CD11b/CD18 (Mac-1) Is a Novel Surface for Extracellular Double-Stranded RNA To Mediate Cellular Inflammatory Responses This information is current as of September 25, 2021. Hui Zhou, Jieying Liao, Jim Aloor, Hui Nie, Belinda C. Wilson, Michael B. Fessler, Hui-Ming Gao and Jau-Shyong Hong J Immunol 2013; 190:115-125; Prepublished online 3 December 2012; Downloaded from doi: 10.4049/jimmunol.1202136 http://www.jimmunol.org/content/190/1/115 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2012/12/03/jimmunol.120213 Material 6.DC1 References This article cites 54 articles, 25 of which you can access for free at: http://www.jimmunol.org/content/190/1/115.full#ref-list-1

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CD11b/CD18 (Mac-1) Is a Novel Surface Receptor for Extracellular Double-Stranded RNA To Mediate Cellular Inflammatory Responses

Hui Zhou,*,1 Jieying Liao,*,1 Jim Aloor,† Hui Nie,* Belinda C. Wilson,* Michael B. Fessler,† Hui-Ming Gao,*,‡ and Jau-Shyong Hong*

During viral infection, extracellular dsRNA is a potent signaling molecule that activates many innate immune cells, including macrophages. TLR3 is a well-known receptor for extracellular dsRNA, and internalization of extracellular dsRNA is required for endosomal TLR3 activation. Preserved inflammatory responses of TLR3-deficient macrophages to extracellular dsRNA strongly support a TLR3-independent mechanism in dsRNA-mediated immune responses. The present study demonstrated that CD11b/

CD18 (Mac-1 [macrophage-1 Ag]), a surface integrin receptor, recognized extracellular dsRNA and induced macrophage im- Downloaded from mune responses. CD11b deficiency reduced inflammatory induction elicited by polyinosinic:polycytidylic acid (poly I:C; a synthetic dsRNA) in mouse sera and livers, as well as in cultured peritoneal macrophages. dsRNA-binding assay and confocal immunofluorescence showed that Mac-1, especially the CD11b subunit, interacted and colocalized with poly I:C on the surface of macrophages. Further mechanistic studies revealed two distinct signaling events following dsRNA recognition by Mac-1. First, Mac-1 facilitated poly I:C internalization through the activation of PI3K signaling and enhanced TLR3-dependent activation of

IRF3 in macrophages. Second, poly I:C induced activation of phagocyte NADPH oxidase in a TLR3-independent, but Mac-1– http://www.jimmunol.org/ dependent, manner. Subsequently, phagocyte NADPH oxidase–derived intracellular reactive oxygen species activated MAPK and NF-kB pathways. Our results indicate that extracellular dsRNA activates Mac-1 to enhance TLR3-dependent signaling and to trigger TLR3-independent, but Mac-1–dependent, inflammatory oxidative signaling, identifying a novel mechanistic basis for macrophages to recognize extracellular dsRNA to regulate innate immune responses. This study identifies Mac-1 as a novel surface receptor for extracellular dsRNA and implicates it as a potential therapeutic target for -related inflammatory diseases. The Journal of Immunology, 2013, 190: 115–125.

