CXCL1 and CXCL2 Regulate NLRP3 Inflammasome Activation Via G-Protein–Coupled Receptor CXCR2

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

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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 macrophages. 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 therapeutic 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) were isolated in from Thermo Fisher Scientific and L chain–specific anti-rabbit IgG (211- sterile ice-cold PBS 4 d post injection. Adherent cells were used as 032-171) was purchased from Jackson ImmunoResearch. macrophages for in vitro experiments. Bone marrow–derived cells were isolated from femurs of 4 wk old male or female BALB/c mice and were then differentiated into bone marrow–derived macrophages (BMDMs) by In vitro and in vivo treatment with pharmacological reagents culturing with 30% culture supernatant obtained from L-929 fibroblast and recombinant proteins cells. The purity of PMs and BMDMs was checked by staining for F4/80 . In vitro. The pharmacological reagents were obtained from Calbiochem or using FACS and was found to be 95% pure. Transfection experiments Sigma-Aldrich and used at the following working concentrations: MCC950/ were carried out in mouse PMs. All cells were cultured in DMEM (Life NLRP3 inhibitor (10 mM) (PZ0280) was obtained from Sigma-Aldrich, Technologies) supplemented with 10% heat-inactivated FBS (Gibco Life PKCa/Safingol inhibitor (10 mM) (559300), PKCb inhibitor/3-[1-(3- Downloaded from Technologies). Mice were purchased from the Jackson Laboratory and Imidazol-1-ylpropyl)-1H-indol-3-yl]-4-anilino-1H-pyrrole-2,5-dione (10 nM) maintained in the Central Animal Facility, Indian Institute of Science. All (539654), PKCd inhibitor/Rottlerin (6 mM) (557370), PKCε inhibitor (50 studies involving mice were performed after the approval from the Insti- mM) (539522), PKCz inhibitor/ PKCz pseudosubstrate inhibitor (50 mM) tutional Ethics Committee for animal experimentation as well as from (539624), PKD/PKCm inhibitor (280 nM) (476495), CASPASE1 inhibitor/ Institutional Biosafety Committee. Ac-YVAD-AOM (10 mM) (400015), CXCR2 antagonist/3-[2-(cyclo- Reagents and Abs pentylamino)-3,4-dioxocyclobut-1-enylamino]-2-hydroxy-N,N-dimethylbenza-

mide (145 nM) (239819), and ILK inhibitor/Cpd 22 (600 nM) (407331) http://www.jimmunol.org/ All the general reagents were purchased from Sigma-Aldrich, Merck. were obtained from Calbiochem. DMSO at a 0.1% concentration was used Carrageenan (CGN) (Sc-216084) was purchased from Santa Cruz Bio- as the vehicle control. A tested concentration of each inhibitor was used technology. Monosodium urate (MSU) (U2875) and ATP (A6144) were after careful titration experiments assessing the viability of the macrophages obtained from Sigma-Aldrich, LPS-B5 ultrapure (tlrl-pb5lps) was obtained using MTT assay. Cells were pretreated with the concerned pharmacologi- from InvivoGen. CCL5 (478-MR/CF) and CCL2 (479-JE) were obtained cal reagents for 1 h prior to the requisite experimental subjugations. The from R&D Systems. Murine recombinant CXCL1 (250-11) or CXCL2 in vitro working concentrations of CXCL1 and CXCL2 were 50 ng/ml. by guest on October 1, 2021

FIGURE 1. CXCL1 and CXCL2 trigger inflammasome activation in macrophages. Macrophages were treated as described. (A) Mouse PMs were treated with CXCL1 (50 ng/ml) or CXCL2 (50 ng/ml) for 4 h to assess the expression of the indicated genes by qRT-PCR. (B and C) LPS-primed PMs (B) or LPS- primed BMDMs (C) were treated with CXCL1 (50 ng/ml), CXCL2 (50 ng/ml) for 4 h, or ATP (5 mM) for 1 h, or MSU (100 mg/ml) for 6 h to assess the IL- 1b in the culture supernatant by ELISA. (D) LPS-primed PMs and BMDMs were treated with CXCL1 (50 ng/ml), CXCL2 (50 ng/ml), CCL2 (100 ng/ml), or CCL5 (100 ng/ml) for 4 h, or ATP (5 mM) for 1 h or MSU (100 mg/ml) for 6 h to assess the status of inflammasome activation by immunoblotting for cleaved CASPASE1 and IL-1b on total cell lysate. b-Actin was used as loading control. Blots are representative of three independent experiments. (E) Analysis of ASC speck by confocal microscopy of LPS-primed mouse PMs treated with CXCL1 (50 ng/ml) or CXCL2 (50 ng/ml) for 4 h. Blue stained by DAPI indicates nuclei, green indicates ASC speck. (F) Quantification of (E) from three independent experiments. (A–C and F) Level of significance was measured by performing Student paired t test form three independent experiments (*p , 0.05, **p , 0.005). Med, medium; ns, not significant. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ FIGURE 2. CASPASE1 and NLRP3 are involved in CXCL1- and CXCL2-induced inflammasome activation in macrophages. (A) LPS-primed PMs and BMDMs were pretreated with Ac-YVAD-AOM (10 mM) for 1 h followed by treatment with CXCL1 (50 ng/ml) or CXCL2 (50 ng/ml) for 4 h to assess the IL-1b maturation by immunoblotting. (B) Densitometric analysis of cleaved IL-1b showing level of significance was performed by using one-way ANOVA followed by Bonferroni multiple comparison test of experiment (A) in PMs from three independent experiments. (C) LPS-primed PMs and BMDMs were pretreated with MCC950 (10 mM) for 1 h followed by treatment with CXCL1 (50 ng/ml) or CXCL2 (50 ng/ml) for 4 h to assess the inflammasome status as indicated by immunoblotting. (D) Nlrp3 was knocked down in mouse PMs by using Nlrp3 siRNA to assess the inflammasome activation in response to treatment with CXCL1 or CXCL2 by immunoblotting. (E and F) Densitometric analysis of NLRP3 showing level of significance was performed by using one-way ANOVA followed by Bonferroni multiple comparison test of experiment (D) from three independent experiments. b-Actin was used as loading control. All immunoblotting experiments were performed on the total cell lysate. Immunoblots are representatives from three independent experiments. **p , by guest on October 1, 2021 0.005, ***p , 0.0001. NT, nontargeting.

