of April 10, 2019. This information is current as Responses RNA To Mediate Cellular Inflammatory Receptor for Extracellular Double-Stranded CD11b/CD18 (Mac-1) Is a Novel Surface Hong Wilson, Michael B. Fessler, Hui-Ming Gao and Jau-Shyong Hui Zhou, Jieying Liao, Jim Aloor, Hui Nie, Belinda C. ol.1202136 http://www.jimmunol.org/content/early/2012/12/02/jimmun published online 3 December 2012 J Immunol Material Supplementary 6.DC1 http://www.jimmunol.org/content/suppl/2012/12/03/jimmunol.120213 average * 4 weeks from acceptance to publication Fast Publication! • Every submission reviewed by practicing scientists No Triage! • from submission to initial decision Rapid Reviews! 30 days* • Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved. 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on April 10, 2019 http://www.jimmunol.org/ Downloaded from by guest on April 10, 2019 http://www.jimmunol.org/ Downloaded from
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of April 10, 2019.This information is current as
ResponsesRNA To Mediate Cellular InflammatoryReceptor for Extracellular Double-Stranded CD11b/CD18 (Mac-1) Is a Novel Surface
HongWilson, Michael B. Fessler, Hui-Ming Gao and Jau-Shyong Hui Zhou, Jieying Liao, Jim Aloor, Hui Nie, Belinda C.
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is published twice each month byThe Journal of Immunology
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–
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 virus-related inflammatory
diseases. The Journal of Immunology, 2013, 190: 000–000.
As the first line of host defense, the innate immune systemcombats numerous pathogens through a diverse set ofpattern recognition receptors (PRRs). These PRRs are
capable of identifying pathogen-associated molecular patterns(PAMPs) and initiating the innate immune response. During viralinfections, various virus-associated PAMPs are recognized by, andbind to, their respective PRRs, which induces robust host antiviraland immune responses. It is well known that viral-produced dsRNA
is a critical viral PAMP, which functions as a powerful stimulus ofboth innate and adaptive antiviral immune responses (1). ViraldsRNA is primarily generated during viral replication in virallyinfected cells (2). Once the infected cells die by lysis, the viraldsRNA is released into the extracellular space and becomes astable molecule; extracellular dsRNA is implicated in both localand systemic reactions associated with viral infections and mod-ulates both innate and adaptive immune responses (3–5). Exper-imentally, synthetic dsRNA, like polyinosinic:polycytidylic acid(poly I:C), has been commonly used to induce antiviral responsesin vivo or in vitro (6).The TLR3, RIG-I–like receptors RIG-I/MDA-5/LGP2, and
NOD-like receptor Nalp3 have been identified as PRRs for dsRNA(1). Based on cellular location, the membrane receptor TLR3 canrecognize internalized extracellular dsRNA, whereas both RIG-I–like receptors and NOD-like receptor Nalp3 are the cytoplasmicsensors that are likely to identify intracellular dsRNA generatedduring the intracellular viral life cycle (7–10). Interestingly, TLR3is only able to recognize and to bind dsRNA in acidified endo-somes (11), which suggests that extracellular dsRNA must firstbe internalized before it activates TLR3. Considering the evidencethat extracellular dsRNA is still able to induce a significant num-ber of proinflammatory cytokines in TLR3-knockout macrophagesor microglia (7, 11, 12), we hypothesize that other PRRs on thecell surface can serve as the first-line receptors to sense extra-cellular dsRNA and to mediate cellular responses.Previous studies indicated that the surface receptor integrin
CD11b/CD18, also known asmacrophage-1Ag (Mac-1), complementreceptor 3 (CR3), or aMb2, serves as a PRR to recognize PAMPs
*Laboratory of Toxicology and Pharmacology, National Institutes of EnvironmentalHealth Sciences, Research Triangle Park, NC 27709; †Laboratory of RespiratoryBiology, National Institutes of Environmental Health Sciences, Research TrianglePark, NC 27709; and ‡Laboratory of Neuroimmunology and Neuropharmacology,Model Animal Research Center, Nanjing University, Jiangsu 210061, China
1H.Z. and J.L. contributed equally to this work.
Received for publication August 2, 2012. Accepted for publication October 28, 2012.
This work was supported by the Intramural Research Program of the National In-stitutes of Health and the National Institute of Environmental Health Sciences.
Address correspondence and reprint requests to Dr. Hui-Ming Gao, Laboratory ofToxicology and Pharmacology, National Institutes of Environmental Health Sci-ences, P.O. Box 12233, MD F1-01 Research Triangle Park, NC 27709 or Laboratoryof Neuroimmunology and Neuropharmacology, Model Animal Research Center,Nanjing University, 12 Xuefu Road, Nanjing, Jiangsu 210061, China. E-mail address:[email protected]
The online version of this article contains supplemental material.