s the first line of host defense, the is a critical viral PAMP, which functions as a powerful stimulus of by guest on September 25, 2021 combats numerous pathogens through a diverse set of both innate and adaptive antiviral immune responses (1). Viral A pattern recognition receptors (PRRs). These PRRs are dsRNA is primarily generated during viral replication in virally capable of identifying pathogen-associated molecular patterns infected cells (2). Once the infected cells die by lysis, the viral (PAMPs) and initiating the innate immune response. During viral dsRNA is released into the extracellular space and becomes a infections, various virus-associated PAMPs are recognized by, and stable molecule; extracellular dsRNA is implicated in both local bind to, their respective PRRs, which induces robust host antiviral and systemic reactions associated with viral infections and mod- and immune responses. It is well known that viral-produced dsRNA ulates both innate and adaptive immune responses (3–5). Exper- imentally, synthetic dsRNA, like polyinosinic:polycytidylic acid *Laboratory of Toxicology and Pharmacology, National Institutes of Environmental (poly I:C), has been commonly used to induce antiviral responses Health Sciences, Research Triangle Park, NC 27709; †Laboratory of Respiratory in vivo or in vitro (6). Biology, National Institutes of Environmental Health Sciences, Research Triangle The TLR3, RIG-I–like receptors RIG-I/MDA-5/LGP2, and Park, NC 27709; and ‡Laboratory of Neuroimmunology and Neuropharmacology, Model Animal Research Center, Nanjing University, Jiangsu 210061, China NOD-like receptor Nalp3 have been identified as PRRs for dsRNA 1H.Z. and J.L. contributed equally to this work. (1). Based on cellular location, the membrane receptor TLR3 can recognize internalized extracellular dsRNA, whereas both RIG-I– Received for publication August 2, 2012. Accepted for publication October 28, 2012. like receptors and NOD-like receptor Nalp3 are the cytoplasmic This work was supported by the Intramural Research Program of the National In- stitutes of Health and the National Institute of Environmental Health Sciences. sensors that are likely to identify intracellular dsRNA generated Address correspondence and reprint requests to Dr. Hui-Ming Gao, Laboratory of during the intracellular viral life cycle (7–10). Interestingly, TLR3 Toxicology and Pharmacology, National Institutes of Environmental Health Sci- is only able to recognize and to bind dsRNA in acidified endo- ences, P.O. Box 12233, MD F1-01 Research Triangle Park, NC 27709 or Laboratory somes (11), which suggests that extracellular dsRNA must first of Neuroimmunology and Neuropharmacology, Model Animal Research Center, Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China. E-mail address: be internalized before it activates TLR3. Considering the evidence [email protected] that extracellular dsRNA is still able to induce a significant num- The online version of this article contains supplemental material. ber of proinflammatory in TLR3-knockout macrophages Abbreviations used in this article: BFA, bafilomycin A; CR3, or microglia (7, 11, 12), we hypothesize that other PRRs on the 9 9 3; DCFH-DA, 2 ,7 -dichlorodihydrofluorescein diacetate; iROS, intracellular reac- cell surface can serve as the first-line receptors to sense extra- tive oxygen species; KO, knockout; Mac-1, macrophage-1 Ag; NOX2, phagocyte NADPH oxidase; NP-40, Nonidet P-40; PAMP, pathogen-associated molecular pat- cellular dsRNA and to mediate cellular responses. tern; poly C, polycytidylic acid; poly I:C, polyinosinic:polycytidylic acid; PRR, Previous studies indicated that the surface receptor integrin pattern recognition receptor; RRV, Ross River virus; SOD, superoxide dismutase; SR-A, type A scavenger receptor; WST-1, 2-[4-iodophenyl]-3-[4-nitrophenyl]-5-[2,4- CD11b/CD18, also known as macrophage-1 Ag (Mac-1), complement disulfophenyl]-2H-tetrazolium; WT, wild-type. receptor 3 (CR3), or aMb2, serves as a PRR to recognize PAMPs www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202136 116 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY and damage-associated molecular patterns, such as Gram-nega- poly C–conjugated beads, poly I:C–conjugated beads, or noncoated empty tive bacteria–derived LPS (13), aggregated b-amyloid (14), and beads were equilibrated in lysis buffer containing RNase inhibitor (Invi- damage-associated alarmin HMGB1 (15). Mac-1, expressed on trogen) and incubated overnight with whole-cell lysates at 4˚C on a rotat- ing shaker. After centrifugation, beads were washed extensively and then many innate immune cells, such as monocytes, granulocytes, mac- resuspended in sample buffer (Invitrogen). Samples were boiled for 10 min rophages, and NK cells (16), has been implicated in various im- and centrifuged at 13,000 3 g for 1 min to discard insoluble pellets. mune cell responses, including adhesion, migration, phagocytosis, Samples were then loaded onto SDS-PAGE, electroblotted onto poly- chemotaxis, cellular activation, and cytotoxicity (17, 18). Further- vinylidene difluoride membranes, and probed by immunoblot analysis against CD11b (Abcam, MA). more, Mac-1 was reported to participate in inflammatory diseases associated with Ross River virus infection (19) and to bind some Flow cytometric analysis nucleotides, such as oligodeoxynucleotide (20). These character- Poly I:C was labeled with FITC using a Mirus RNA labeling (24). istics of Mac-1 prompted us to investigate the possibility that it Labeled RNA was then purified with an RNeasy mini kit (QIAGEN, CA). serves as a PRR for extracellular dsRNA to regulate the innate Peritoneal macrophages or RAW 264.7 cells were washed twice and sus- 6 immune response. pended in HBSS (Invitrogen). The cells (1 3 10 cells/ml) were kept on In this study, we identified Mac-1 as a novel surface receptor ice for determination of surface binding of poly I:C by incubating with 10 mg/ml FITC-labeled poly I:C for 20 min (20). After washing three times mediating extracellular dsRNA-elicited cellular immune responses. with cold HBSS, poly I:C–bound macrophages were detected by a BD Our results demonstrate that Mac-1 can recognize extracellular LSR II Flow Cytometer. A live cell gate was made using forward versus dsRNA on the cell surface and then mediate outside-in signaling, side scatter plot. The surface binding was expressed as the mean fluores- regulate dsRNA internalization, and mediate activation of phago- cence intensities of the total calculated population. Internalization analysis of FITC-labeled poly I:C was performed using the same procedure as cyte NADPH oxidase (NOX2) to induce cellular immune responses described above for the surface binding process, with the exception that Downloaded from in macrophages. Our results provide new insight into how the cells were incubated at 37˚C instead of on ice (20), and trypan blue (1 mg/ macrophage recognizes extracellular signals associated with lytic ml) was used to quench extracellular surface-bound fluorescence, as de- virus infections and mediates the innate immune response. scribed previously (25). The internalization of poly I:C was expressed as the mean fluorescence intensities of the total calculated population. Materials and Methods Confocal microscopy Animal study Poly I:C and poly C were labeled with Cy3 for confocal observation (24). http://www.jimmunol.org/