In vivo. BALB/c 4 wk old mice were intravenously injected with CXCR2 Immunoblotting antagonist (5 mg/kg), ILK inhibitor (5 mg/kg), or PKCm inhibitor (10 mg/kg), and kept for 12 h followed by i.v. injection of CXCL1 (500 ng per 20 g of Treated or untreated cells were lysed in radioimmunoprecipitation assay mouse body weight) as indicated. After 36 h mice were sacrificed to isolate buffer containing 50 mM Tris-HCl (pH 7.4), 1% NP-40, 0.25% sodium m the lymph nodes, spleens, and lungs. These organs were then processed deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 g/ml each of and used for experiments. Each experimental group had five to six mice. aprotinin, leupeptin, and pepstatin, 1 mM Na3VO4, and 1 mM NaF. An equal amount of protein from each sample was loaded and resolved on a Transient transfection studies 12% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (Millipore) by the semidry transfer (Bio-Rad) method. Nonspecific Mouse PMs were transfected with the relevant siRNAs (150 nM) for 24 h by binding was blocked with 5% nonfat dry milk powder in TBST (20 mM using low m.w. polyethylenimine (Sigma-Aldrich), followed by experi- Tris-HCl [pH 7.4], 137 mM NaCl, 0.1% Tween 20) for 60 min. The blots mental treatments. Transfection efficiency was more than 50% in all ex- were incubated overnight at 4˚C with primary Ab, followed by probing periments as determined by counting the number of siGLOLamin A/C with goat anti-rabbit/anti-mouse HRP-conjugated secondary Ab in 5% nonfat positive cells in a microscopic field using a fluorescence microscope. milk for 2 h. After washing in TBST, the immunoblots were developed with Knock-down efficiency was confirmed by immunoblotting for the re- an ECL detection system (PerkinElmer) as per the manufacturer’s protocol. spective molecules. Immunoprecipitation assay Quantitative real-time PCR Immunoprecipitation assays were carried out following a modified version Total RNA was isolated from untreated or CXCL1- or CXCL2-treated of the protocol provided by Millipore. In brief, LPS-primed macrophages mouse PMs by TRI reagent (Sigma-Aldrich). Two micrograms of RNA treated with CXCL1 or PBS were gently suspended and lysed in ice-cold was converted into cDNA by using a first-strand cDNA synthesis kit radioimmunoprecipitation assay buffer. The cell lysates obtained were (Bioline). Quantitative real-time PCR (qRT-PCR) was performed using a incubated with IgG or anti-ILK Ab for 2 h at 4˚C. The immune complexes SYBR Green PCR mix (Kapa Biosystems) for the quantification of tar- were captured on protein A agarose beads at 4˚C for 4 h. The beads were get gene expression. Expression of Gapdh was used as the normalization separated, washed, and boiled in Laemmli buffer for 10 min. These bead- control for qRT-PCR analysis. The primer sequences used for the current free samples were analyzed for respective target molecules by immuno- study are as follows: Nlrp3 forward 59-ATCAACAGGCGAGACCTCTG- blotting after separation by SDS-PAGE. L chain–specific secondary Ab 39, Nlrp3 reverse 59-GTCCTCCTGGCATACCATAGA-39; Pycard for- was used for immunoblotting after immunoprecipitation. ward 59-GACAGTGCAACTGCGAGAAG-39, Pycard reverse 59-CGA- CTCCAGATAGTAGCTGACAA-39; Caspase1 forward 59-CTTGGAGAC- Inflammasome assay in pharmacological agent or ATCCTGTCAGGG-39; Caspase1 reverse 59-AGTCACAAGACCAGGC- inhibitor-treated macrophages ATATTCT-39; Il-1b forward 59-GAAATGCCACCTTTTGACAGTG-39, Il-1b reverse 59-TGGATGCTCTCATCAGGACAG-39; Gapdh forward 59-GAGC- Mouse PMs and BMDMs were primed with LPS (100 ng/ml) for 3 h. CAAACGGGTCATCATCT-39, Gapdh reverse 59- GAGGGGCCATCCA- Medium was removed and replaced with fresh DMEM containing inhibitors CAGTCTT-39. for CASPASE1, NLRP3, ILK, PKCm, or CXCR2 antagonist. After 1 h 4 CXCR2 ORCHESTRATES THE ACTIVATION OF NLRP3 INFLAMMASOME Downloaded from http://www.jimmunol.org/ FIGURE 3. CXCR2 is required for NLRP3 inflammasome activation by CXCL1 in macrophages. (A) Expression of CXCR2 was checked by immunoblotting of total cell lysates obtained from CXCL1 (50 ng/ml) treated PMs or BMDMs, (B) LPS-primed mouse PMs and BMDMs were pretreated with CXCR2 antagonist (145 nM) for 1 h followed by treatment with CXCL1 (50 ng/ml) for 4 h, ATP (5 mM) for 1 h, or MSU (100 mg/ml) for 6 h to assess the maturation of pro-CASPASE1 and pro–IL-1b by immunoblotting. (C and D) The culture supernatants harvested from experiment (B) were used to detect active IL-1b by ELISA. (E) Cxcr2 was knocked down in mouse PMs by using Cxcr2 siRNA, followed by the indicated treatment with CXCL1 to assess the maturation of pro-CASPASE1 and pro–IL-1b by immunoblotting. (F) Densitometric analysis of CXCR2 showing level of significance was performed by using one-way ANOVA followed by Bonferroni multiple comparison test of experiment (E) from three independent experiments (*p , 0.05, **p , 0.005, ***p , 0.0001). (G) The culture supernatants harvested from experiment (E) were used for performing ELISA to detect mature IL-1b. b-Actin was used as loading control. All immunoblotting experiments were performed on the total cell lysate. Blots are representative of three independent experiments. The

ELISA data represent the mean 6 SE (n = 3), **p , 0.005, ***p , 0.0001 (one-way ANOVA followed by Bonferroni multiple comparison test). anta, by guest on October 1, 2021 antagonist; ns, not significant; NT, nontargeting. these cells were treated with CXCL1 (50 ng/ml) or CXCL2 (50 ng/ml) for venously infected with 1000 CFU of Mycobacterium tuberculosis.After 4 h, ATP (5 mM) for 1 h, or MSU (100 mg/ml) for 6 h. 36 h, mice were sacrificed to isolate lymph nodes, spleens, and lungs to perform the respective experiments. For vehicle control, 10 mg isotypic ASC speck staining IgG Ab was used. Control mice were injected with PBS. Each experimental group had four mice. LPS-primed mouse PMs were treated with CXCL1 or CXCL2 for 4 h followed by treatment with 4% para formaldehyde for 15 min and blocking with 5% BSA in 13 PBS for 1 h at room temperature. Following this, In vivo CGN model of inflammation in footpads and air fixed cells were treated with anti-ASC Ab in 0.2% saponin and kept over- pouches night at 4˚C. After staining with anti-ASC Ab, cells were stained with Footpads. BALB/c 4 wk old mice were intravenously injected with CXCR2 DyLight 488 conjugated secondary Ab. DAPI was used to stain the nu- antagonist (5 mg/kg) or CASPASE1 inhibitor (50 mg/kg) and kept for 12 h, clei. Confocal images (Z-stacks) were taken on a Zeiss LSM 710 Meta followedbycoinjectionof25ml of 1% CGN and 500 ng of CXCL1 into confocal laser scanning microscope (Carl Zeiss) using a plan-Apochromat the footpad. After 36 h mice were sacrificed to isolate the footpad ex- 633/1.4 Oil DIC objective (Carl Zeiss), and images were analyzed using udate for studying inflammasome activation by immunoblotting. Control ZEN2 software. mice were injected with CGN alone. Each experimental group had six mice. ELISA Air pouches. To generate air pouches, 5 ml sterile air was injected s.c. in the Sandwich ELISA was performed in 96-well microtiter plates (Nunc, back of each mouse. The pouches were allowed to settle for 3 d to permit the Denmark) using cell-free supernatants of respectively treated samples. healing of the wound. The pouches were then reinjected with 5 ml of sterile CXCL1 (900-K127) and CXCL2 (900-K152) ELISA kits were purchased air. Meanwhile, these mice were intravenously injected with CXCR2 an- from PeproTech and the IL-1b ELISA kit (559603) was obtained from BD tagonist (5 mg/kg) or CASPASE1 inhibitor (50 mg/kg). After 12 h, 25 mlof Biosciences. ELISA was performed according to the manufacturer’s in- 1% CGN and 500 ng of CXCL1 were coinjected into the air pouch and left structions. Briefly, assay plates were coated with the capture Ab at 4˚C for 36 h, followed by isolation of air pouch exudate in ice-cold PBS for overnight. After blocking with 1% BSA for 1 h at room temperature, wells studying inflammasome activation by immunoblotting. Control mice were were incubated with cell-free supernatant for 2 h followed by probing injected with CGN alone. Each experimental group had six mice. with biotinylated detection Ab for 2 h at 37˚C. After incubation with streptavidin-HRP for 1 h at 37˚C, reactions were developed with 3, 39,5, Densitometric analysis of immunoblots 59-tetramethylbenzidine (Promega). The absorbance was measured at The intensity of the band from each lane was obtained after subtracting the 450 nm using an ELISA reader (Molecular Devices). background intensity. Following this, the band intensity of the target protein In vivo neutralization study was normalized with the band intensity of loading control b-actin. To measure the intensity of phosphorylated proteins, normalization was per- BALB/c 4 wk old mice were intravenously injected with 10 mg each of formed by their respective total proteins as shown in the figures. Further, CXCL1- and CXCL2-neutralizing Ab. After 3 h, these mice were intra- the intensity of the band from experimental treatments were calculated as The Journal of Immunology 5 fold change over control. All the densitometric analysis was performed by induced the expression of Nlrp3 and Il-1b (Fig. 1A). However, using ImageQuant software from GE Healthcare Life Sciences. the expression of Asc and Caspase1 remained unaltered (Fig. 1A). Statistical analysis Because the inflammasome-dependent generation of active IL- 1b requires the priming of macrophages with LPS, we primed The normality of the data was checked by the Shapiro–Wilk normality test. mouse PMs and BMDMs with LPS before analyzing the possible Based on the Shapiro–Wilk test (p . 0.05) and the visual analysis of their histograms, normal Q-Q plots showed that our data are normally distrib- inflammasome activation by CXCL1, CXCL2, or other well-known uted. The Shapiro–Wilk test was performed using IBM-SPSS software. inflammasome activators such as ATP or MSU as positive con- Levels of significance for comparison between samples were determined trol throughout our experiments. Surprisingly, treatment of PMs or by Student paired t test or one-way ANOVA (Bonferroni multiple com- 6 BMDMs with CXCL1 or CXCL2 induced the secretion of bioactive parison test). The data in the graphs are expressed as the mean SE from b three independent experiments and p values , 0.05 were defined as sig- IL-1 in the culture supernatants (Fig. 1B, 1C). Because the gen- nificant. GraphPad Prism 5.0 software (GraphPad Software) was used for eration of active IL-1b requires the activation of CASPASE1, the all the statistical analysis. status of CASPASE1 was assessed in CXCL1- or CXCL2-treated PMs and BMDMs. In this context, we observed induced generation Results of active CASPASE1 and IL-1b in CXCL1- or CXCL2-treated PMs Chemokines CXCL1 and CXCL2 are involved in inflammasome and BMDMs (Fig. 1D). Further, the inflammasome activation was activation and subsequent IL-1b production in macrophages found to be specific for CXCL1 and CXCL2 as the other chemo- As inflammatory mediators, chemokines have been increasingly kines such as CCL2 and CCL5 known to regulate macrophages implicated in processes other than their conventional role in or- (23–25) were not able to activate inflammasome (Fig. 1D). A core component of the inflammasome pathway, ASC, was found to form chestrating directed immune cell migration in response to immu- Downloaded from nologic cues. Recently, we reported the involvement of chemokines ASC speck upon CXCL1 or CXCL2 treatment of macrophages, CXCL1 and CXCL2 in the generation of defensive molecules, suggesting that the concerned chemokines activate ASC and hence including defensins, lysozymes, and proinflammatory cytokines inflammasome (Fig. 1E, 1F). Altogether, these results demonstrate TNF-a etc. during mycobacterial infection (22). It was intriguing that chemokines CXCL1 and CXCL2 induce inflammasome acti- to assess if these chemokines involve themselves in regulating vation in macrophages. the prime inflammatory pathways, including the coordinated as- http://www.jimmunol.org/ sembly and activation of inflammasome. In this context, we first CXCL1- and CXCL2-induced inflammasome activation in analyzed the expression of core components of the inflamma- macrophages was dependent on NLRP3 and CASPASE1 some pathway such as Nlrp3, Il-1b, Asc,andCaspase1 at the To test whether CASPASE1 is required for IL-1b maturationinre- RNA level. Treatment of mouse PMs with CXCL1 or CXCL2 sponse to CXCL1 or CXCL2 treatment, CASPASE1 was specifically by guest on October 1, 2021

