Caffeine Inhibits NLRP3 Inflammasome Activation by Suppressing MAPK/NF-κB and A2aR Signaling in LPS-Induced THP-1 Macrophages
Identifieur interne : 000912 ( Pmc/Checkpoint ); précédent : 000911; suivant : 000913Caffeine Inhibits NLRP3 Inflammasome Activation by Suppressing MAPK/NF-κB and A2aR Signaling in LPS-Induced THP-1 Macrophages
Auteurs : Weiming Zhao ; Li Ma ; Cheng Cai ; Xiaohui GongSource :
- International Journal of Biological Sciences [ 1449-2288 ] ; 2019.
Abstract
Excessive inflammation induced by various risk factors is associated with the development of bronchopulmonary dysplasia (BPD). Caffeine exerts potent anti-inflammatory effects as a clinical preventive medicine for BPD. Recently, NLRP3 inflammasome activation has been demonstrated to be essential for the pathogenesis of BPD. In the present study, we aimed to investigate the effects of caffeine on NLRP3 inflammasome activation in LPS-induced THP-1 macrophages and to explore the underlying the detailed mechanism. We found that caffeine significantly reduced NLRP3 expression, ASC speck formation, and caspase 1 cleavage and therefore decreased IL-1β and IL-18 secretion in THP-1 macrophages. Caffeine also markedly decreased the phosphorylation levels of MAPK and NF-κB pathway members, further suppressing the translocation of NF-κB in THP-1 macrophages. Moreover, silencing of the caffeine-antagonized adenosine A2a receptor (A2aR) significantly decreased cleaved caspase 1 expression in THP-1 macrophages by reducing ROS production. Given these findings, we conclude that caffeine inhibits NLRP3 inflammasome activation by suppressing MAPK/NF-κB signaling and A2aR-associated ROS production in LPS-induced THP-1 macrophages.
Url:
DOI: 10.7150/ijbs.34211
PubMed: 31360100
PubMed Central: 6643212
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Caffeine Inhibits NLRP3 Inflammasome Activation by Suppressing MAPK/NF-κB and A2aR Signaling in LPS-Induced THP-1 Macrophages</title><author><name sortKey="Zhao, Weiming" sort="Zhao, Weiming" uniqKey="Zhao W" first="Weiming" last="Zhao">Weiming Zhao</name></author><author><name sortKey="Ma, Li" sort="Ma, Li" uniqKey="Ma L" first="Li" last="Ma">Li Ma</name></author><author><name sortKey="Cai, Cheng" sort="Cai, Cheng" uniqKey="Cai C" first="Cheng" last="Cai">Cheng Cai</name></author><author><name sortKey="Gong, Xiaohui" sort="Gong, Xiaohui" uniqKey="Gong X" first="Xiaohui" last="Gong">Xiaohui Gong</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">31360100</idno><idno type="pmc">6643212</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6643212</idno><idno type="RBID">PMC:6643212</idno><idno type="doi">10.7150/ijbs.34211</idno><date when="2019">2019</date><idno type="wicri:Area/Pmc/Corpus">000593</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000593</idno><idno type="wicri:Area/Pmc/Curation">000593</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Curation">000593</idno><idno type="wicri:Area/Pmc/Checkpoint">000912</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Checkpoint">000912</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Caffeine Inhibits NLRP3 Inflammasome Activation by Suppressing MAPK/NF-κB and A2aR Signaling in LPS-Induced THP-1 Macrophages</title><author><name sortKey="Zhao, Weiming" sort="Zhao, Weiming" uniqKey="Zhao W" first="Weiming" last="Zhao">Weiming Zhao</name></author><author><name sortKey="Ma, Li" sort="Ma, Li" uniqKey="Ma L" first="Li" last="Ma">Li Ma</name></author><author><name sortKey="Cai, Cheng" sort="Cai, Cheng" uniqKey="Cai C" first="Cheng" last="Cai">Cheng Cai</name></author><author><name sortKey="Gong, Xiaohui" sort="Gong, Xiaohui" uniqKey="Gong X" first="Xiaohui" last="Gong">Xiaohui Gong</name></author></analytic><series><title level="j">International Journal of Biological Sciences</title><idno type="eISSN">1449-2288</idno><imprint><date when="2019">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Excessive inflammation induced by various