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Monocytes Production in Β -Induced IL-1 Δ Kinase C Associated Kinase-1

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Monocytes Production in Β -Induced IL-1 Δ Kinase C Associated Kinase-1 IL-1R−Associated Kinase-1 Mediates Protein Kinase C δ-Induced IL-1β Production in Monocytes This information is current as Rajiv Lochan Tiwari, Vishal Singh, Ankita Singh and Manoj of October 1, 2021. Kumar Barthwal J Immunol published online 29 July 2011 http://www.jimmunol.org/content/early/2011/07/29/jimmun ol.1002526 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2011/07/29/jimmunol.100252 Material 6.DC1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 29, 2011, doi:10.4049/jimmunol.1002526 The Journal of Immunology IL-1R–Associated Kinase-1 Mediates Protein Kinase Cd-Induced IL-1b Production in Monocytes Rajiv Lochan Tiwari, Vishal Singh, Ankita Singh, and Manoj Kumar Barthwal The role of IL-1R–associated kinase (IRAK)1 and its interaction with protein kinase C (PKC)d in monocytes to regulate IL-1b production has not been reported so far. The present study thus investigates such mechanisms in the THP1 cell line and human monocytes. PMA treatment to THP1 cells induced CD11b, TLR2, TLR4, CD36, IRAK1, IRAK3, and IRAK4 expression, IRAK1 kinase activity, PKCd and JNK phosphorylation, AP-1 and NF-kB activation, and secretory IL-1b production. Moreover, PMA- induced IL-1b production was significantly reduced in the presence of TLR2, TLR4, and CD11b Abs. Rottlerin, a PKCd-specific inhibitor, significantly reduced PMA-induced IL-1b production as well as CD11b, TLR2 expression, and IRAK1–JNK activation. In PKCd wild-type overexpressing THP1 cells, IRAK1 kinase activity and IL-1b production were significantly augmented, whereas recombinant inactive PKCd and PKCd small interfering RNA significantly inhibited basal and PMA-induced IRAK1 activation and IL-1b production. Endogenous PKCd–IRAK1 interaction was observed in quiescent cells, and this interaction was Downloaded from regulated by PMA. IRAK1/4 inhibitors, their small interfering RNAs, and JNK inhibitor also attenuated PMA-induced IL-1b production. NF-kB activation inhibitor and SN50 peptide inhibitor, however, failed to affect PMA-induced IL-1b production. A similar role of IRAK1 in IL-1b production and its regulation by PKCd was evident in the primary human monocytes, thus signifying the importance of our finding. To our knowledge, the results obtained demonstrate for the first time that IRAK1 and PKCd functionally interact to regulate IL-1b production in monocytic cells. A novel mechanism of IL-1b production that involves TLR2, CD11b, and the PKCd/IRAK1/JNK/AP-1 axis is thus being proposed. The Journal of Immunology, 2011, 187: 000–000. http://www.jimmunol.org/ nterleukin-1b is an important proinflammatory cytokine that aa region in their cytoplasmic tail called the Toll-IL-1R domain, has a role in varied types of diseases, including type 1 and which plays an important role in inducing the signals from this I type 2 diabetes, atherosclerosis, metabolic syndrome, and receptor (9). Consequently, signaling induced by these receptors is autoimmune diseases such as rheumatoid arthritis and inflam- very similar and culminates into activation of the NF-kB and JNK/ matory bowel disease (1–3). IL-1b is induced by TLRs or by AP-1 pathways (9–11). IL-1b induces the activation of NF-kBand cytokines such as TNF or IL-1b itself (2, 3). Before secretion, the JNK pathways by recruiting MyD88, IRAK1, TNFR-associated inactive form of the cytokine is processed by caspase-1, leading factor 6, and TGF-b–activated kinase 1 (1, 9). IL-1b–induced by guest on October 1, 2021 to its maturation and secretion (2, 3). Blood monocytes, tissue JNK and NF-kB activation diverges at IRAK (12). The IRAK macrophages, and dendritic cells are the major source of IL-1b; family consists of four members, namely IRAK1, IRAK2, IRAK3 however, NK cells and B lymphocytes also produce this cytokine (IRAKM), and IRAK4. IRAK1, IRAK2, and IRAK4 positively (1). Monocyte-derived resident macrophages often play a crucial regulate the immune response, and IRAKM usually antagonizes role in the generation of inflammatory responses in atherosclerosis their effect by disrupting the IRAK1/TNFR-associated factor 6 (4) and diabetes (1–3). Cytokines such as IL-1b secreted by these complex (7, 9). Out of all of these kinases, IRAK1 and IRAK4 are cells often amplify the inflammatory response leading to lesion more worked-out proteins and are said to be true kinases, although progression and b cell death. IL-1b Ab is positioned for the