FIGURE 4. Blocking of CXCL1 and CXCL2 signiﬁcantly reduced M. tuberculosis–induced IL-1b secretion. Mice were intravenously injected with M. tuberculosis for 36 h followed by isolation of spleens, lungs, and lymph nodes to detect the secreted levels of CXCL1 (A) or CXCL2 (B) by ELISA. The ELISA data represent the mean 6 SE (n = 3), *p , 0.05, **p , 0.005, ***p , 0.0001 (Student paired t test). (C) LPS-primed mouse PMs were treated for 4 h with culture sup obtained from M. tuberculosis–infected macrophages treated with control isotype Ab or with CXCL1 and CXCL2 neutralizing Ab; or treated for 1 h with ATP and for 6 h with MSU to assess the inﬂammasome activation by immunoblotting on the total cell lysate. b-Actin was used as loading control. Blots are representative of three independent experiments. (D) Mice were intravenously coinjected with CXCL1- and CXCL2- neutralizing Abs. After 3 h these mice were intravenously challenged with M. tuberculosis for 36 h followed by isolation of spleens, lungs, and lymph nodes to detect the secreted level of IL-1b by ELISA (C). For in vivo experiments, four mice were used for each group. Med, medium; Mtb, M. tuberculosis; sup, supernatant. 6 CXCR2 ORCHESTRATES THE ACTIVATION OF NLRP3 INFLAMMASOME Downloaded from