risk factors is associated with the development of bronchopulmonary dysplasia (BPD). Caffeine exerts potent anti-inflammatory effects as a clinical preventive medicine for BPD. Recently, NLRP3 inflammasome activation has been demonstrated to be essential for the pathogenesis of BPD. In the present study, we aimed to investigate the effects of caffeine on NLRP3 inflammasome activation in LPS-induced THP-1 macrophages and to explore the underlying the detailed mechanism. We found that caffeine significantly reduced NLRP3 expression, ASC speck formation, and caspase 1 cleavage and therefore decreased IL-1β and IL-18 secretion in THP-1 macrophages. Caffeine also markedly decreased the phosphorylation levels of MAPK and NF-κB pathway members, further suppressing the translocation of NF-κB in THP-1 macrophages. Moreover, silencing of the caffeine-antagonized adenosine A2a receptor (A2aR) significantly decreased cleaved caspase 1 expression in THP-1 macrophages by reducing ROS production. Given these findings, we conclude that caffeine inhibits NLRP3 inflammasome activation by suppressing MAPK/NF-κB signaling and A2aR-associated ROS production in LPS-induced THP-1 macrophages.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Steinhorn, R" uniqKey="Steinhorn R">R Steinhorn</name></author><author><name sortKey="Davis, Jm" uniqKey="Davis J">JM Davis</name></author><author><name sortKey="Gopel, W" uniqKey="Gopel W">W Göpel</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Owen, Ls" uniqKey="Owen L">LS Owen</name></author><author><name sortKey="Manley, Bj" uniqKey="Manley B">BJ Manley</name></author><author><name sortKey="Davis, Pg" uniqKey="Davis P">PG Davis</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Matta, P" uniqKey="Matta P">P Matta</name></author><author><name sortKey="Sherrod, Sd" uniqKey="Sherrod S">SD Sherrod</name></author><author><name 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Int J Biol Sci</journal-id><journal-id journal-id-type="iso-abbrev">Int. J. Biol. Sci</journal-id><journal-id journal-id-type="publisher-id">ijbs</journal-id><journal-title-group><journal-title>International Journal of Biological Sciences</journal-title></journal-title-group><issn pub-type="epub">1449-2288</issn><publisher><publisher-name>Ivyspring International Publisher</publisher-name><publisher-loc>Sydney</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">31360100</article-id><article-id pub-id-type="pmc">6643212</article-id><article-id pub-id-type="doi">10.7150/ijbs.34211</article-id><article-id pub-id-type="publisher-id">ijbsv15p1571</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Paper</subject></subj-group></article-categories><title-group><article-title>Caffeine Inhibits NLRP3 Inflammasome Activation by Suppressing MAPK/NF-κB and A2aR Signaling in LPS-Induced THP-1 Macrophages</article-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Zhao</surname><given-names>Weiming</given-names></name><xref ref-type="author-notes" rid="FNA_number">#</xref></contrib><contrib contrib-type="author"><name><surname>Ma</surname><given-names>Li</given-names></name><xref ref-type="author-notes" rid="FNA_number">#</xref></contrib><contrib contrib-type="author"><name><surname>Cai</surname><given-names>Cheng</given-names></name></contrib><contrib contrib-type="author"><name><surname>Gong</surname><given-names>Xiaohui</given-names></name><xref ref-type="corresp" rid="FNA_envelop">✉</xref></contrib></contrib-group><aff>Department of Neonatology, Shanghai Children's Hospital, Shanghai Jiao Tong University, Shanghai, China.</aff><author-notes><corresp id="FNA_envelop">✉ Corresponding author: Xiaohui Gong, E-mail: <email>gongxh@shchildren.com.cn</email>.</corresp><fn fn-type="equal" id="FNA_number"><p># These authors contributed equally to this work</p></fn><fn fn-type="COI-statement"><p>Competing Interests: The authors have declared that no competing interest exists.