Abbreviations used in this article: BFA, bafilomycin A; CR3, complement receptor3; DCFH-DA, 29,79-dichlorodihydrofluorescein diacetate; iROS, intracellular reac-tive oxygen species; KO, knockout; Mac-1, macrophage-1 Ag; NOX2, phagocyteNADPH oxidase; NP-40, Nonidet P-40; PAMP, pathogen-associated molecular pat-tern; poly C, polycytidylic acid; poly I:C, polyinosinic:polycytidylic acid; PRR,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-disulfophenyl]-2H-tetrazolium; WT, wild-type.
www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202136
Published December 3, 2012, doi:10.4049/jimmunol.1202136 by guest on A
and damage-associated molecular patterns, such as Gram-nega-tive bacteria–derived LPS (13), aggregated b-amyloid (14), anddamage-associated alarmin HMGB1 (15). Mac-1, expressed onmany innate immune cells, such as monocytes, granulocytes, mac-rophages, and NK cells (16), has been implicated in various im-mune cell responses, including adhesion, migration, phagocytosis,chemotaxis, cellular activation, and cytotoxicity (17, 18). Further-more, Mac-1 was reported to participate in inflammatory diseasesassociated with Ross River virus infection (19) and to bind somenucleotides, such as oligodeoxynucleotide (20). These character-istics of Mac-1 prompted us to investigate the possibility that itserves as a PRR for extracellular dsRNA to regulate the innateimmune response.In this study, we identified Mac-1 as a novel surface receptor
mediating extracellular dsRNA-elicited cellular immune responses.Our results demonstrate that Mac-1 can recognize extracellulardsRNA on the cell surface and then mediate outside-in signaling,regulate dsRNA internalization, and mediate activation of phago-cyte NADPH oxidase (NOX2) to induce cellular immune responsesin macrophages. Our results provide new insight into how themacrophage recognizes extracellular signals associated with lyticvirus infections and mediates the innate immune response.
Materials and MethodsAnimal study
CD11b2/2 mice (Mac-1–deficient), gp912/2 mice (NADPH oxidase–de-ficient), and their age-matched wild-type (WT) control (C57BL/6J) wereobtained from The Jackson Laboratory (Bar Harbor, ME). TLR32/2 miceand their age-matched WT control (TLR+/+, C57BL/6NJ) were also ob-tained from The Jackson Laboratory. Housing and breeding of the animalswere performed humanely and with regard for alleviation of sufferingfollowing the National Institutes of Health’s Guide for the Care and Use ofLaboratory Animals (Institute of Laboratory Animal Resources 1996). Six-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-ronmental Health Sciences Animal Care and Use Committee.
An in vivo animal model involved immune activation by poly I:C(Sigma-Aldrich, St. Louis, MO; an average size of 300–750 bp). WT miceand CD11b2/2 (CD11b-knockout [KO]) mice were injected i.p. with poly I:C(5 mg/kg). Serum was collected 2 h later for cytokine measurement usingcommercially available ELISA kits (R&D Systems, Minneapolis, MN),and liver tissues were harvested for mRNA isolation and cytokine assayby quantitative real-time PCR.
Preparation of peritoneal macrophages and culture ofmacrophage cell line
Peritoneal macrophages from different strains were induced and har-vested, as previously described (21). Briefly, WT mice, CD11b-KO mice,and gp912/2 (gp91-KO) mice were injected i.p. with 2 ml 3% sterile thio-glycollate. After 4 d, peritoneal exudate macrophages were collected bylavage in 5 ml ice-cold RPMI 1640 medium (Invitrogen, CA), washedtwice in RPMI 1640, and preincubated in serum-free medium for 1 h. Thecells were then washed twice to remove nonadherent cells. Adherentmacrophages were cultured in RPMI 1640 medium containing 10% FBS(Invitrogen), 50 U/ml penicillin, and 50 mg/ml streptomycin at 37˚C ina humidified atmosphere of 5% CO2.
The murine macrophage cell line RAW 264.7 (ATCC TIB-71) wassuspension cultured in DMEM containing 10% FBS, 2 mM L-glutamine, 50U/ml penicillin, and 50 mg/ml streptomycin at 37˚C in a humidified at-mosphere of 5% CO2.
dsRNA-binding assay
The dsRNA-binding assay was performed as described previously (22,23). Briefly, poly I:C–conjugated agarose beads were prepared by incu-bating polycytidylic acid (poly C) agarose beads with polyinosinic acid(both from Sigma-Aldrich) at 4˚C overnight. Cyanogen bromide–activatedagarose beads (Sigma-Aldrich) were used as controls. Cell lysates wereprepared in Nonidet P-40 (NP-40) lysis buffer (10 mM Tris-HCl [pH 7.5],1% NP-40, 0.15 M NaCl, 1 mM EDTA, and protease inhibitor mixture)and centrifuged at 16,000 3 g for 15 min at 4˚C. For pull-down assays,
poly C–conjugated beads, poly I:C–conjugated beads, or noncoated emptybeads were equilibrated in lysis buffer containing RNase inhibitor (Invi-trogen) and incubated overnight with whole-cell lysates at 4˚C on a rotat-ing shaker. After centrifugation, beads were washed extensively and thenresuspended in sample buffer (Invitrogen). Samples were boiled for 10 minand centrifuged at 13,000 3 g for 1 min to discard insoluble pellets.Samples were then loaded onto SDS-PAGE, electroblotted onto poly-vinylidene difluoride membranes, and probed by immunoblot analysisagainst CD11b (Abcam, MA).