their kinase activity is still under investigation (9). IRAK4 activates treatment of such disorders (5). IRAK1 by phosphorylating Thr387 and Ser376 present in the latter The IL-1R–associated kinase (IRAK) family of proteins repre- activation loop (13). Both IRAKM and IRAK2 are predicted to be sents important mediators of innate immunity and plays a crucial inactive kinases (9, 14), although recent work has shed more light role in the signaling cascade induced by the TLR/IL-1R family (6– on IRAK2 activity and its role in TLR signaling (14, 15). IRAK1- 9). Members of the TLR and IL-1R family possess a common 200- deficient macrophages and fibroblasts show impaired cytokine pro- duction in response to LPS and IL-1b (8, 16, 17). Division of Pharmacology, CSIR-Central Drug Research Institute, Council of Scien- Although previous studies demonstrate the role of IRAK1 in tific and Industrial Research, Lucknow 226 001, India TLR/IL-1R–induced signaling events (17), its role and regulation Received for publication July 28, 2010. Accepted for publication June 24, 2011. during IL-1b production are not known. It has been demonstrated This work was supported by the Department of Science and Technology, India (to that IRAK1 interacts and phosphorylates PKCi and regulates the M.K.B.) and by Fellowships from the Indian Council of Medical Research (to V.S.), NF-kB pathway (18). At the same time, PKCz also plays an im- the Council of Scientific and Industrial Research (to R.L.T.), and the University Grants Commission (to A.S.). This is CDRI communication number 8090. portant role in endotoxin-induced macrophage activation by acti- vating the TLR4/IRAK1 pathway (19). Furthermore, previous Address correspondence and reprint requests to Dr. Manoj Kumar Barthwal, Division of Pharmacology, CSIR-Central Drug Research Institute, 1 M.G. Marg, Lucknow 226 studies suggest a role of PKCd in IL-1b production from mono- 001, India. E-mail address: [email protected] cytes (20), but its interaction with IRAK1 is not known. Rottlerin, The online version of this article contains supplemental material. a PKCd-specific inhibitor, affects signaling events and cytokine Abbreviations used in this article: EGFP, enhanced GFP; INH, inhibitor; IRAK, IL- production in human monocytes stimulated by PMA and LPS 1R–associated kinase; MBP, myelin basic protein; PKC, protein kinase C; siRNA, (20). Therefore, the present study was undertaken to elucidate the small interfering RNA; WT, wild-type. role of the IRAK family of proteins in IL-1b production. At the Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 same time, we checked the hypothesis that PKCd could regulate www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002526 2 IRAK1 REGULATES IL-1b PRODUCTION Downloaded from FIGURE 1. PMA induces time-dependent CD11b, TLR2, TLR4, and CD36 expression and IL-1b production. A, THP1 cells were stimulated with PMA http://www.jimmunol.org/ for different times and IL-1b production was measured in culture media by ELISA. B, CD11b, (C) TLR2, (D) TLR4, and (E) CD36 expression on THP1 cells was measured by flow cytometry after PMA stimulation for 24 and 72 h. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control; ##p , 0.01, ###p , 0.001. IRAK1 for IL-1b production. In this study we report the role of regulated by PKCd. With this finding IRAK1 can be positioned as IRAK family members in PMA-induced IL-1b production and an attractive target for regulating IL-1b production in various a novel IRAK1-mediated pathway of IL-1b production that is metabolic and autoimmune disorders. by guest on October 1, 2021 FIGURE 2. Time-dependent expression of IRAKs. A, THP1 cells stimulated with PMA for different time points were analyzed for IRAK1, IRAK2, IRAK3, and IRAK4 expressions by Western blotting. B, Densitometric analysis of the expressed IRAK isoforms in relative image quant units. C, IRAK1 kinase activity measured in an in vitro kinase assay at different times of PMA treatment. Cells were lysed, and immunoprecipitated IRAK1 was subjectedto kinase assay in the presence of [g-32P]ATP and MBP as substrate. Values represent means 6 SE. *p , 0.05, **p , 0.01, ***p , 0.001 versus control. The Journal of Immunology 3 Materials and Methods Human monocyte isolation, cell culture, and treatments Materials Human primary circulating monocytes were isolated as described earlier (21) with slight modification from healthy donors after their informed Pharmacological inhibitors, including IRAK1/4 inhibitor (INH), JNK INH 3 II, rottlerin, NF-kB activation INH, SN50, Go6976, Ro-31-8220, caspase- consent.
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