cleavage. The observed IC50s of 2-AP and C13H8N4OS were 0.5 mM and 0.25 M respectively, which acknowledge closely with their known IC50 against Molidustat PKR (Fig. 2c, Supplementary Fig. 14). PKR inhibition significantly reduced ATP- and ALU-induced inflammasome activation in murine macrophages (Supplementary Fig. 15, 16), and in human being monocytic THP-1 cells (Supplementary Fig. 17). IL-18 launch was significantly decreased in PKR-/- macrophages as compared with PKR+/+ macrophages stimulated with ATP, MSU or ALU, whereas TNF and IL-6 were not suppressed (Supplementary Fig. 18). Addition of 2-AP reduced MSU-induced IL-18 launch, but not TNF and IL-6 (Supplementary Fig. 18). 2-AP failed to inhibit MSU-induced caspase-1 activation and IL-1 cleavage in PKR-/- macrophages (Supplementary Fig.19). Collectively, these findings establish a essential part for PKR in activating the NLRP3 inflammasome. PKR+/+ and PKR-/- mice were then exposed to live in order to activate the NLRP3 inflammasome Immunoprecipitation (IP) and Western-blot (WB) analysis of the physical connection 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.Lau, D. 1f, Supplementary Fig. 2) also significantly inhibited HMGB1 launch. Open in a separate windowpane Fig. 1 Part of PKR in pyroptosis-mediated HMGB1 releaseCells 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. 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. LPS-primed PKR+/+ or PKR-/- macrophages were stimulated or treated with 2-AP as indicated. HMGB1 levels in the supernatant were determined by Traditional western blot. Cytotoxicity was dependant on LDH assay. Data proven are means SD of 3 indie tests. #, p<0.05 vs. wild-type activated groups. Mass-spectrometric evaluation of acetylation position of nuclear area sequences (NLS) of HMGB1. Pyroptosis, a kind of designed, inflammatory cell loss of life, takes place with macrophage inflammasome activation, and we noticed that deletion of PKR considerably inhibited LDH discharge (Fig. 1g). Evaluation by tandem mass spectrometry of HMGB1 released in response to ATP, MSU, or ALU indicated that HMGB1 was extremely acetylated in the nuclear area series (NLS) (Fig. 1h, Supplementary Fig. 3-6). On the other hand, HMGB1 released from macrophages put through freeze/thaw cycles had not been acetylated in the NLS (Fig. 1h). As well as proof that inflammasome activation participates in the nuclear translocation of HMGB1 4, these outcomes indicated that HMGB1 hyperacetylation and discharge, and inflammasome activation, are governed by PKR. To handle the function of PKR in activating the NLRP3 inflammasome, we assessed caspase-1 Molidustat activation and IL-1 cleavage in peritoneal macrophages from PKR+/+ and PKR-/- mice. Caspase-1 activation and IL-1 cleavage had been considerably inhibited in PKR-/- macrophages activated by contact with ATP, MSU and ALU (Fig. 2a). Equivalent results had been attained in bone-marrow-derived dendritic cells (Supplementary Fig. 7) and macrophages (Supplementary Fig. 8). The appearance of NLRP3 and pro-IL-1 didn't differ considerably in PKR-/- macrophages when compared with PKR+/+ macrophages (Fig. 2a, Supplementary Fig. 9), but IL-1 secretion by macrophages subjected to live LPS-primed PKR+/+ or PKR-/- macrophages had been activated as indicated. PKR+/+ macrophages had been activated or treated with 2-AP or C13H8N4OS (CNS) as indicated. PKR+/+ or PKR-/- mice (n=5) had been injected with live HEK293A cells had been transfected as indicated. Caspase-1 activation and IL-1 cleavage had been evaluated by Western-blot. Data are representative of at least three indie experiments. Degrees of IL-1, IL-18, HMGB1, and IL-6, in the supernatant (b) or serum (d) had been dependant on ELISA. Peritoneal lavage liquid was gathered and neutrophil articles measured by stream cytometry (e). Data proven are means SD. #, p<0.05 vs. wild-type contaminated groupings. Transfection with Poly I:C and RNA in bone-marrow produced dendritic cells considerably turned on caspase-1 and activated IL-1 cleavage in PKR+/+, however, not PKR-/- cells (Supplementary Fig. 11). Equivalent observations had been attained in PKR-/- and PKR+/+macrophages activated by rotenone, which induces mitochondrial ROS creation and PKR phosphorylation (Supplementary Fig. 12, 13). Pharmacological inhibition of PKR dose-dependently suppressed MSU-induced caspase-1 activation and IL-1 cleavage. The noticed IC50s of 2-AP and C13H8N4OS had been 0.5 mM and 0.25 M respectively, which recognize closely using their known IC50 against PKR (Fig. 2c, Supplementary Fig. 14). PKR inhibition considerably decreased ATP- and ALU-induced inflammasome activation in murine macrophages (Supplementary Fig. 15, 16), and in individual monocytic THP-1 cells (Supplementary Fig. 17). IL-18 discharge was considerably reduced in PKR-/- macrophages in comparison with PKR+/+ macrophages activated with ATP, MSU or ALU, whereas TNF and IL-6 weren't suppressed (Supplementary Fig. 18). Addition of 2-AP decreased MSU-induced IL-18 discharge, however, not TNF and IL-6 (Supplementary Fig. 18). 