</p></fn></author-notes><pub-date pub-type="collection"><year>2019</year></pub-date><pub-date pub-type="epub"><day>2</day><month>6</month><year>2019</year></pub-date><volume>15</volume><issue>8</issue><fpage>1571</fpage><lpage>1581</lpage><history><date date-type="received"><day>18</day><month>2</month><year>2019</year></date><date date-type="accepted"><day>2</day><month>5</month><year>2019</year></date></history><permissions><copyright-statement>© Ivyspring International Publisher</copyright-statement><copyright-year>2019</copyright-year><license license-type="open-access"><license-p>This is an open access article distributed under the terms of the Creative Commons Attribution (CC BY-NC) license (<ext-link ext-link-type="uri" xlink:href="https://creativecommons.org/licenses/by-nc/4.0/">https://creativecommons.org/licenses/by-nc/4.0/</ext-link>). See <ext-link ext-link-type="uri" xlink:href="http://ivyspring.com/terms">http://ivyspring.com/terms</ext-link> for full terms and conditions.</license-p></license></permissions><abstract><p>Excessive inflammation induced by various risk factors is associated with the development of bronchopulmonary dysplasia (BPD). Caffeine exerts potent anti-inflammatory effects as a clinical preventive medicine for BPD. Recently, NLRP3 inflammasome activation has been demonstrated to be essential for the pathogenesis of BPD. In the present study, we aimed to investigate the effects of caffeine on NLRP3 inflammasome activation in LPS-induced THP-1 macrophages and to explore the underlying the detailed mechanism. We found that caffeine significantly reduced NLRP3 expression, ASC speck formation, and caspase 1 cleavage and therefore decreased IL-1β and IL-18 secretion in THP-1 macrophages. Caffeine also markedly decreased the phosphorylation levels of MAPK and NF-κB pathway members, further suppressing the translocation of NF-κB in THP-1 macrophages. Moreover, silencing of the caffeine-antagonized adenosine A2a receptor (A2aR) significantly decreased cleaved caspase 1 expression in THP-1 macrophages by reducing ROS production. Given these findings, we conclude that caffeine inhibits NLRP3 inflammasome activation by suppressing MAPK/NF-κB signaling and A2aR-associated ROS production in LPS-induced THP-1 macrophages.</p></abstract><kwd-group><kwd>Caffeine</kwd><kwd>NLRP3 inflammasome</kwd><kwd>MAPK</kwd><kwd>NF-κB</kwd><kwd>A2aR</kwd><kwd>THP-1 macrophages</kwd></kwd-group></article-meta></front><floats-group><fig id="F1" position="float"><label>Figure 1</label><caption><p><bold> Effects of caffeine on THP-1 macrophage survival and apoptosis.</bold> (A) Cell survival was measured in THP-1 macrophages treated with caffeine (100-800 μM) for 6 h using Annexin V-PI staining and flow cytometry. (B) Bar graph showing the survival rates of all cell lines. (C) THP-1 macrophages were treated with caffeine (100-800 μM) for 6 h. The protein levels of cleaved Caspase 3 and Caspase 3 were analyzed by western blotting. (D) Grayscale analysis was used to quantify the relative expression of cleaved Caspase 3 and Caspase 3. The results represent the mean ± SD (n=3).</p></caption><graphic xlink:href="ijbsv15p1571g001"></graphic></fig><fig id="F2" position="float"><label>Figure 2</label><caption><p><bold> Caffeine attenuates production of the cytokines IL-1β and IL-18 in THP-1 macrophages stimulated by LPS and ATP.</bold> (A) IL-1β levels were evaluated in THP-1 macrophages pretreated with caffeine for 1 h and then incubated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. (B) IL-18 levels were evaluated in THP-1 macrophages pretreated with caffeine for 1 h and then incubated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. (C) The mRNA levels of <italic>il-1β</italic> were analyzed by qRT-PCR. (D) The mRNA levels of <italic>il-18</italic> were analyzed by qRT-PCR. The results represent the mean ± SD for three experiments. *** <italic>p</italic>< 0.001 vs. the control group. # <italic>p</italic><0.05, ## <italic>p</italic><0.01 and ### <italic>p</italic>< 0.001 vs. the LPS and ATP group.