2/2 2/2 Peritoneal macrophages were seeded and grown in glass-bottom microwell CD11b mice (Mac-1–deficient), gp91 mice (NADPH oxidase–de- dishes (MatTek, MA). Cells were then treated with 10 mg/ml Cy3-labeled ficient), and their age-matched wild-type (WT) control (C57BL/6J) were poly I:C or poly C, either on ice for 20 min to observe the surface binding obtained from The Jackson Laboratory (Bar Harbor, ME). TLR32/2 mice +/+ or at 37˚C for 15 or 30 min to determine the internalization. Stained and their age-matched WT control (TLR , C57BL/6NJ) were also ob- macrophages were washed three times with ice-cold PBS and fixed with tained from The Jackson Laboratory. Housing and breeding of the animals 4% paraformaldehyde for further observation on a Zeiss LSM510 Laser were performed humanely and with regard for alleviation of suffering Scanning Confocal Microscope (Carl Zeiss Microimaging, Germany). following the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources 1996). Six- Quantitative real-time PCR to eight-week-old male mice of different strains were used in all experi- ments. All procedures were approved by the National Institutes of Envi- Total RNA of cells or tissues was isolated using TRIzol reagent and then by guest on September 25, 2021 ronmental Health Sciences Animal Care and Use Committee. first-strand cDNA was synthesized using SuperScript Reverse Transcrip- m An in vivo animal model involved immune activation by poly I:C tase (Invitrogen), according to the manufacturer’s protocols. After 0.5 g m (Sigma-Aldrich, St. Louis, MO; an average size of 300–750 bp). WT mice total RNA was subjected to a reverse-transcription reaction, 2 l cDNA and CD11b2/2 (CD11b-knockout [KO]) mice were injected i.p. with poly I:C was amplified by quantitative real-time PCR analysis for the induction (5 mg/kg). Serum was collected 2 h later for cytokine measurement using of inflammatory cytokines using SYBR Green Master mix (Bio-Rad, CA) m a commercially available ELISA kits (R&D Systems, Minneapolis, MN), in a final volume of 25 l. The following primers were used: mouse TNF- : 9 9 9 and liver tissues were harvested for mRNA isolation and cytokine assay 5 -CATCTTCTCAAAATTCGAGTGGACAA-3 (forward), 5 -TGGGAG- 9 9 by quantitative real-time PCR. TAGACAAGGTAC-AACCC-3 (reverse); mouse IL-12p40: 5 -GGAAG- CACGGCAGCAGAATA-39 (forward), 59-AACTTGAGGGAGAAGTAGG- Preparation of peritoneal macrophages and culture of AATGG-39 (reverse); mouse IFN-b:59-ATGAGTGGTGGTTGCAGGC-39 9 9 macrophage cell line (forward), 5 -TGACCTTTCAAATGCAGTAGATTCA-3 (reverse); mouse IL-6: 59-TTGCCTTCTTGGGACTGATGCT-39 (forward), 59-GTATCTC- Peritoneal macrophages from different strains were induced and har- TCTGAAGGACTCTG-G-39 (reverse); mouse GAPDH: 59-TTCACCAC- vested, as previously described (21). Briefly, WT mice, CD11b-KO mice, CATGGAGAAGGC-39 (forward), 59-GGCATGGACTGTGGTCATGA-39 and gp912/2 (gp91-KO) mice were injected i.p. with 2 ml 3% sterile thio- (reverse). Data were normalized to GAPDH expression. glycollate. After 4 d, peritoneal exudate macrophages were collected by lavage in 5 ml ice-cold RPMI 1640 medium (Invitrogen, CA), washed Measurement of extracellular superoxide and intracellular twice in RPMI 1640, and preincubated in serum-free medium for 1 h. The reactive oxygen species cells were then washed twice to remove nonadherent cells. Adherent macrophages were cultured in RPMI 1640 medium containing 10% FBS The release of superoxide was determined by measuring the superoxide (Invitrogen), 50 U/ml penicillin, and 50 mg/ml streptomycin at 37˚C in dismutase (SOD)–inhibitable reduction of tetrazolium salt (2-[4-iodo- phenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium [WST-1]) a humidified atmosphere of 5% CO2. 3 5 The murine macrophage cell line RAW 264.7 (ATCC TIB-71) was as described (26). Briefly, peritoneal macrophages (1 10 /well) were grown overnight in 96-well plates in DMEM medium containing 10% FBS suspension cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 50 m U/ml penicillin, and 50 mg/ml streptomycin at 37˚C in a humidified at- and switched to phenol red–free HBSS (100 l/well). Fifty microliters of mosphere of 5% CO . HBSS containing vehicle, poly I:C, or PMA was then added to each well, 2 followed by the addition of 50 ml WST-1 (1 mM) in HBSS, with or without dsRNA-binding assay 600 U/ml SOD. The cells were incubated for 30 min at 37˚C, and the absorbance was read at 450 nm with a SpectraMax Plus microplate spec- The dsRNA-binding assay was performed as described previously (22, trophotometer (Molecular Devices, CA). 23). Briefly, poly I:C–conjugated agarose beads were prepared by incu- The production of intracellular reactive oxygen species (iROS) was bating polycytidylic acid (poly C) agarose beads with polyinosinic acid measured by the fluorescence probe 29,79-dichlorofluorescin diacetate (both from Sigma-Aldrich) at 4˚C overnight. Cyanogen bromide–activated (DCFH-DA), as described (26). Briefly, macrophages cultured overnight in agarose beads (Sigma-Aldrich) were used as controls. Cell lysates were 96-well plates were incubated with 10 mM DCFH-DA (Invitrogen) for prepared in Nonidet P-40 (NP-40) lysis buffer (10 mM Tris-HCl [pH 7.5], 30 min at 37˚C. After two washes with HBSS buffer, cells were switched to 1% NP-40, 0.15 M NaCl, 1 mM EDTA, and protease inhibitor mixture) HBSS containing 1% FBS. After the cells were incubated with vehicle or and centrifuged at 16,000 3 g for 15 min at 4˚C. For pull-down assays, poly I:C (50 mg/ml) at 37˚C for 30 min, the fluorescence density was read The Journal of Immunology 117 at 488 nm for excitation and at 525 nm for emission using a SpectraMax Measurement of serum inflammatory factors 2 h after poly I:C Gemini XS fluorescence microplate reader (Molecular Devices). injection revealed decreased circulating TNF-a, IFN-b, and IL- 2/2 Nuclear extraction, gel electrophoresis, and Western blotting 12p40 in CD11b mice compared with WT controls (Fig. 1A). analysis Similarly, the mRNA level of TNF-a, IL-12p40, IFN-b, and IL-6 2/2 2/2 in the liver of CD11b mice was reduced (Fig. 1B). Collec- Peritoneal macrophages from WT and CD11b mice were incubated with tively, Mac-1–deficient (CD11b2/2) mice displayed an impaired poly I:C (50 mg/ml) or vehicle at 37˚C for 60 or 120 min and washed twice with cold PBS. Nuclear extraction was performed at 4˚C with a nuclear immune response to poly I:C. extraction kit from Affymetrix, following the manufacturer’s instructions. concentrations were determined using the DC protein assay (Bio- Mac-1 deficiency impaired immune responses to poly I:C in Rad). The whole-cell lysates from cultured cells were homogenized in macrophages radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8], 150 mM NaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, Because Mac-1 is expressed dominantly on macrophages that have and 1:100 protease inhibitor mixture) with phosphatase inhibitors (10 mM a central role in the innate immune response, we next compared NaF, 1 mM Na4P3O7, and 1 mM sodium orthovanadate), sonicated, and inflammatory cytokine induction and investigated inflammatory boiled for 10 min. Protein concentrations were determined using the signaling cascades after poly I:C stimulation in CD11b2/2 and WT bicinchoninic acid assay (Pierce). Protein samples were resolved on 4–12% SDS-PAGE, and immunoblot analysis was performed using Abs against the peritoneal macrophages. Consistent with our in vivo results, the indicated signaling molecules (Cell Signaling Technology). An Ab against mRNA level of TNF-a, IL-12p40, IFN-b, and IL-6 was signifi- 2 2 GAPDH (Sigma-Aldrich) or HDAC2 (Santa Cruz Biotech) was included as cantly lower in CD11b / macrophages than in WT controls (Fig. an internal standard to monitor loading errors. 2A). Secreted TNF-a and IL-12p40 in the supernatant showed 2 2