2-AP didn't inhibit MSU-induced caspase-1 activation and IL-1 cleavage in PKR-/- macrophages (Supplementary Fig.19). Collectively, these results establish a vital function for PKR in activating the NLRP3 inflammasome. PKR+/+ and PKR-/- mice had been then subjected to live in purchase to activate the NLRP3 inflammasome Immunoprecipitation (IP) and Western-blot (WB) evaluation from the physical relationship of PKR and NLRP3 in cell-free program using recombinant proteins (a) or LPS-primed macrophages activated with ATP or treated with of 2-AP or C13H8N4OS (CNS) as indicated (b). Rabbit Polyclonal to GIMAP5 The.performed the tests; B.L., S.We.V., P.S.O., H.Con., S.S.C., J.R., T.K., G.S.H., U.A., and K.J.T. potassium-substituted moderate (KCl) as indicated. Cells had been lysed at indicated period factors and PKR activation was supervised by autophosphorylation. LPS-primed PKR+/+ or PKR-/- macrophages had been activated or treated with 2-AP as indicated. HMGB1 amounts in the supernatant had been determined by Traditional western blot. Cytotoxicity was dependant on LDH assay. Data proven are means SD of 3 indie tests. #, p<0.05 vs. wild-type activated groups. Mass-spectrometric evaluation of acetylation position of nuclear area sequences (NLS) of HMGB1. Pyroptosis, a kind of designed, inflammatory cell loss of life, takes place with macrophage inflammasome activation, and we noticed that deletion of PKR considerably inhibited LDH discharge (Fig. 1g). Evaluation by tandem mass spectrometry of HMGB1 released in response to ATP, MSU, or ALU indicated that HMGB1 was extremely acetylated in the nuclear area series (NLS) (Fig. 1h, Supplementary Fig. 3-6). On the other hand, HMGB1 released from macrophages put through freeze/thaw cycles had not been acetylated in the NLS (Fig. 1h). As well as proof that inflammasome activation participates in the nuclear translocation of HMGB1 4, these outcomes indicated that HMGB1 hyperacetylation and discharge, and inflammasome activation, are governed by PKR. To handle the function of PKR in activating the NLRP3 inflammasome, we assessed caspase-1 activation and IL-1 cleavage in peritoneal macrophages from PKR+/+ and PKR-/- mice. Caspase-1 activation and IL-1 cleavage had been considerably inhibited in PKR-/- macrophages activated by contact with ATP, MSU and ALU (Fig. 2a). Equivalent results had been attained in bone-marrow-derived dendritic cells (Supplementary Fig. 7) and macrophages (Supplementary Fig. 8). The appearance of NLRP3 and pro-IL-1 didn't differ considerably in PKR-/- macrophages when compared with PKR+/+ macrophages (Fig. 2a, Supplementary Fig. 9), but IL-1 secretion by macrophages subjected to live LPS-primed PKR+/+ or PKR-/- macrophages had been activated as indicated. PKR+/+ macrophages had been activated or treated with 2-AP or C13H8N4OS (CNS) as indicated. PKR+/+ or PKR-/- mice (n=5) had been injected with live HEK293A cells had been transfected as indicated. Caspase-1 activation and IL-1 cleavage had been evaluated by Western-blot. Data are representative of at least three indie experiments. Degrees of IL-1, IL-18, HMGB1, and IL-6, in the supernatant (b) or serum (d) had been dependant on ELISA. Peritoneal lavage liquid was gathered and neutrophil content material measured by movement cytometry (e). Data demonstrated are means SD. #, p<0.05 vs. wild-type contaminated organizations. Transfection with Poly I:C and RNA in bone-marrow produced dendritic cells considerably triggered caspase-1 and activated IL-1 cleavage in PKR+/+, however, not PKR-/- cells (Supplementary Fig. 11). Identical observations had been acquired in PKR-/- and PKR+/+macrophages activated by rotenone, which induces mitochondrial ROS creation and PKR phosphorylation (Supplementary Fig. 12, 13). Pharmacological inhibition of PKR dose-dependently suppressed MSU-induced caspase-1 activation and IL-1 cleavage. The noticed IC50s of 2-AP and C13H8N4OS had been 0.5 mM and 0.25 M respectively, which consent closely using their known IC50 against PKR (Fig. 2c, Supplementary Fig. 14). PKR inhibition considerably decreased ATP- and ALU-induced inflammasome activation in murine macrophages (Supplementary Fig. 15, 16), and in human being monocytic THP-1 cells (Supplementary Fig. 17). IL-18 launch was considerably reduced in PKR-/- macrophages in comparison with PKR+/+ macrophages activated with ATP, MSU or ALU, whereas TNF and IL-6 weren't suppressed (Supplementary Fig. 18). Addition of 2-AP decreased MSU-induced IL-18 launch, however, not TNF and IL-6 (Supplementary Fig. 18). 2-AP didn't inhibit MSU-induced caspase-1 activation and IL-1 cleavage in PKR-/- macrophages (Supplementary Fig.19). Collectively, these results establish a important part for PKR in activating the NLRP3 inflammasome. PKR+/+ and PKR-/- mice had been then subjected to.