FIGURE 5. CXCL1 amplifies inflammasome activation in a mouse model of CGN-induced inflammation. (A and F) Mice were intravenously injected with (A) CXCR2 antagonist (5 mg/kg) or (F) CASPASE1 inhibitor (50 mg/kg) for 12 h, followed by coinjection of 25 ml of 1% CGN and 500 ng of CXCL1 into the footpad or air pouch. After 36 h mice were sacrificed to isolate the footpad and air pouch exudates for studying inflammasome activation by http://www.jimmunol.org/ immunoblotting. Densitometric analysis of cleaved CASPASE1 (B and D) and cleaved IL-1b (C and E) from footpad and air pouch experiment (A). (G and H) Densitometric analysis of cleaved IL-1b from footpad and air pouch experiment (F). b-Actin was used as loading control. Blots are representative of three independent experiments. (B–E, G, and H) Level of significance was measured by performing one-way ANOVA followed by Bonferroni multiple comparison test (*p , 0.05, **p , 0.005, ***p , 0.0001) from three independent experiments. anta, antagonist; inhi, inhibitor. inhibited in PMs and BMDMs. Inhibition of CASPASE1 led to the inflammasome activation (Fig. 3E–G). Although, CXCR2 is in- significant reduction in CXCL1- or CXCL2-induced generation of volved in CXCL1-triggered inflammasome activation, ATP- or b active IL-1 in PMs and BMDMs (Fig. 2A, 2B). Further, inhibition MSU-inducedinflammasomeactivationarefoundtobeCXCR2 by guest on October 1, 2021 of NLRP3 by MCC950 also blocked the CXCL1- or CXCL2- independent (Fig. 3B–D). Altogether, these results clearly dem- induced activation of CASPASE1 and IL-1b (Fig. 2C). The role onstrate the involvement of the CXCL1–CXCR2–NLRP3– of NLRP3 in this context was also assessed by siRNA-mediated CASPASE1 axis in regulating the generation of bioactive IL-1b knockdown of NLRP3, which compromised the ability of CXCL1 in macrophages. and CXCL2 to activate CASPASE1 and IL-1b (Fig. 2D, 2F). Al- Blocking of CXCL1 and CXCL2 significantly reduces together, these results suggest that CXCL1- and CXCL2-regulated M. tuberculosis-induced IL-1b secretion IL-1b maturation in macrophages is dependent on NLRP3 and CASPASE1. Results obtained so far indicate the participation of CXCL1/ CXCL2-CXCR2 in the intricate process of inflammasome as- CXCR2, a G-protein–coupled receptor, mediates CXCL1- sembly and activation. As introduced, inflammasomes are formed induced NLRP3 inflammasome activation in macrophages in response to many immunologic cues, NLRP3 being responsive Having observed that chemokines CXCL1 and CXCL2 lead to the to a wide array of them. In this context, we found that CXCL1 and activation of NLRP3 inflammasome, the membrane proximal mo- CXCL2 modulate the production of inflammatory mediators lecular intermediates transducing this assembly were speculated. during mycobacterial infection and that mycobacterial infection It is reported that both CXCL1 and CXCL2 bind to the same re- leads to enhanced production and secretion of IL-1b along with ceptor, CXCR2, a G-protein–coupled receptor (GPCR), to bring chemokines CXCL1 (Fig. 4A) and CXCL2 (Fig. 4B) (4, 22, 30), about their effect on target cells (26–28) and GPCRs have been among others. With this prelude, it was hypothesized that if reported to mediate inflammasome functions (29). This gave a cue M. tuberculosis–induced–IL-1b production is dependent on to assess the contribution of CXCR2 in the process. In this regard, M. tuberculosis–induced chemokine expression. In this context, we first checked whether CXCR2 is expressed on macrophages. we obtained culture supernatants from M. tuberculosis–infected Interestingly, we observed expression of CXCR2 on mouse PMs macrophages, which were then treated with CXCL1- and CXCL2- and BMDMs (Fig. 3A). Because both CXCL1 and CXCL2 offer neutralizing Abs. Neutralization of CXCL1 and CXCL2 in the similar trends in inflammasome activation (Figs. 1 and 2), CXCL1 supernatant obtained from M. tuberculosis–infected macrophages was narrowed down to be used for further experiments. To ana- rendered it incapable of activating inflammasome as compared lyze a possible role for CXCR2, PMs and BMDMs were treated with the isotype control Ab-treated supernatant obtained from with CXCL1 in the presence of CXCR2 antagonist. Inhibition of M. tuberculosis–infected macrophages (Fig. 4C). Further, for in CXCR2 compromised the ability of CXCL1 to activate CASPASE1 vivo relevance mice were treated with CXCL1- and CXCL2- and subsequent production of mature IL-1b (Fig.3B–D).This neutralizing Abs in the presence of M. tuberculosis infection. It was was further confirmed by utilizing siRNA against Cxcr2,wherein found that blocking CXCL1 and CXCL2 using neutralizing Abs the loss of function of the GPCR concerned led to failure of significantly reduced the levels of secreted IL-1b in the spleens, The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. CXCL1 activates ILK via CXCR2. (A) Mouse PMs were treated with the indicated concentrations of CXCL1 for 4 h or (B) CXCL1 (50 ng/ml) for the indicated time points to analyze the level of pILK and ILK by immunoblotting on the total cell lysate. (C) Densitometric analysis of pILK from experiment (B). (D) PMs and BMDMs were pretreated with CXCR2 antagonist (145 nM) for 1 h followed by treatment with CXCL1 (50 ng/ml) for 4 h to detect the level of pILK and ILK on the total cell lysates by immunoblotting. (E and F) Densitometric analysis of pILK from experiment with PMs and BMDMs (D) respectively. (G) Mice were injected with CXCR2 antagonist (5 mg/kg) i.v. for 12 h followed by i.v. injection of CXCL1 (500 ng). After 36 h mice were sacrificed to isolate lymph nodes, spleens, and lungs to analyze the status of pILK and ILK on the total cell lysate by immunoblotting. (H–J) Densitometric analysis of pILK from experiment (G) with lymph nodes, spleens, and lungs, respectively. (K) Mouse PMs were transfected with Cxcr2 siRNA for 24 h followed by treatment with CXCL1 for 4 h to detect the level of pILK, ILK, and CXCR2 on the total cell lysate by immunoblotting. (L) Densitometric analysis of pILK and (M) CXCR2 from experiment (K). b-Actin was used as loading control. Blots are representative of three inde- by guest on October 1, 2021 pendent experiments. (C, E, F, H, I, J, L,andM) Level of significance was measured by performing one-way ANOVA followed by Bonferroni multiple comparison test (*p , 0.05, **p , 0.005, ***p , 0.0001) from three independent experiments. anta, antagonist; LN, lymph node; Med, medium; NT, nontargeting. lungs, and lymph nodes of M. tuberculosis–infected mice (Fig. (Fig. 5A–E). Because the generation of active IL-1b during 4D). These observations suggest that CXCL1, CXCL2, and inflammasome activation requires CASPASE1, the role of CASPASE1 M. tuberculosis synergistically regulate the induction and secretion in CXCL1- and CGN-induced IL-1b activation was assessed. of IL-1b. However, the failure to reduce IL-1b secretion to a basal Inhibition of CASPASE1 using a specific CASPASE1 inhibitor level upon blocking of M. tuberculosis–induced CXCL1 and completely blocked the IL-1b activation in the footpads and air CXCL2 may be attributed to the contribution of CXCL1-/CXCL2- pouches of mice that received CGN and CXCL1 together (Fig. 5F– independent mechanisms in the secretion of IL-1b. Together, these H), demonstrating that CASPASE1 is absolutely required for gen- results demonstrate that during immune insult, CXCL1/CXCL2 erating active IL-1b. Together, these results suggest that CXCL1 could potentially provide a positive feedback loop to sustain the could amplify the CGN-induced inflammasome activation, impli- secretion of proinflammatory IL-1b. cating CXCL1 as one of the basic inflammatory molecules capable of fine-tuning inflammasome functions. CXCL1 amplifies inflammasome activation in a mouse model of CGN-induced inflammation CXCL1 activates ILK in a CXCR2-dependent manner Our previous observations suggest CXCL1 and CXCL2 play an The obtained results provide a prelude that CXCR2, a GPCR, essential role in amplifying the M. tuberculosis–induced IL-1b mediates CXCL1-induced inflammasome activation. However, the production. To further implicate the same during a general call of molecular events that bridge the membrane-proximal events of the inflammatory stimulation, a well-established model of CGN-induced chemokine-chemokine receptor connecting to the intracellular inflammation was adopted (31–33). CGN alone or CXCL1 and CGN assembly of inflammasome require elucidation. In this context, it were coinjected into the footpads and air pouches of mice. CGN was observed that ILK, a multifunctional serine/threonine kinase, by itself activates inflammasome and it could be observed that plays a significant role in transducing multiple signaling pathways CXCL1 amplifies CGN-induced inflammasome activation in the linked to cell-matrix interactions (34–36). Further, several reports footpad as well as air pouch model. Further, blocking CXCR2 have also indicated ILK is a significant regulator of inflammatory using CXCR2 antagonist compromised the ability of CXCL1 to processes (37, 38) and to our interest, ILK was found to be associ- amplify CGN-induced inflammasome activation. However, CGN- ated with GPCR-driven responses (39, 40). This evidence prompted induced inflammasome activation was not dependent on CXCR2 us to speculate the possible involvement of ILK in transducing 8 CXCR2 ORCHESTRATES THE ACTIVATION OF NLRP3 INFLAMMASOME Downloaded from

FIGURE 7. CXCL1-triggered NLRP3 inflammasome activation in macrophages requires ILK. (A) LPS-primed PMs and BMDMs were pretreated with ILK inhibitor (600 nM) for 1 h followed by treatment with CXCL1 (50 ng/ml) for 4 h, ATP (5 mM) for 1 h, or MSU (100 mg/ml) for 6 h. (B) Mouse PMs were transfected with Ilk siRNA for 24 h followed by treatment with CXCL1 for 4 h. In each case cell pellets (A and B) were collected to assess for inflammasome activation by detecting the levels of pro-CASPASE1, CASPASE1, pro–IL-1b, IL-1b, and ILK by immunoblotting. (C) Densitometric http://www.jimmunol.org/ analysis of ILK showing level of significance was performed by using one-way ANOVA followed by Bonferroni multiple comparison test (***p , 0.0001) of experiment (B) from three independent experiments. (D) Cell free supernatants harvested from experiment (B) were subjected to ELISA to detect the levels of secretory IL-1b. The ELISA data represent the mean 6 SE (n = 3), ***p , 0.0001 (one-way ANOVA followed by Bonferroni multiple comparison test). (E) LPS-primed mouse PMs and BMDMs were treated with CXCL1 (50 ng/ml) or left untreated to perform immuno-pulldown assay to assess the interaction of ILK with NLRP3. b-Actin was used as loading control. Blots are representative of three independent experiments. IB, immunoblotting; IP, immuno-pulldown; NT, nontargeting.