/ Downloaded from Immunofluorescence staining a significant reduction in CD11b macrophages (Fig. 2B). IL- 12p70 was detected after macrophages were challenged with 50 Macrophages were grown and treated on glass coverslips, fixed in 4% mg/ml poly I:C for 24 h, reaching 2.8 6 0.3 pg/ml in WT mac- paraformaldehyde, permeabilized with 0.4% Triton X-100, and blocked with 6 2/2 5% goat serum. Samples were incubated sequentially with primary Abs and rophages and 0.8 0.6 pg/ml in CD11b macrophages. A the corresponding fluorescence probe–conjugated secondary Abs (Invitrogen) similar pattern was observed for the poly I:C–elicited release of and then analyzed using a Zeiss LSM510 Confocal Microscope. To study NO, an inflammatory molecule (Fig. 2C). In contrast, stimulation the distribution and colocalization of desired , we analyzed images of cultured peritoneal macrophages with TNF-a (50 ng/ml) or http://www.jimmunol.org/ from $10 random fields from three independent experiments. PMA (100 nM) induced indistinguishable immune responses Statistical analysis in CD11b2/2 macrophages and WT macrophages (Supplemental 2/2 Data are expressed as mean 6 SEM. Statistical significance was assessed Fig. 1). In addition, TLR3 expression in CD11b and WT mac- by ANOVA, followed by the Bonferroni t test, using GraphPad Prism rophages was not different (Supplemental Fig. 2). Together, these software (GraphPad Software, CA). A p value , 0.05 was considered data suggest that the observed impairment in the immune response statistically significant. of CD11b2/2 macrophages to poly I:C is due to the functional deficiency of Mac-1 receptor to poly I:C stimulation and is not due Results to a general immune deficit in these cells.

Mac-1–deficient mice exhibited impaired immune response to by guest on September 25, 2021 poly I:C Poly I:C bound to Mac-1 receptor on the cell surface of To determine whether Mac-1 is a potential candidate in sensing macrophages extracellular dsRNA and regulating the immune response, mice A dsRNA-binding assay using cell extracts from peritoneal mac- deficient in the CD11b subunit of Mac-1 and age-matched WT rophages or RAW 264.7 cells (a mouse macrophage–like cell mice were injected i.p. with synthetic dsRNA poly I:C (5 mg/kg). line) was used to test the possibility that poly I:C binds to Mac-1.

FIGURE 1. Mac-1–deficient mice exhibited impaired immune response after poly I:C injection. WT or CD11b2/2 mice were injected i.p. with 5 mg/kg poly I:C. After 2 h, blood and livers were collected for analysis of inflammatory cytokine induction. The amount of TNF-a, IL-12p40, and IFN-b in serum was detected by ELISA (A), and the mRNA level of TNF-a, IFN-b, IL-6, and IL-12p40 in livers was measured by RT-PCR (B). Data are mean 6 SEM from five to seven pairs of WT and CD11b2/2 mice. *p , 0.01 versus vehicle-injected WT mice, #p , 0.01 versus poly I:C–injected WT mice. 118 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 2. Mac-1 deficiency impaired immune responses to poly I:C in macrophages. Poly I:C was added to macrophages prepared from WT or CD11b2/2 mice with the indicated dose range. (A) After 2 h, total RNA was extracted and used in real-time PCR. (B) The release of TNF-a and IL-12p40 from cultured macrophages was determined by ELISA at 4 or 24 h after the treatment. (C) NO production was measured at 24 h after the treatment. Data are mean 6 SEM from three independent experiments in triplicate. #p , 0.01 versus poly I:C–treated WT mice.

