CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein− Coupled Receptor CXCR2 This information is current as Monoranjan Boro and Kithiganahalli Narayanaswamy Balaji of October 1, 2021. J Immunol published online 24 July 2017 http://www.jimmunol.org/content/early/2017/07/22/jimmun ol.1700129 Downloaded from Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision http://www.jimmunol.org/ • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription by guest on October 1, 2021 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 © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 24, 2017, doi:10.4049/jimmunol.1700129 The Journal of Immunology CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation via G-Protein–Coupled Receptor CXCR2 Monoranjan Boro and Kithiganahalli Narayanaswamy Balaji Inflammation is an extensively concerted process that confers protection to the host encountering immune insult. The major inflammatory mediators include IL-1 family members, such as IL-1b, and the functional activation of such molecules is arbitrated by their regulated cleavage brought about by components of a multiprotein complex called inflammasome. In this context, NLR family pyrin domain containing 3 (NLRP3) inflammasome activation often acts as a rate-limiting step in regulating critical cell- fate decisions in various inflammatory scenarios. In this study, we identify the G-protein–coupled receptor CXCR2 (recognizing chemokines CXCL1 and CXCL2) as another arm feeding into the regulated activation of NLRP3 inflammasome in macro- phages. We demonstrate that in vivo blocking of CXCL1 and CXCL2 can significantly reduce the Mycobacterium tuberculosis– induced bioactive IL-1b production. Further, CXCL1 could amplify the inflammasome activation in in vivo mouse models of carrageenan-induced inflammation in footpads and air pouches. The mechanistic insights revealed CXCR2-driven protein kinase Downloaded from C m–dependent integrin-linked kinase to be essential for CXCL1-mediated activation of NLRP3 inflammasome. Blocking the activity of integrin-linked kinase or protein kinase C m either by small interfering RNA–mediated knockdown or pharmacological inhibitor compromised inflammasome activation and subsequent production of bioactive IL-1b. Taken together, our study demonstrates CXCR2-driven activation of NLRP3 inflammasome in macrophages and indicates a potential host-directed thera- peutic target to limit the damaging inflammation associated with overt production of proinflammatory IL-1b. The Journal of Immunology, 2017, 199: 000–000. http://www.jimmunol.org/ hemokines, as inflammatory mediators, contribute sig- been implicated in the differentiation of T cells and in modu- nificantly in effectuating immune responses toward an im- lating the magnitude and cytokine polarity of T cell responses C munity breach (1–6). As known conventionally, chemokines (9). CXCL1 has been reported to be important for Th17 differ- have their role in orchestrating a concerted recruitment of immune entiation, and has been found to be essential for reactive oxygen cells to the site of inflammation or injury. Importantly, chemokines species production and neutrophil extracellular trap formation such as CXCL8 direct the recruitment of neutrophils whereas other (10). One of the cardinal responses to foreign particles or en- chemokines like CCL2, RANTES, and CXCL1 are known potent dogenous danger signals is constituted by the assembly and ac- by guest on October 1, 2021 chemoattractants for T cells, monocytes, and neutrophils, respectively tivation of inflammasome. (7). Apart from their role in immune cell recruitment, mounting Inflammasomes are multiprotein complexes that mediate the evidence implicates various chemokines in processes of cellular innate immune response by generating effectors of the proin- homeostasis. For instance, CCL2 has been reported to negatively flammatory IL-1 family of cytokines such as IL-1b and IL-18 (11). regulate processes including autophagy and necrosis in luminal Many pattern recognition receptors are integral to inflammasome B breast cancer cells (8). In the same manner, CCL2 has also assembly. Some of these include NOD-like receptors and absent in melanoma 2–like receptors (12). Cognate stimulation leads to their activation, oligomerization, and formation of a CASPASE- Department of Microbiology and Cell Biology, Indian Institute of Science, activating scaffold containing apoptosis-associated