CXCR2-driven inflammasome activation. Initially, it was observed various proteins can modulate NLRP3 activation (41–43). With by guest on October 1, 2021 that treatment of macrophages with CXCL1 leads to the activation the prelude of this literature evidence, it was speculated if upon of ILK in a dose- and time-dependent manner (Fig. 6A–C). Further, treatment of macrophages with CXCL1, ILK interacts with as for the inflammasome, activation of ILK was found to be NLRP3 for mediating the activation of the latter. To this end, an CXCR2-dependent as loss of function of CXCR2 by utilizing siRNA immuno-pulldown experiment was performed to detect the in- against Cxcr2 compromised the ability of CXCL1 to activate ILK teraction of ILK with NLRP3. Interestingly, treatment of mouse (Fig. 6K–M). In vitro blocking of CXCR2 by CXCR2 antagonist PMs and BMDMs with CXCL1 induced the interaction of ILK also inhibited the CXCL1-induced ILK activation in PMs and with NLRP3 as compared with that in untreated macrophages BMDMs (Fig. 6D–F). Corroborating the in vitro findings, CXCL1 (Fig. 7E). Together, these results suggested that the introduction could induce in vivo activation of ILK in lymph nodes, spleens, of CXCL1 to the macrophages triggers the interaction of ILK and lungs of CXCL1-treated mice in a CXCR2-dependent manner with NLRP3, which might form the probable mechanism by as the same result could not be observed in the presence of CXCR2 which NLRP3 is activated by ILK in response to stimulation of antagonist (Fig. 6G–J). These results demonstrate that CXCL1 cells with CXCL1. signals via CXCR2 to activate ILK in vitro as well as in vivo. PKCm forms a bridge to transduce signals from CXCL1 to ILK orchestrates CXCL1-driven activation of NLRP3 drive ILK activation inflammasome in macrophages Having established that ILK triggers NLRP3 inflammasome ac- To assess if CXCL1-induced NLRP3 inflammasome activation is tivation in response to CXCL1 treatment, the molecular mechanism dependent on ILK activity, mouse PMs and BMDMs were treated of ILK activation was speculated. In this context, GPCRs have been with a selective pharmacological inhibitor of ILK. It was observed generally reported to interact with distinct protein kinases in a that in the presence of ILK inhibitor, the ability of CXCL1 to stimulus-dependent manner. Therefore, a panel of PKC inhibitors activate pro-CASPASE1 and subsequent maturation of IL-1b was were employed to uncover the specific PKC that might play a role compromised (Fig. 7A). The requirement of ILK for inflamma- in activating CXCL1-responsive ILK (Fig. 8A–I). This revealed some activation was specific for CXCL1 because other known PKCm as a plausible candidate because the specific inhibition of activators of inflammasome such as ATP or MSU could activate PKCm by PKCm inhibitor blocked the CXCL1-triggered ILK inflammasome in ILK-inhibited macrophages (Fig. 7A). Loss of activation in PMs and BMDMs (Fig. 8C–E). PKCm/PKD/PRKD1, ILK function by utilizing Ilk siRNA further confirmed the cru- a serine/threonine kinase, is involved in a wide aspect of cellular cial role for ILK in mediating CXCL1-induced inflammasome signaling including membrane trafficking, cell proliferation, ap- activation (Fig. 7B–D). Next, we assessed the mechanism of optosis, and oxidative stress (44–46), and in the regulation of inflammasome activation by ILK in CXCL1-treated macrophages. inflammatory processes (47). Interestingly, it was also shown that Reports had also indicated that the interaction of NLRP3 with stimulation of certain GPCRs modulate PKCm activity (48). The Journal of Immunology 9 Downloaded from http://www.jimmunol.org/ FIGURE 8. PKCm is necessary for ILK activation in response to CXCL1 treatment. (A) Mouse PMs were pretreated with inhibitors for PKCa (10 mM), PKCb (10 nM), PKCd (6 mM), PKCε (50 mM), or PKCz (50 mM) for 1 h followed by treatment with CXCL1 (50 ng/ml) for 4 h to detect pILK and ILK on the total cell lysate by immunoblotting. (B) Densitometric analysis of pILK from experiment (A). (C) Mouse PMs and BMDMs were pretreated with PKCm inhibitor (280 nM) for 1 h followed by treatment with CXCL1 (50 ng/ml) for 4 h to detect pILK and ILK on the total cell lysate by immunoblotting. (D and E) Densitometric analyses of pILK from experiment with PMs and BMDMs (C). (F) Mice were intravenously injected with PKCm inhibitor (10 mg/kg) for 12 h followed by i.v. injection of CXCL1 (500 ng). After 36 h mice were sacrificed to isolate lymph node, spleens, and lungs to analyze the status of pILKand ILK on the total cell lysate by immunoblotting. (G–I) Densitometric analyses of pILK from experiment with lymph nodes, spleens, and lungs (F). (J)MousePMs were transfected with Prkd1 siRNAfor24hfollowedbytreatmentwithCXCL1(50ng/ml)for4htodetectthepILK,ILKandPKCm/ PRKD1 on the total cell lysate by immunoblotting. (K) Densitometric analysis of pILK, PKCm (L) from experiment (J). b-Actin was used for loading control. Blots are representative of three independent experiments. (B, D, E, G–I, K,andL) Level of significance was measured by performing one-way ANOVA followed by Bonferroni multi- by guest on October 1, 2021 ple comparison test (**p , 0.005, ***p , 0.0001) from three independent experiments. inhi, inhibitor; LN, lymph node; Med, medium; NT, nontargeting.

However, the signaling cross-talk between PKCm and ILK has CXCL1 triggers PKCm activation via CXCR2 m not been elucidated so far. Also, the status of PKC in response With the prelude that PKCm selectively activates ILK and orches- to CXCL1 treatment is not known. With this prelude, the role for trates inflammasome activation in response to CXCL1 stimulation, m PKC in regulating CXCL1-driven ILK activation was further it was pertinent to assess the status of PKCm in CXCL1-stimulated validated using Prkd1 siRNA (Fig. 8J–L). Substantiating these cells. It was observed that PKCm was indeed activated in PMs and m findings, in vivo inhibition of PKC was found to hamper the BMDMs treated with CXCL1 in a CXCR2-dependent manner activation of ILK in the lymph nodes, spleens, and lungs of mice (Fig. 10A–C). This was further confirmed by transiently expressing treated with CXCL1 (Fig. 8F–I). Together, these results dem- Cxcr2 siRNA in cells treated with CXCL1. In such a scenario, m onstrate that PKC selectively modulates ILK activation in mac- CXCL1 failed to activate PKCm (Fig. 10H–J). Corroborating the rophages stimulated with CXCL1. in vitro data, it was observed that CXCL1-driven PKCm activation PKCm is involved in CXCL1-driven inflammasome activation in lymph nodes, spleens, and lungs was compromised in mice in macrophages treated with pharmacological inhibitor of CXCR2 (Fig. 10D–G). Overall, the results suggest that the CXCL1–CXCR2–PKCm–ILK– Because ILK is required for inflammasome activation in CXCL1- NLRP3–CASPASE1 axis regulates maturation of IL-1b and that treated macrophages (Fig. 7) and ILK activation is dependent on chemokines like CXCL1 may form prime factors regulating the PKCm in response to CXCL1 treatment (Fig. 8), the role for crucial inflammatory processes initiated in response to an immunity PKCm in modulating inflammasome activation was analyzed. In breach (Fig. 11). this context, inhibition of PKCm by PKCm inhibitor blocked the CXCL1-induced inflammasome activation in PMs and BMDMs (Fig. 9A). However, ATP- or MSU-induced inflammasome ac- Discussion tivation was found to be independent of PKCm (Fig.9A),sug- Inflammation is a highly coordinated process brought about by gesting that PKCm-mediated inflammasome activation is specific orchestrated priming and subsequent activation of multiprotein for CXCL1. Further, siRNA-mediated knockdown of PKCm complexes called inflammasomes, which produce moieties cru- blocked the CXCL1-induced inflammasome activation (Fig. 9B, cial for immune functions. Canonical inflammasome that are 9C). Together, the results obtained demonstrated that in response activated during pathogenic stimulation include those harboring to treatment of macrophages with CXCL1, PKCm drives ILK NLRPs, AIM, and NLRCs, and they function through the ac- activation to subsequently induce inflammasome activation. tivation of CASPASE1 (12, 19). Several endogenous signals 10 CXCR2 ORCHESTRATES THE ACTIVATION OF NLRP3 INFLAMMASOME Downloaded from