Poly I:C–conjugated agarose beads pulled down the CD11b subunit mediate immune responses to poly I:C through outside-in sig- of Mac-1, whereas unconjugated beads or beads conjugated with naling. single-stranded poly C failed to do so (Fig. 3A). These results indicate that Mac-1 can specifically recognize double-stranded Mac-1 facilitated the internalization of poly I:C poly I:C. To further confirm the interaction between poly I:C It is well known that internalization of extracellular dsRNA is a and Mac-1, a surface binding assay was performed using flow crucial step to subsequent antiviral responses induced by endo- cytometry and confocal imaging. FITC-labeled poly I:C bound somal TLR3 activation (6, 11, 24). Our confocal imaging analysis to the surface of WT macrophages, whereas such surface binding detected intracellular poly I:C within 15 min after Cy3-labeled was dramatically decreased in CD11b2/2 macrophages (Fig. 3B, poly I:C was added to macrophages, and further accumulation of 3C). Furthermore, the surface binding was also significantly re- intracellular poly I:C was observed with extended time (30 min). duced in WT macrophages or RAW 264.7 cells pretreated with However, the uptake of poly I:C was significantly attenuated in fibrinogen, an endogenous ligand of Mac-1, whereas such an in- CD11b2/2 macrophages compared with WT macrophages (Fig. hibitory effect of fibrinogen was not observed in CD11b2/2 4A). In contrast to the differential internalization of poly I:C, macrophages (Fig. 3B, 3C). Consistent with this result, confocal the uptake of Cy3-labeled poly C (ssRNA) was indistinguish- imaging (Fig. 3D) showed that CD11b deletion or fibrinogen able in WT and CD11b2/2 peritoneal macrophages, indicating the pretreatment reduced the surface binding of poly I:C in macro- specificity of CD11b on dsRNA poly I:C (Fig. 4A). The flow phages. In addition, immunofluorescence staining of CD11b re- cytometric analysis revealed ∼40% reduction in the uptake of vealed colocalization of CD11b with surface-bound Cy3–poly I:C FITC-labeled poly I:C into CD11b2/2 macrophages compared in WT macrophages (Fig. 3E). Further overlapping analysis with WT macrophages (Fig. 4B). These results indicate a critical showed .75% overlap of Mac-1 with Cy3–poly I:C on the cell role for Mac-1 in the internalization of poly I:C into macrophages. surface (Fig. 3E). Overall, these data provide strong evidence Such a conclusion was confirmed by the finding that Mac-1– that Mac-1 can recognize and bind double-stranded poly I:C blocking Ab significantly attenuated the uptake of poly I:C into on the surface of the macrophage, indicating that Mac-1 may WT macrophages but not CD11b2/2 macrophages (Fig. 4C). The Journal of Immunology 119 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 3. Poly I:C bound to Mac-1 receptor on the cell surface. dsRNA-binding assay was performed using poly I:C–coated agarose beads, un- conjugated beads, or poly C–coated beads. (A) Only poly I:C–coated beads can pull down the CD11b subunit of Mac-1 in either primary macrophages or RAW264.7 macrophage cells. (B–E) Surface-binding assay further delineated the interaction between poly I:C and Mac-1 on the cell surface. (B and C) Flow cytometry showed that WT macrophages had higher surface binding of FITC-labeled poly I:C than did CD11b2/2 macrophages, and the poly I:C binding was significantly inhibited by fibrinogen (1 mM) in WT macrophages and RAW264.7 cells but not in CD11b2/2 macrophages. (D) The confocal experiments revealed that more Cy3-labeled poly I:C bound to the surface of WT macrophages than to the surface of CD11b2/2 macrophages, and the binding of Cy3-labeled poly I:C was inhibited by fibrinogen only in WT macrophages. (E) CD11b immunostaining showed that Mac-1 colocalized with surface-bound Cy3–poly I:C. Data are mean 6 SEM from three independent experiments in triplicate. *p , 0.01 versus poly I:C–treated groups, #p , 0.01 versus poly I:C–treated WT groups.

As a common cellular signaling pathway of integrins (27, 28), activation in CD11b2/2 macrophages suggest that Mac-1 pro- the activation of PI3K was specifically described in previous motes the internalization of poly I:C through activating the PI3K studies on Mac-1–signaling transduction (29, 30). PI3K was also pathway. Thus, Mac-1 may facilitate immune responses in mac- implicated in the regulation of endocytosis and intracellular mem- rophages through enhancing the uptake of extracellular poly I:C brane trafficking in macrophages (31, 32). In this study, we found into the endosome, where TLR3 resides. that wortmannin, a PI3K inhibitor, blocked the uptake of poly I:C into the macrophage (Fig. 4D). Importantly, when challenged with Mac-1 altered poly I:C–induced downstream signaling poly I:C, the phosphorylation of AKT, a key kinase downstream of It is well known that poly I:C activates IRF3 signaling via TLR3, PI3K, was significantly impaired in CD11b2/2 macrophages (Fig. leading to the induction of IFN and IFN-inducible . The 4E). Taken together, the reduced uptake of poly I:C in the setting attenuation of poly I:C–elicited IFN-b induction in CD11b2/2 of PI3K inhibition in WT macrophages and the diminished PI3K macrophages and mice (Fig. 1) suggests that Mac-1 might par- 120 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY Downloaded from