</p></caption><graphic xlink:href="ijbsv15p1571g002"></graphic></fig><fig id="F3" position="float"><label>Figure 3</label><caption><p><bold> Caffeine inhibits NLRP3 inflammasome activation in THP-1 macrophages stimulated by LPS and ATP/nigericin.</bold> (A) The expression of NLRP3 inflammasome component proteins was evaluated by western blotting in THP-1 macrophages pretreated with caffeine for 1 h and then incubated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. (B) Densitometric analysis was used to quantify the levels of NLRP3 inflammasome protein expression. The mRNA levels of <italic>nlrp3</italic> (C), <italic>asc</italic> (D) and<italic> caspase 1</italic> (E) were analyzed by qRT-PCR. The results represent the mean ± SD for three experiments. *** <italic>p</italic>< 0.001 vs. the control group. # <italic>p</italic><0.05, ## <italic>p</italic><0.01 and ### <italic>p</italic>< 0.001 vs. the LPS and ATP group. (F) ASC speck formation was visualized by immunofluorescence analysis with an anti-ASC (red) antibody in THP-1 macrophages pretreated with caffeine (800 μM) for 1 h and then treated with LPS (1 μg/ml) and nigericin (10 μM) for 90 min. DAPI (blue) was used as a nuclear marker. Scale bar: 50 μm.</p></caption><graphic xlink:href="ijbsv15p1571g003"></graphic></fig><fig id="F4" position="float"><label>Figure 4</label><caption><p><bold> Caffeine inhibits NF-κB activation in THP-1 macrophages stimulated by LPS.</bold> (A) The protein levels of IκB-α, p65 and their phosphorylated forms (P-IκB-α and P-p65) were analyzed by western blotting in THP-1 macrophages pretreated with caffeine for 1 h and then incubated with LPS (1 μg/ml) for 3 h. (B) Densitometric analysis was used to quantify the phosphorylation of IκB-α at Ser32. (C) Densitometric analysis was used to quantify the phosphorylation of p65 at Ser536. The results represent the mean ± SD for three experiments. *** <italic>p</italic>< 0.001 vs. the control group. # <italic>p</italic><0.05, ## <italic>p</italic><0.01, and ### <italic>p</italic>< 0.001 vs. the LPS group. (D) p65 nuclear localization was visualized by immunofluorescence analysis with an anti-p65 (green) antibody in THP-1 macrophages pretreated with caffeine (800 μM) for 1 h followed by LPS (1 μg/ml) for 3 h. DAPI (blue) was used as a nuclear marker. Scale bar: 50 μm.</p></caption><graphic xlink:href="ijbsv15p1571g004"></graphic></fig><fig id="F5" position="float"><label>Figure 5</label><caption><p><bold> Caffeine suppresses MAPK pathway activation in THP-1 macrophages stimulated by LPS.</bold> (A) The protein levels of JNK, ERK, p38, and their phosphorylated forms (P-JNK, P-ERK, and P-p38) were evaluated by western blotting in THP-1 macrophages pretreated with caffeine for 1 h and then incubated with LPS (1 μg/ml) for 3 h. (B) Densitometric analysis was used to quantify the phosphorylation of JNK at Thr183/Tyr185. (C) Densitometric analysis was used to quantify the phosphorylation of ERK1/2 at Thr202/Tyr1204. (D) Densitometric analysis was used to quantify the phosphorylation of p38 at Thr180/Tyr182. The results represent the mean ± SD for three experiments. *** <italic>p</italic>< 0.001 vs. the control group. # <italic>p</italic><0.05, ## <italic>p</italic><0.01, and ### <italic>p</italic>< 0.001 vs. the LPS group.</p></caption><graphic xlink:href="ijbsv15p1571g005"></graphic></fig><fig id="F6" position="float"><label>Figure 6</label><caption><p><bold> Caffeine inhibits A2aR activity to reduce the generation of ROS required for NLRP3 activation in THP-1 macrophages.