m A

FIGURE 9. PKC is involved in CXCL1-induced inflammasome activation in macrophages. ( ) LPS-primed mouse PMs and BMDMs were pretreated http://www.jimmunol.org/ with PKCm inhibitor (280 nM) for 1 h followed by treatment with CXCL1 (50 ng/ml) for 4 h, ATP (5 mM) for 1 h, or MSU (100 mg/ml) for 6 h to assess the maturation of pro-CASPASE1 and pro–IL-1b by immunoblotting on the total cell lysate. (B) Mouse PMs were transfected with Prkd1 siRNA for 24 h followed by treatment with CXCL1 (50 ng/ml) for 4 h to assess the pro-CASPASE1, CASPASE1, pro–IL-1b, IL-1b, and PKCm on the total cell lysate by immunoblotting. b-Actin was used as loading control. Blots are representative of three independent experiments. (C) Culture supernatants harvested from experiment (B) was used for ELISA to detect IL-1b. The ELISA data represent the mean 6 SE (n = 3), ***p , 0.0001 (one-way ANOVA followed by Bonferroni multiple comparison test). NT, nontargeting. such as mitochondrial reactive oxygen species (49), ATP (50), regulatory nodes. Through this study, we propose that CXCL1 by guest on October 1, 2021 and uric acid crystals (51) were shown to be significant regu- and CXCL2, produced during certain immune insults, can sus- lators of inflammasome activation. To our interest, GPCR li- tain a proinflammatory state by activating inflammasomes. We gands such as extracellular Ca2+ (29) and LTB4 (52) are known used a mouse model of M. tuberculosis infection and found to modulate inflammasome activation. In this study, we investigated that chemokines CXCL1 and CXCL2 are abundantly produced the molecular players executing the activation of NLRP3 inflam- in the lymph nodes, lungs, and spleens of mice infected with masome by crucial chemokines, CXCL1 and CXCL2. Chemokines M. tuberculosis. Further, the released chemokines could acti- are key regulators governing the outcome of an array of physio- vate NLRP3 inflammasomes to intensify the immune response, logical states, and have been conventionally associated with their as neutralization of CXCL1 and CXCL2 compromised the ability of ability to direct the recruitment of immune cells to the site of im- M. tuberculosis to do the same. This was also corroborated in a mune insult. However, emerging reports endorse their role in CGN-induced mouse model of inflammation, where we found that cellular homeostatic processes such as autophagy, production of CGN-induced activation of inflammasome was amplified in the reactive oxygen species, and T cell functions (8–10). In this presence of CXCL1, both in air pouches and footpad models of study, we show for the first time, to our knowledge, that CXCL1 inflammation. In line, blocking CXCR2 (CXCL1 receptor) com- and CXCL2 are capable of activating NLRP3 inflammasome promised the activation of pro-CASPASE1 and subsequent mat- through the GPCR CXCR2. To our knowledge, this is the first uration of pro–IL-1b. report to implicate chemokines as endogenous signals in the As priming of inflammasomes is innate to their activation, it initial steps of inflammasome activation. The responses gener- is becoming apparent that several posttranslational modifica- ated through inflammasomes require two signals: one leading to tions are essential, along with their transcriptional regulation. the production of NLRP3 and pro–IL-1b; and a second mediating For instance, deubiquitination of NLRP3 has been identified as the activation of CASPASE 1 and subsequent pro–IL-1b matura- a prerequisite for its activation (53). Also, a recent report sug- tion. It was found that CXCL1 and CXCL2 led to the activation of gests a probable PKCd-mediated phosphorylation events on NLRP3 inflammasome in LPS-primed macrophages. The NLRP3 NLRC4 are significant for inflammasome activation (54). We find inflammasome activation in macrophages was observed to be spe- that CXCR2-activated PKCm is essential for the activation of cific for CXCL1 and CXCL2, as other chemokines such as CCL2 NLRP3 inflammasome. PKCm activates ILK in macrophages in (24) and CCL5 (25) known to act on macrophages were not able to response to CXCL1 and thereby leads to its interaction with NLRP3 bring about this activation. and subsequent CASPASE1-dependent IL-1b production (Fig. 11). The maintenance of inflammation after the loss of the initial However, the domains in NLRP3 responsible for binding ILK re- signal is cardinal in controlling infections and conversely, may main unexplored, and it would be interesting to uncover such sometimes lead to overt inflammation. Therefore, the modulators of interacting partners and identify specific classes of molecules feed-forward loops of consistent immune responses form key that can finely regulate inflammasome function in a particular The Journal of Immunology 11 Downloaded from

FIGURE 10. CXCL1 triggers PKCm activation via CXCR2. (A) Mouse PMs and BMDMs were pretreated with CXCR2 antagonist (145 nM) for 1 h followed by treatment with CXCL1 (50 ng/ml) for 4 h to analyze the status of pPKCm and PKCm on the total cell lysate by immunoblotting. (B)Densitometric analysis of pPKCm from experiment with PMs (A)and(C) from experiment with BMDMs (A). (D) Mice were intravenously injected with CXCR2 antagonist (5 mg/kg) and kept for 12 h followed by i.v. injection of CXCL1 (500 ng). After 36 h mice were sacriﬁced to isolate lymph node, spleens, and lungs to analyze the status of pPKCm and PKCm on the total cell lysate by immunoblotting. (E) Densitometric analysis of pPKCm from experiment with LN (D), (F)from D G D H experiment with spleen ( ), and ( ) from experiments with lungs ( ). ( ) Mouse PMs were transfected with Cxcr2 siRNA for 24 h followed by treatment with http://www.jimmunol.org/ CXCL1 (50 ng/ml) for 4 h to detect the pPKCm,PKCm, and CXCR2 on the total cell lysate by immunoblotting. Densitometric analysis of pPKCm (I)and CXCR2 (J) from experiment (H). b-Actin was used for loading control. Blots are representative of three independent experiments. (B, C, E–G, I,andJ)Levelof signiﬁcance was measured by performing one-way ANOVA followed by Bonferroni multiple comparison test (*p , 0.05, **p , 0.005, ***p , 0.0001) from three independent experiments. anta, antagonist; LN, lymph node; Med, medium; NT, nontargeting.

way. Although CXCL1-induced NLRP3 inflammasome activation required the involvement of CXCR2, ILK, and PKCm,other known activators of inflammasome such as ATP and MSU acted independently of the CXCR2–PKCm–ILK axis, suggesting that by guest on October 1, 2021 the requirement of CXCR2, ILK, and PKCm for inflammasome activation was specific for CXCL1. Taken together, to our knowledge we identify a previously unknown role of the CXCL1–CXCR2 axis in regulating NLRP3 inflammasome activation in macrophages in a PKCm- and ILK-dependent manner.

Acknowledgments We thank the Central Animal Facility, Indian Institute of Science for pro- viding mice for experimentation. We acknowledge Tanushree Mukherjee for kind help during manuscript preparation.

Disclosures The authors have no ﬁnancial conﬂicts of interest.