FIGURE 4. Mac-1 facilitates the internalization of poly I:C. Cultured peritoneal macrophages from WT and CD11b2/2 mice were incubated with Cy3-labeled poly I:C (10 mg/ml) at 37˚C for 15 or 30 min or Cy3-labeled poly C (10 mg/ml) for 30 min. (A) Confocal imaging shows decreased in- tracellular Cy3 fluorescence in CD11b2/2 macrophages compared with WT macrophages after these cells were treated with Cy3-labeled poly I:C but not with Cy3-labeled poly C. (B) Macrophages from WT and CD11b2/2 mice were incubated with FITC-labeled poly I:C (10 mg/ml) at 37˚C for 30 min. http://www.jimmunol.org/ After the fluorescence of extracellular surface-bound FITC-labeled poly I:C was quenched by trypan blue (1 mg/ml), the fluorescence density of the internalized FITC-labeled poly I:C was measured by flow cytometry and was present after the subtraction of nonspecific autofluorescence in untreated control cells. (C) After pretreatment with normal IgG (2.5 mg/ml) or anti-CD11b Ab (2.5 mg/ml) for 15 min, macrophages from WT and CD11b2/2 mice were incubated with Cy3-labeled poly I:C for 30 min. Confocal imaging showed reduced fluorescence density in the Ab-treated group. (D) The pre- treatment of WT macrophages for 15 min with PI3K inhibitor wortmannin attenuated the uptake of Cy3-labeled poly I:C. (E) Western blot analysis on poly I:C–challenged WT and CD11b2/2 macrophages at different time points. Impaired AKT phosphorylation was observed in CD11b2/2 macrophages. Data are mean 6 SEM from three independent experiments in triplicate. *p , 0.01 versus vehicle-treated controls, #p , 0.01 versus poly I:C–treated WT groups. by guest on September 25, 2021 ticipate in TLR3-dependent IRF3 activation. IRF3 is present in hibitor (Compound A; 1 mM) (33) for 30 min and then challenged an inactive form in the cytoplasm in unstimulated innate immune the cells with poly I:C. Both inhibitors significantly suppressed the cells; upon the phosphorylation of its serine, the active IRF3 release of TNF-a and IL-12p40, which further demonstrated the translocates to the nucleus, leading to the transcription of IFN and importance of the activation of JNK and NF-kB in the poly I:C– IFN-inducible genes. We next examined the phosphorylation and induced immune response (Fig. 5D). Together, these results sug- the nuclear translocation of IRF3 protein by using the nuclear gest that Mac-1 contributes to the poly I:C–induced immune re- fraction of WT and CD11b2/2 macrophages. To rule out possible sponse in macrophages through promoting the activation of the interference from any contaminated cytoplasmic IRF3 in the MAPK and NF-kB pathways. nuclear fraction, we used phospho-IRF3 (Ser396) Ab to determine In response to extracellular dsRNA, NF-kB activation is thought IRF3 activation. As expected, we observed decreases in poly I:C– to be involved in the downstream signaling of TLR3 activation (7), induced phosphorylation and nuclear translocation of IRF3 in but the engagement of TLR3 does not seem to be required for the CD11b2/2 macrophages compared with WT macrophages (Fig. activation of MAPK signaling in macrophages (34). Thus, our 5A), indicating that Mac-1 enhanced TLR3-dependent IRF3 ac- findings described above (Fig. 5) suggest that, in addition to the tivation. contribution to the TLR3–NF-kB pathway by facilitating dsRNA To further determine the downstream-signaling cascades of internalization, Mac-1 may induce TLR3-independent signaling poly I:C–elicited Mac-1 activation, the MAPK and NF-kBpath- pathways, such as the MAPK pathway. Bafilomycin A (BFA), an ways, two major signaling pathways responsible for proinflam- inhibitor of vacuolar-type H+-ATPase, blocks the acidification of matory cytokine induction, were assayed in WT and CD11b2/2 the endosome and, therefore, inhibits TLR3 activation (35). In the macrophages. WT macrophages exhibited time-dependent phos- presence of BFA, considerable induction of the proinflammatory phorylation in the MAPK and NF-kB pathways (Fig. 5B, 5C) after cytokines TNF-a and IL-6 persisted after poly I:C treatment, poly I:C (50 mg/ml) stimulation. CD11b2/2 macrophages displayed whereas the induction of IFN-b was abolished (Supplemental Fig. a significant reduction in the phosphorylation of JNK1/2, the p65 3). These data support the premise that type I IFN induction by subunit of NF-kB, and IkB, as well as the degradation of IkB-a extracellular poly I:C is TLR3 dependent (36, 37), but they also (Fig. 5B, 5C). The phosphorylation of p38 was also impaired in suggest that other proinflammatory cytokines can be induced by CD11b2/2 macrophages, although its reduction was less prom- extracellular poly I:C in a TLR3-independent manner. Interest- inent than the reduction in JNK phosphorylation (Fig. 5B). These ingly, the persisting levels of TNF-a andIL-6inthesettingof results indicate an important role for Mac-1 in the activation of BFA were significantly lower in CD11b2/2 macrophages than in JNK and NF-kB by poly I:C. We next treated WT macrophages WT controls, further supporting the involvement of Mac-1 in a with either a JNK inhibitor (SP600125; 5 mM) or an NF-kB in- TLR3-independent response to extracellular poly I:C. The Journal of Immunology 121 Downloaded from http://www.jimmunol.org/

FIGURE 5. Mac-1 altered the poly I:C–induced downstream signaling pathway. (A) Peritoneal macrophages from WT and CD11b2/2 mice were treated with 50 mg/ml poly I:C. The nuclear fraction was extracted from these cells 60 or 120 min after the treatment. The phosphorylation and the nuclear translocation of IRF3 protein were examined by using phospho-IRF3 (Ser396) Ab. Ab specific for nuclear marker HDAC2 was included to monitor loading errors. (B) MAPK pathway activation was determined by the phosphorylation of its major kinases p38 and JNK at the indicated time points (0, 15, 30, or C k k a k

60 min) in the whole-cell lysates. ( ) The activation of NF- B was indicated by the phosphorylation of p65 and I b (an inhibitory cofactor of NF- B) and by guest on September 25, 2021 the degradation of Ikba in the whole-cell lysis. (D) Peritoneal macrophages from WT mice were pretreated with the JNK inhibitor SP600125 and the NF-kB inhibitor Compound A for 30 min, followed by vehicle or poly I:C challenge. Secreted TNF-a and IL-12p40 were determined by ELISA after 24 h. Both inhibitors significantly reduced poly I:C–stimulated cytokine production. All immunoblot lanes are representative results from three independent experiments. Data are mean 6 SEM from three independent experiments in triplicate. *p , 0.05 versus WT controls, #p , 0.05 versus poly I:C–treated WT macrophages.

Mac-1–dependent, TLR3-independent activation of NOX2 Next, we demonstrated the contribution of NOX2 activation enhanced immune responses to poly I:C to poly I:C–elicited induction of proinflammatory cytokines. The a Previous studies showed that Mac-1 ligands can induce the pro- production of TNF- and IL-12p40 in poly I:C–treated macro- duction of superoxide free radicals in macrophages and neu- phages was significantly attenuated by the genetic deletion of trophils where NOX2 acts as a major source of superoxide during gp91 (Fig. 6C) or by the pharmacological inhibition of NOX2 inflammation (20, 30). Therefore, we examined the effect of the activity by apocynin, a widely used NOX2 inhibitor (Fig. 6D). The interaction of poly I:C and Mac-1 on NOX2 activation in mac- downstream signaling of NOX2 activation was determined in WT 2/2 rophages. As shown in Fig. 6A, poly I:C (50 mg/ml) induced and gp91 macrophages. The phosphorylation of p38, JNK, and 2/2 significant production of extracellular superoxide in WT macro- p65 was attenuated in gp91 macrophages compared with WT phages. On the contrary, poly I:C failed to do so in macrophages macrophages (Fig. 6E). Such reduced phosphorylation of p38, 2/2 deficient in CD11b or gp91 (the catalytic subunit of NOX2). JNK, and p65 in gp91 macrophages was also observed in However,PMA(acommonlyusedNOX2stimulator)triggered Mac-1–deficient macrophages (Fig. 5B, 5C). These results, com- robust superoxide production in both WT and CD11b2/2 mac- bined with the finding that Mac-1 is required for poly I:C–induced rophages; as expected, PMA was inactive in gp912/2 macro- NOX2 activation (Fig. 6A, 6B), indicated that poly I:C–elicited phages. Although superoxide radical is membrane impermeable, activation of Mac-1 activated NOX2 and, thereby, stimulated the k its downstream products, H2O2 and peroxynitrite (the reaction MAPK and NF- B pathways to promote proinflammatory cy- product of superoxide and NO), are membrane permeable. Con- tokine production. sistent with extracellular superoxide production, iROS were also Interestingly, poly I:C–induced superoxide production was not 2 2 elevated after poly I:C treatment in WT macrophages, whereas altered in TLR3 / macrophages compared with WT cells (Fig. deficiency in either Mac-1 or NOX2 abolished such iROS pro- 6F). In addition, the inhibition of NOX2 activity by apocynin led duction (Fig. 6B). These results indicate that poly I:C acti- to a similar reduction in the induction of TNF-a and IL-12p40 2 2 vates NOX2 to secrete superoxide anion in a Mac-1–dependent in TLR3 / and WT peritoneal macrophages (Fig. 6G). These manner. results indicate that TLR3 was not involved in the activation of 122 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY Downloaded from http://www.jimmunol.org/