speck-like pro- Bangalore 560012, India tein containing a CARD (ASC). Canonically, activated CASPASE1 Received for publication January 26, 2017. Accepted for publication June 21, 2017. subsequently cleaves the proinflammatory cytokines pro–IL-1b and This work was supported by funds from the Department of Biotechnology and the Department of Science and Technology (under the Government of India). Infrastruc- pro–IL-18 to their respective bioactive forms (13). Hence, these ture support was provided by the Indian Council of Medical Research (Center for inflammasomes form a major arm of innate immune effectors Advanced Study in Molecular Medicine), the Department of Science and Technology and their dysfunction has been increasingly linked to a variety of (Fund for Improvement of Science and Technology Infrastructure in Universities and Higher Educational Institutions), and the University Grants Commission (special autoinflammatory and autoimmune diseases like Alzheimer’s dis- assistance to K.N.B.). K.N.B. is a J.C. Bose National Fellow of the Department of ease, atherosclerosis, and so on (14–18). Because chemokines also Science and Technology, New Delhi, India. M.B. is a recipient of a fellowship from play an important role in major host inflammatory responses and the Department of Biotechnology. cardinal immune mechanisms, their possible fine regulation of M.B. and K.N.B. designed and performed experiments and analyzed data; K.N.B. wrote the manuscript and supervised the study. the inflammasome appeared intriguing. Address correspondence and reprint requests to Dr. Kithiganahalli Narayanaswamy Among the different inflammasomes, NLR family pyrin domain Balaji, Department of Microbiology and Cell Biology, Indian Institute of Science, containing 3 (NLRP3) inflammasome responds to many different Bangalore 560012, Karnataka, India. E-mail address: [email protected] stimuli (19–21). In this study, to our knowledge it was found for Abbreviations used in this article: ASC, apoptosis-associated speck-like protein con- the first time that the chemokines CXCL1 and CXCL2 could lead taining a CARD; BMDM, bone marrow–derived macrophage; CGN, carrageenan; GPCR, G-protein–coupled receptor; ILK, integrin-linked kinase; MSU, monosodium to the activation of NLRP3 inflammasome in macrophages. This urate; NLRP3, NLR family pyrin domain containing 3; PKC, protein kinase C; PM, effect was mediated by their interaction with their cognate receptor peritoneal macrophage; qRT-PCR, quantitative real-time PCR; siRNA, small inter- CXCR2. Further insights into the molecular mechanism shed light fering RNA. on the role of protein kinase C (PKC) m integrin-linked kinase Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$30.00 (ILK) in transducing the signal from CXCR2 to CASPASE1 and www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700129 2 CXCR2 ORCHESTRATES THE ACTIVATION OF NLRP3 INFLAMMASOME subsequent IL-1b maturation. Because dysregulated inflamma- (250-15) were purchased from PeproTech. Non-targeting small interfering somes may lead to overt inflammation and physiological pathology, RNA (siRNA) (D-001210-01-20), Nlrp3 (M-053455-01-0005), Ilk (M-040115- m delineation of the molecular mechanisms contributing to its acti- 00-0005), Cxcr2 (M-042157-01-0005), and Prkd1/Pkc (M-048415-01-0005) siRNAs were obtained from Dharmacon as siGENOME SMART-pool reagents. vation and modulation demands attention. In this regard, uncover- Anti–b-actin (A3854) Ab was purchased from Sigma-Aldrich and HRP- ing pivotal components in the arsenal of inflammasome regulation conjugated anti-rabbit (111-035-045)/anti-mouse (115-035-003) IgG Abs may pave the way for the development of advanced therapeutic were procured from Jackson ImmunoResearch. Anti-ASC (67824), anti– b strategies to control hyperinflammation and associated disorders. IL-1 (12242), anti-CASPASE1 (3866), anti-ILK (3862), anti-phospho PKCm (Ser916) (2051), and anti-PKCm (2052) Abs were obtained from Cell Signaling Technology. Anti-phospho ILK (Ser 246) (AB1076) Ab Materials and Methods was purchased from Merck-Millipore and anti-NLRP3 (66846) Ab was Cells and mice obtained from Santa Cruz Biotechnology. CXCL1-neutralizing (AF-453- NA) and CXCL2-neutralizing (AF-452-NA) Abs were purchased from Male or female 4 wk old BALB/c mice were injected i.p. with 1 ml of 8% R&D Systems. Goat IgG (02-6202) isotype control Ab was procured Brewer thioglycollate and peritoneal macrophages (PMs)