References 1. Brown, H. J., H. R. Lock, T. G. Wolfs, W. A. Buurman, S. H. Sacks, and M. G. Robson. 2007. Toll-like receptor 4 ligation on intrinsic renal cells contributes to the induction of antibody-mediated glomerulonephritis via CXCL1 and CXCL2. J. Am. Soc. Nephrol. 18: 1732–1739. 2. Cai, S., S. Batra, S. A. Lira, J. K. Kolls, and S. Jeyaseelan. 2010. CXCL1 reg- FIGURE 11. Model of CXCL1- and CXCL2-driven activation of ulates pulmonary host defense to Klebsiella Infection via CXCL2, CXCL5, NF- NLRP3 inflammasome. Chemokines are released upon distinct inflam- kappaB, and MAPKs. J. Immunol. 185: 6214–6225. 3. De Filippo, K., A. Dudeck, M. Hasenberg, E. Nye, N. van Rooijen, K. Hartmann, matory challenges, like during mycobacterial infection or in in vivo in- M. Gunzer, A. Roers, and N. Hogg. 2013. Mast cell and macrophage chemokines flammatory mouse models. Chemokines CXCL1 and CXCL2 lead to the CXCL1/CXCL2 control the early stage of neutrophil recruitment during tissue activation of a prime inflammatory response (i.e., assembly and activation inflammation. Blood 121: 4930–4937. of inflammasome). CXCL1 and CXCL2 activate a selective signaling 4. Doz, E., R. Lombard, F. Carreras, D. Buzoni-Gatel, and N. Winter. 2013. cascade by interacting with their receptor CXCR2, a GPCR, which Mycobacteria-infected dendritic cells attract neutrophils that produce IL-10 m and specifically shut down Th17 CD4 T cells through their IL-10 receptor. transduces its signal through PKC and ILK to activate pro-CASPASE1, J. Immunol. 191: 3818–3826. leading to the subsequent production and secretion of the mature proin- 5. Roche, J. K., T. R. Keepers, L. K. Gross, R. M. Seaner, and T. G. Obrig. 2007. flammatory cytokine IL-1b. CXCL1/KC and CXCL2/MIP-2 are critical effectors and potential targets for 12 CXCR2 ORCHESTRATES THE ACTIVATION OF NLRP3 INFLAMMASOME

therapy of Escherichia coli O157:H7-associated renal inflammation. Am.J.Pathol. 30. Wu, F., Y. Zhao, T. Jiao, D. Shi, X. Zhu, M. Zhang, M. Shi, and H. Zhou. 2015. 170: 526–537. CXCR2 is essential for cerebral endothelial activation and leukocyte recruitment 6. Sakitani, K., Y. Hirata, Y. Hayakawa, T. Serizawa, W. Nakata, R. Takahashi, during neuroinflammation. J. Neuroinflammation 12: 98. H. Kinoshita, K. Sakamoto, H. Nakagawa, M. Akanuma, et al. 2012. Role of 31. Barth, C. R., G. A. Funchal, C. Luft, J. R. de Oliveira, B. N. Porto, and interleukin-32 in Helicobacter pylori-induced gastric inflammation. Infect. M. V. Donadio. 2016. Carrageenan-induced inflammation promotes ROS gen- Immun. 80: 3795–3803. eration and neutrophil extracellular trap formation in a mouse model of perito- 7. Meńdez-Samperio, P. 2008. Expression and regulation of chemokines in my- nitis. Eur. J. Immunol. 46: 964–970. cobacterial infection. J. Infect. 57: 374–384. 32. Itoh, K., M. Mochizuki, Y. Ishii, T. Ishii, T. Shibata, Y. Kawamoto, V. Kelly, 8. Fang, W. B., M. Yao, I. Jokar, N. Alhakamy, C. Berkland, J. Chen, D. Brantley- K. Sekizawa, K. Uchida, and M. Yamamoto. 2004. Transcription factor Nrf2 Sieders, and N. Cheng. 2015. The CCL2 chemokine is a negative regulator of regulates inflammation by mediating the effect of 15-deoxy-Delta(12,14)- autophagy and necrosis in luminal B breast cancer cells. Breast Cancer Res. prostaglandin j(2). Mol. Cell. Biol. 24: 36–45. Treat. 150: 309–320. 33. Matsui, T. C., G. M. Coura, I. S. Melo, C. R. Batista, P. S. Augusto, A. M. Godin, 9. Chensue, S. W., K. S. Warmington, J. H. Ruth, P. S. Sanghi, P. Lincoln, and D. P. Arau´jo, I. C. Ce´sar, L. S. Ribeiro, D. G. Souza, et al. 2015. Nicorandil S. L. Kunkel. 1996. Role of monocyte chemoattractant protein-1 (MCP-1) in Th1 inhibits neutrophil recruitment in carrageenan-induced experimental pleurisy in (mycobacterial) and Th2 (schistosomal) antigen-induced granuloma formation: mice. Eur. J. Pharmacol. 769: 306–312. relationship to local inflammation, Th cell expression, and IL-12 production. 34. McDonald, P. C., A. B. Fielding, and S. Dedhar. 2008. Integrin-linked kinase– J. Immunol. 157: 4602–4608. essential roles in physiology and cancer biology. J. Cell Sci. 121: 3121–3132. 10. Jin, L., S. Batra, D. N. Douda, N. Palaniyar, and S. Jeyaseelan. 2014. CXCL1 35. Troussard, A. A., C. Tan, T. N. Yoganathan, and S. Dedhar. 1999. Cell- contributes to host defense in polymicrobial sepsis via modulating T cell and extracellular matrix interactions stimulate the AP-1 transcription factor in an neutrophil functions. J. Immunol. 193: 3549–3558. integrin-linked kinase- and glycogen synthase kinase 3-dependent manner. Mol. 11. Gabay, C., C. Lamacchia, and G. Palmer. 2010. IL-1 pathways in inflammation Cell. Biol. 19: 7420–7427. and human diseases. Nat. Rev. Rheumatol. 6: 232–241. 36. Wu, C., and S. Dedhar. 2001. Integrin-linked kinase (ILK) and its interactors: a 12. Wen, H., E. A. Miao, and J. P. Ting. 2013. Mechanisms of NOD-like receptor- new paradigm for the coupling of extracellular matrix to actin cytoskeleton and associated inflammasome activation. Immunity 39: 432–441. signaling complexes. J. Cell Biol. 155: 505–510. 13. Netea, M. G., A. Simon, F. van de Veerdonk, B. J. Kullberg, J. W. Van der Meer, 37. Ahmed, A. U., S. T. Sarvestani, M. P. Gantier, B. R. Williams, and G. E. Hannigan. 2014. Integrin-linked kinase modulates lipopolysaccharide- and

and L. A. Joosten. 2010. IL-1beta processing in host defense: beyond the Downloaded from k a inﬂammasomes. PLoS Pathog. 6: e1000661. Helicobacter pylori-induced nuclear factor B-activated tumor necrosis factor- 14. Fogal, B., and S. J. Hewett. 2008. Interleukin-1beta: a bridge between inﬂam- production via regulation of p65 serine 536 phosphorylation. J. Biol. Chem. 289: mation and excitotoxicity? J. Neurochem. 106: 1–23. 27776–27793. 15. Gherardi, R. K., L. Be´lec, G. Fromont, M. Divine, D. Malapert, P. Gaulard, and 38. Hortelano, S., R. Lo´pez-Fontal, P. G. Trave´s, N. Villa, C. Grashoff, L. Bosca´,and J. D. Degos. 1994. Elevated levels of interleukin-1 beta (IL-1 beta) and IL-6 in A. Luque. 2010. ILK mediates LPS-induced vascular adhesion receptor ex- serum and increased production of IL-1 beta mRNA in lymph nodes of patients pression and subsequent leucocyte trans-endothelial migration. Cardiovasc. Res. with polyneuropathy, organomegaly, endocrinopathy, M protein, and skin 86: 283–292. changes (POEMS) syndrome. Blood 83: 2587–2593. 39. Chang, L. H., S. L. Pan, C. Y. Lai, A. C. Tsai, and C. M. Teng. 2013. Activated