FIGURE 6. Mac-1–dependent activation of NOX2 enhanced poly I:C–elicited immune response. Macrophages from WT, CD11b2/2,andgp912/2 mice were treated with vehicle, poly I:C (50 mg/ml), or PMA (50 nM). (A) Production of extracellular superoxide was measured by the SOD-inhibitable reduction 2 2 of WST-1. (B) iROS was determined by using the fluorescent DCFH-DA probe. (C) Secreted TNF-a and IL-12p40 were detected in WT and gp91 / by guest on September 25, 2021 macrophages treated with 50 mg/ml poly I:C for 24 h. (D) WT macrophages were pretreated with vehicle or apocynin (0.1 or 0.25 mM) for 15 min, and secreted TNF-a and IL-12p40 were measured after a 24-h treatment with 50 mg/ml poly I:C. (E) Western blot analysis showed impaired phosphorylation of p38, JNK, and p65 in gp912/2 macrophages challenged with 50 mg/ml poly I:C. (F) After stimulation with poly I:C (50 mg/ml) or PMA (50 nM), TLR32/2 and WT (TLR3+/+) macrophages produced a similar amount of extracellular superoxide. (G) TLR32/2 and WT (TLR3+/+) macrophages were pretreated with vehicle or apocynin (0.1 mM) for 15 min, and secreted TNF-a and IL-12p40 were measured 24 h after the cells were treated with 50 mg/ml of poly I:C. Data are mean 6 SEM from three independent experiments in triplicate. *p , 0.01 versus vehicle-treated controls, #p , 0.01 versus WT mice.

NOX2. Altogether, our findings demonstrated that poly I:C acti- findings suggest that Mac-1 acts as a bona fide PRR for extra- vates NOX2 to release superoxide anion and to participate in the cellular dsRNA to signal downstream inflammatory responses. induction of proinflammatory cytokines in a TLR3-independent, As a member of b2 integrins, Mac-1 has long been recognized but Mac-1–dependent, manner (Fig. 7). In addition, through fa- to participate in immune responses to infection and was sug- cilitating the internalization of poly I:C, Mac-1 activation may gested to be a therapeutic target (18, 38). For instance, Mac-1 (also amplify TLR3-dependent proinflammatory responses to dsRNA known as CR3) can bind complement components (such as iC3b) (Fig. 7). induced by invading pathogens to activate innate and adaptive immune responses (39, 40). CD11b-deficient mice were used to Discussion uncover the specific contribution of Mac-1 to many immune re- Extracellular dsRNA, as a potent stimulator of the innate immune sponses, such as FcR-triggered inflammation (41), phagocytosis of response, induces inflammatory cytokines and chemokines in many complement-opsonized particles (42), and immune reactions to cell types. However, the precise mechanism underlying its extra- bacteria or LPS (13, 21). Recent studies of Ross River virus cellular recognition and intracellular signaling remains largely (RRV)–associated arthritis/myositis indicated a role for Mac-1 in unknown. In this study, we demonstrate that Mac-1 functions as virus-induced inflammation (19). RRV causes severe leukocyte- a novel surface receptor for dsRNA in macrophages, as summa- mediated inflammatory responses in joint and skeletal muscle rized in Fig. 7. Specifically, Mac-1 binds extracellular dsRNA on tissues, leading to a chronic inflammatory manifestation similar 2 2 the surface of macrophages to mediate cellular immune responses. to arthritis/myositis. CD11b / mice exhibit decreased proin- Further mechanistic studies indicate that Mac-1 activation facili- flammatory and cytotoxic effectors (e.g., S100A9/S100A8 and tates the internalization of dsRNA and also activates NOX2, which IL-6) and less severe tissue damage compared with WT mice, enhances TLR3-dependent proinflammatory responses and trig- indicating the contribution of Mac-1 to RRV-induced chronic in- gers TLR3-independent oxidative immune responses. Thus, Mac- flammation. Although the investigators considered the activated 1 plays a distinct role in dsRNA-induced immune responses. Our complement components to be the stimulus for Mac-1, a direct The Journal of Immunology 123