</bold> (A) The protein levels of A2aR, NLRP3, ASC, Caspase 1 and Caspase 1 (p20) were evaluated by western blotting in THP-1 macrophages preincubated with A2aR-siRNA or negative control (NC)-siRNA for 12 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. (B) Densitometric analysis was used to quantify the levels of A2aR, NLRP3, ASC, Caspase 1 and Caspase 1 (p20). (C) THP-1 macrophages were preincubated with A2aR-siRNA or NC-siRNA for 12 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. ROS production was detected by flow cytometry. (D) Densitometric analysis was used to quantify the levels of ROS. (E) The protein levels of A2aR were evaluated by western blotting in THP-1 macrophages pretreated with caffeine for 1 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. (F) The mRNA levels of <italic>a2ar</italic> were detected by qRT-PCR. (G) Densitometric analysis was used to quantify the levels of A2aR protein expression. (H) THP-1 macrophages were preincubated with caffeine for 1 h and then treated with LPS (1 μg/ml) for 3 h and ATP (5 mM) for 30 min. ROS production was detected by flow cytometry. (I) Densitometric analysis was used to quantify the levels of ROS. The results represent the mean ± SD for three experiments. *** <italic>p</italic>< 0.001 vs. the control group. #<italic> p</italic><0.05, ## <italic>p</italic><0.01, and ### <italic>p</italic>< 0.001 vs. the LPS and ATP group.</p></caption><graphic xlink:href="ijbsv15p1571g006"></graphic></fig><fig id="F7" position="float"><label>Figure 7</label><caption><p>Schematic diagram showing the effect of caffeine on LPS-stimulated NLRP3 inflammasome activation in THP-1 macrophages.</p></caption><graphic xlink:href="ijbsv15p1571g007"></graphic></fig><table-wrap id="T1" position="float"><label>Table 1</label><caption><p>Primers used in this study</p></caption><table frame="hsides" rules="groups"><thead valign="top"><tr><th rowspan="1" colspan="1">Gene</th><th rowspan="1" colspan="1">GenBank accession no.</th><th rowspan="1" colspan="1">Primer sequence (5'-3')</th><th rowspan="1" colspan="1">Product size (bp)</th></tr></thead><tbody valign="top"><tr><td rowspan="1" colspan="1">GAPDH</td><td rowspan="1" colspan="1">NM_001115114.1</td><td rowspan="1" colspan="1">F-GGACTCATGACCACAGTCCA<break></break>R-TCAGCTCAGGGATGACCTTG</td><td rowspan="1" colspan="1">157</td></tr><tr><td rowspan="1" colspan="1">NLRP3</td><td rowspan="1" colspan="1">NM_001079821.2</td><td rowspan="1" colspan="1">F-AACATGCCCAAGGAGGAAGA<break></break>R-GGCTGTTCACCAATCCATGA</td><td rowspan="1" colspan="1">115</td></tr><tr><td rowspan="1" colspan="1">Caspase 1</td><td rowspan="1" colspan="1">NM_001257118.3</td><td rowspan="1" colspan="1">F-GCACACGTCTTGCTCTCATT<break></break>R-GCCTCCAGCTCTGTAGTCAT</td><td rowspan="1" colspan="1">169</td></tr><tr><td rowspan="1" colspan="1">ASC</td><td rowspan="1" colspan="1">NM_013258.5</td><td rowspan="1" colspan="1">F-GTCACAAACGTTGAGTGGCT<break></break>R-AAGTCCTTGCAGGTCCAGTT</td><td rowspan="1" colspan="1">152</td></tr><tr><td rowspan="1" colspan="1">IL-1β</td><td rowspan="1" colspan="1">NM_000576.2</td><td rowspan="1" colspan="1">F-TGCTCAAGTGTCTGAAGCAG<break></break>R-TGGTGGTCGGAGATTCGTAG</td><td rowspan="1" colspan="1">189</td></tr><tr><td rowspan="1" colspan="1">IL-18</td><td rowspan="1" colspan="1">NM_001562.4</td><td rowspan="1" colspan="1">F-TCGGGAAGAGGAAAGGAACC<break></break>R-TTCTACTGGTTCAGCAGCCA</td><td rowspan="1" colspan="1">96</td></tr><tr><td rowspan="1" colspan="1">A2aR</td><td rowspan="1" colspan="1">NM_000675.6</td><td rowspan="1" colspan="1">F-AGGCAGCAAGAACCTTTCAA<break></break>R-CTAAGGAGCTCCACGTCTGG</td><td rowspan="1" colspan="1">236</td></tr></tbody></table></table-wrap></floats-group></pmc><affiliations><list></list><tree><noCountry><name sortKey="Cai, Cheng" sort="Cai, Cheng" uniqKey="Cai C" first="Cheng" last="Cai">Cheng Cai</name><name sortKey="Gong, Xiaohui" sort="Gong, Xiaohui" uniqKey="Gong X" first="Xiaohui" last="Gong">Xiaohui Gong</name><name sortKey="Ma, Li" sort="Ma, Li" uniqKey="Ma L" first="Li" last="Ma">Li Ma</name><name sortKey="Zhao, Weiming" sort="Zhao, Weiming" uniqKey="Zhao W" first="Weiming" last="Zhao">Weiming Zhao</name></noCountry></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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