PAR-2 regulates pancreatic cancer progression through ILK/HIF-a-induced http://www.jimmunol.org/ 16. Heneka, M. T., M. P. Kummer, A. Stutz, A. Delekate, S. Schwartz, A. Vieira- a Saecker, A. Griep, D. Axt, A. Remus, T. C. Tzeng, et al. 2013. NLRP3 is ac- TGF- expression and MEK/VEGF-A-mediated angiogenesis. Am. J. Pathol. 183: 566–575. tivated in Alzheimer’s disease and contributes to pathology in APP/PS1 mice. 40. Shafiei, M. S., and D. C. Rockey. 2012. The function of integrin-linked kinase in Nature 493: 674–678. normal and activated stellate cells: implications for fibrogenesis in wound 17. Kay, J., and L. Calabrese. 2004. The role of interleukin-1 in the pathogenesis of healing. Lab. Invest. 92: 305–316. rheumatoid arthritis. Rheumatology 43(Suppl. 3): iii2–iii9. 41. He, Y., M. Y. Zeng, D. Yang, B. Motro, and G. Nuń˜ez. 2016. NEK7 is an es- 18. Rothwell, N. 2003. Interleukin-1 and neuronal injury: mechanisms, modification, sential mediator of NLRP3 activation downstream of potassium efflux. Nature and therapeutic potential. Brain Behav. Immun. 17: 152–157. 530: 354–357. 19. Guo, H., J. B. Callaway, and J. P. Ting. 2015. Inflammasomes: mechanism of 42. Mao, K., S. Chen, Y. Wang, Y. Zeng, Y. Ma, Y. Hu, H. Zhang, S. Sun, X. Wu, action, role in disease, and therapeutics. Nat. Med. 21: 677–687. G. Meng, et al. 2015. b-arrestin1 is critical for the full activation of NLRP3 and 20. Lee, G. S., N. Subramanian, A. I. Kim, I. Aksentijevich, R. Goldbach-Mansky, NLRC4 inflammasomes. J. Immunol. 194: 1867–1873. D. B. Sacks, R. N. Germain, D. L. Kastner, and J. J. Chae. 2012. The calcium- 43. Subramanian, N., K. Natarajan, M. R. Clatworthy, Z. Wang, and R. N. Germain. by guest on October 1, 2021 sensing receptor regulates the NLRP3 inflammasome through Ca2+ and cAMP. 2013. The adaptor MAVS promotes NLRP3 mitochondrial localization and Nature 492: 123–127. inflammasome activation. Cell 153: 348–361. 21. Zhou, R., A. S. Yazdi, P. Menu, and J. Tschopp. 2011. A role for mitochondria in 44. Rozengurt, E. 2011. Protein kinase D signaling: multiple biological functions in NLRP3 inflammasome activation. [Published erratum appears in 2011 Nature health and disease. Physiology 26: 23–33. 475: 122.] Nature 469: 221–225. 45. Rozengurt, E., O. Rey, and R. T. Waldron. 2005. Protein kinase D signaling. 22. Boro, M., V. Singh, and K. N. Balaji. 2016. Mycobacterium tuberculosis-trig- J. Biol. Chem. 280: 13205–13208. gered Hippo pathway orchestrates CXCL1/2 expression to modulate host im- 46. Rykx, A., L. De Kimpe, S. Mikhalap, T. Vantus, T. Seufferlein, J. R. Vandenheede, mune responses. Sci. Rep. 6: 37695. and J. Van Lint. 2003. Protein kinase D: a family affair. FEBS Lett. 546: 81–86. 23. Keophiphath, M., C. Rouault, A. Divoux, K. Cle´ment, and D. Lacasa. 2010. 47. Ivison, S. M., N. R. Graham, C. Q. Bernales, A. Kifayet, N. Ng, L. A. Shobab, and CCL5 promotes macrophage recruitment and survival in human adipose tissue. T. S. Steiner. 2007. Protein kinase D interaction with TLR5 is required for in- Arterioscler. Thromb. Vasc. Biol. 30: 39–45. flammatory signaling in response to bacterial flagellin. J. Immunol. 178: 5735– 24. Sierra-Filardi, E., C. Nieto, A. Domıńguez-Soto, R. Barroso, P. Sańchez-Mateos, 5743. A. Puig-Kroger, M. Lo´pez-Bravo, J. Joven, C. Ardavıń, J. L. Rodrı´guez-Fernańdez, 48. Rey, O., J. R. Reeve, Jr., E. Zhukova, J. Sinnett-Smith, and E. Rozengurt. 2004. et al. 2014. CCL2 shapes macrophage polarization by GM-CSF and M-CSF: G protein-coupled receptor-mediated phosphorylation of the activation loop of identification of CCL2/CCR2-dependent gene expression profile. J. Immunol. protein kinase D: dependence on plasma membrane translocation and protein 192: 3858–3867. kinase Cepsilon. J. Biol. Chem. 279: 34361–34372. 25. Tyner, J. W., O. Uchida, N. Kajiwara, E. Y. Kim, A. C. Patel, M. P. O’Sullivan, 49. Heid, M. E., P. A. Keyel, C. Kamga, S. Shiva, S. C. Watkins, and R. D. Salter. M. J. Walter, R. A. Schwendener, D. N. Cook, T. M. Danoff, and M. J. Holtzman. 2013. Mitochondrial reactive oxygen species induces NLRP3-dependent lyso- 2005. CCL5-CCR5 interaction provides antiapoptotic signals for macrophage somal damage and inflammasome activation. J. Immunol. 191: 5230–5238. survival during viral infection. Nat. Med. 11: 1180–1187. 50. Mariathasan, S., D. S. Weiss, K. Newton, J. McBride, K. O’Rourke, M. Roose- 26. Ritzman, A. M., J. M. Hughes-Hanks, V. A. Blaho, L. E. Wax, W. J. Mitchell, Girma, W. P. Lee, Y. Weinrauch, D. M. Monack, and V. M. Dixit. 2006. Cryopyrin and C. R. Brown. 2010. The chemokine receptor CXCR2 ligand KC (CXCL1) activates the inflammasome in response to toxins and ATP. Nature 440: 228– mediates neutrophil recruitment and is critical for development of experimental 232. Lyme arthritis and carditis. Infect. Immun. 78: 4593–4600. 51. Martinon, F., V.Pe´trilli, A. Mayor, A. Tardivel, and J. Tschopp. 2006. Gout-associated 27. Tsai, H. H., E. Frost, V. To, S. Robinson, C. Ffrench-Constant, R. Geertman, uric acid crystals activate the NALP3 inflammasome. Nature 440: 237–241. R. M. Ransohoff, and R. H. Miller. 2002. The chemokine receptor CXCR2 52. Amaral, F. A., V. V. Costa, L. D. Tavares, D. Sachs, F. M. Coelho, C. T. Fagundes, controls positioning of oligodendrocyte precursors in developing spinal cord by F. M. Soriani, T. N. Silveira, L. D. Cunha, D. S. Zamboni, et al. 2012. NLRP3 arresting their migration. Cell 110: 373–383. inflammasome-mediated neutrophil recruitment and hypernociception de- 28. Wang, D., H. Wang, J. Brown, T. Daikoku, W. Ning, Q. Shi, A. Richmond, pend on leukotriene B(4) in a murine model of gout. Arthritis Rheum. 64: R. Strieter, S. K. Dey, and R. N. DuBois. 2006. CXCL1 induced by prosta- 474–484. glandin E2 promotes angiogenesis in colorectal cancer. J. Exp. Med. 203: 941– 53. Juliana, C., T. Fernandes-Alnemri, S. Kang, A. Farias, F. Qin, and E. S. Alnemri. 951. 2012. Non-transcriptional priming and deubiquitination regulate NLRP3 29. Rossol, M., M. Pierer, N. Raulien, D. Quandt, U. Meusch, K. Rothe, K. Schubert, inflammasome activation. J. Biol. Chem. 287: 36617–36622. T. Scho¨neberg, M. Schaefer, U. Krugel,€ et al. 2012. Extracellular Ca2+ is a 54. Qu, Y., S. Misaghi, A. Izrael-Tomasevic, K. Newton, L. L. Gilmour, M. Lamkanfi, danger signal activating the NLRP3 inflammasome through G protein-coupled S. Louie, N. Kayagaki, J. Liu, L. Ko¨muves,€ et al. 2012. Phosphorylation of NLRC4 calcium sensing receptors. Nat. Commun. 3: 1329. is critical for inflammasome activation. Nature 490: 539–542.