volvement of Mac-1 in the phagocytosis of its ligands (20, 42), but little is known about the underlying mechanism. For dsRNA entry, type A scavenger receptors (SR-As) can mediate its internaliza- tion in fibroblasts (6) or epithelial cells (24). Consistent with these findings, inhibitors of SR-As (fucoidan or dextran sulfate) blocked the uptake of poly I:C into macrophages (Supplemental Fig. 4). The reduction in poly I:C internalization in macrophages deficient in CD11b or pretreated with an anti-CD11b Ab (Fig. 4A–C) in- dicates the participation of Mac-1 in the endocytosis of poly I:C. Thus, Mac-1 may assist or regulate the function of SR-As in the uptake of dsRNA. A recent study reported that Mac-1 and SR-As mediated the phagocytosis of degenerated myelin by macrophages through a PI3K-dependent pathway (45). Blockage of poly I:C entry into macrophages by a PI3K inhibitor (Fig. 4D) and attenuation of PI3K activation in CD11b2/2 macrophages (Fig. 4E) imply that Mac-1 facilitates the endocytosis of poly I:C through activation FIGURE 7. The role of Mac-1 in dsRNA-mediated signaling in the of the PI3K pathway. The endocytosis of extracellular dsRNA is macrophage. The membrane receptor TLR3 recognizes and binds inter- a critical step for antiviral TLR3 activation to induce type I IFN nalized extracellular dsRNA only in acidified endosomes and then acti- production via IRF3 (36, 37, 46). Although TLR3 partially lo- Downloaded from vated TLR3 induces type I IFN production via IRF3 and proinflammatory cates to the surface in some cell types (47–49), it only binds cytokine generation via the NF-kB pathway. The present study identifies dsRNA in the low-pH endosome (11). The reduction in poly I:C Mac-1 (CD11b/CD18 or CR3) as a novel PRR on the surface of macro- internalization (Fig. 4A, 4B), IRF3 activation (Fig. 5A), and pro- phages. Mac-1 senses and binds extracellular dsRNA to facilitate the en- duction of type I IFN (IFN-b)inCD11b2/2 macrophages (Fig. docytosis of extracellular dsRNA, thereby amplifying the TLR3-dependent 2A) indicates that Mac-1–mediated endocytosis of poly I:C par- signaling, or to activate oxidative NOX2 to produce ROS, thereby ticipates in the TLR3-dependent antiviral response. http://www.jimmunol.org/ activating the MAPK and NF-kB pathways to induce proinflammatory TLR3 is a well-established receptor for extracellular dsRNA. cytokine production in a TLR3-independent manner. However, extracellular dsRNA is still able to induce a significant amount of multiple proinflammatory cytokines in TLR32/2 mac- rophages or microglia (7, 12). The TLR3-independent inflamma- link between complement and Mac-1 was not demonstrated (19). tory responses triggered by dsRNA were confirmed by our similar Viral-produced dsRNA is a powerful viral PAMP, and it stimulates findings (Fig. 6G). Most importantly, the current study delineated both innate and adaptive antiviral immune responses (1). Previous a TLR3-independent, but Mac-1–dependent, mechanism that un- studies showed that Mac-1 acts as a membrane-bound PRR and derlines NOX2-associated oxidative immune responses to extra- binds a number of PAMPs and damage-associated molecular cellular dsRNA. First, poly I:C stimulated WT macrophages but by guest on September 25, 2021 patterns (22, 42, 43). Further studies to define the role of viral not macrophages deficient in Mac-1 or gp91 (the catalytic subunit dsRNA and Mac-1 activation in virus infection and host antiviral of NOX2) to generate extracellular superoxide and iROS (Fig. 6A, inflammatory response will identify novel disease mechanisms re- 6B). Second, TLR32/2 and WT macrophages released the same lated to virus pathogens. amount of extracellular superoxide upon poly I:C challenge (Fig. CD11b2/2 mice displayed impaired immune responses to poly I:C, 6F). Third, the genetic deletion or the pharmacological inhibition suggesting that Mac-1 may contribute to viral dsRNA-induced of NOX2 reduced poly I:C–elicited proinflammatory cytokine inflammation (Fig. 1). A dsRNA-binding assay and a cell sur- production (Fig. 6C, 6D). Fourth, poly I:C–mediated production face–binding assay revealed that Mac-1 can recognize poly I:C of proinflammatory cytokines in TLR32/2 macrophages was de- on the surface of macrophages, indicating that it is a novel surface creased by NOX2 inhibition (Fig. 6G). Last, the impaired acti- PRR for dsRNA (Fig. 3). The ligand-binding ability of Mac-1 may vation of MAPKs and NF-kB in macrophages deficient in Mac-1 be due to intrinsic properties of the I-domain (the major binding (Fig. 5B, 5C) or gp91 (Fig. 6E) further implies that Mac-1 can site for Mac-1 ligands) in the CD11b subunit (44). The I-domain induce inflammatory signaling through NOX2-induced iROS. In- is known to interact with a large array of ligands. However, the deed, growing evidence has indicated an important role for iROS binding profile of the I-domain is still unclear. The blockage of in inducing cellular immune responses to viral infection (50–52). poly I:C binding by the I-domain–binding ligand fibrinogen (Fig. Two independent groups showed recently that, in airway epithelial 3B–D) and the attenuation in poly I:C internalization by the anti- cells treated with either poly I:C or respiratory syncytial virus, CD11b Ab specific for residues 250–350 within the I-domain (Fig. NADPH oxidase is the major source of iROS and plays a crucial 4C) suggest that poly I:C may bind to the I-domain of CD11b. role in mediating downstream cellular immune responses (53, 54). Structural analysis of the I-domain reveals a metal ion–dependent In this article, we reported an important role for NOX2-associated adhesion site (44); this site is thought to prefer negative-charged ROS in dsRNA-mediated innate immune responses in macro- groups, such as aspartate or glutamate residues of ligands (e.g., phages. ICAM-1) (18). Such coordination may also be important for In summary, the current study demonstrated that Mac-1 acts as the binding of the negative-charged phosphate group of poly I:C a novel signaling PRR on the cell surface, sensing extracellular to this domain. However, our dsRNA-binding assay indicated dsRNA. We further elucidate that poly I:C activates inflammatory that the recognition was specific for dsRNA (poly I:C), but not oxidative enzyme NOX2 to produce ROS and to participate in the for ssRNA (poly C), which showed that the negative-charged induction of proinflammatory cytokines in a TLR3-independent, group is not a determining factor for the binding of poly I:C to but Mac-1–dependent manner. Through facilitating the internali- Mac-1. zation of poly I:C, Mac-1 activation also amplifies TLR3-depen- We next demonstrated that Mac-1 promotes endocytosis of dent proinflammatory responses to dsRNA. Our results provide extracellular poly I:C. 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