Flow cytometric analysis
Poly I:C was labeled with FITC using a Mirus RNA labeling kit (24).Labeled RNA was then purified with an RNeasy mini kit (QIAGEN, CA).Peritoneal macrophages or RAW 264.7 cells were washed twice and sus-pended in HBSS (Invitrogen). The cells (1 3 106 cells/ml) were kept onice for determination of surface binding of poly I:C by incubating with 10mg/ml FITC-labeled poly I:C for 20 min (20). After washing three timeswith cold HBSS, poly I:C–bound macrophages were detected by a BDLSR II Flow Cytometer. A live cell gate was made using forward versusside scatter plot. The surface binding was expressed as the mean fluores-cence intensities of the total calculated population. Internalization analysisof FITC-labeled poly I:C was performed using the same procedure asdescribed above for the surface binding process, with the exception thatcells were incubated at 37˚C instead of on ice (20), and trypan blue (1 mg/ml) was used to quench extracellular surface-bound fluorescence, as de-scribed previously (25). The internalization of poly I:C was expressed asthe mean fluorescence intensities of the total calculated population.
Confocal microscopy
Poly I:C and poly C were labeled with Cy3 for confocal observation (24).Peritoneal macrophages were seeded and grown in glass-bottom microwelldishes (MatTek, MA). Cells were then treated with 10 mg/ml Cy3-labeledpoly I:C or poly C, either on ice for 20 min to observe the surface bindingor at 37˚C for 15 or 30 min to determine the internalization. Stainedmacrophages were washed three times with ice-cold PBS and fixed with4% paraformaldehyde for further observation on a Zeiss LSM510 LaserScanning Confocal Microscope (Carl Zeiss Microimaging, Germany).
Quantitative real-time PCR
Total RNA of cells or tissues was isolated using TRIzol reagent and thenfirst-strand cDNA was synthesized using SuperScript Reverse Transcrip-tase (Invitrogen), according to the manufacturer’s protocols. After 0.5 mgtotal RNA was subjected to a reverse-transcription reaction, 2 ml cDNAwas amplified by quantitative real-time PCR analysis for the inductionof inflammatory cytokines using SYBR Green Master mix (Bio-Rad, CA)in a final volume of 25 ml. The following primers were used: mouse TNF-a:59-CATCTTCTCAAAATTCGAGTGGACAA-39 (forward), 59-TGGGAG-TAGACAAGGTAC-AACCC-39 (reverse); mouse IL-12p40: 59-GGAAG-CACGGCAGCAGAATA-39 (forward), 59-AACTTGAGGGAGAAGTAGG-AATGG-39 (reverse); mouse IFN-b: 59-ATGAGTGGTGGTTGCAGGC-39(forward), 59-TGACCTTTCAAATGCAGTAGATTCA-39 (reverse); mouseIL-6: 59-TTGCCTTCTTGGGACTGATGCT-39 (forward), 59-GTATCTC-TCTGAAGGACTCTG-G-39 (reverse); mouse GAPDH: 59-TTCACCAC-CATGGAGAAGGC-39 (forward), 59-GGCATGGACTGTGGTCATGA-39(reverse). Data were normalized to GAPDH expression.
Measurement of extracellular superoxide and intracellularreactive oxygen species
The release of superoxide was determined by measuring the superoxidedismutase (SOD)–inhibitable reduction of tetrazolium salt (2-[4-iodo-phenyl]-3-[4-nitrophenyl]-5-[2,4-disulfophenyl]-2H-tetrazolium [WST-1])as described (26). Briefly, peritoneal macrophages (1 3 105/well) weregrown overnight in 96-well plates in DMEM medium containing 10% FBSand switched to phenol red–free HBSS (100 ml/well). Fifty microliters ofHBSS containing vehicle, poly I:C, or PMA was then added to each well,followed by the addition of 50 ml WST-1 (1 mM) in HBSS, with or without600 U/ml SOD. The cells were incubated for 30 min at 37˚C, and theabsorbance was read at 450 nm with a SpectraMax Plus microplate spec-trophotometer (Molecular Devices, CA).
The production of intracellular reactive oxygen species (iROS) wasmeasured by the fluorescence probe 29,79-dichlorofluorescin diacetate(DCFH-DA), as described (26). Briefly, macrophages cultured overnight in96-well plates were incubated with 10 mM DCFH-DA (Invitrogen) for30 min at 37˚C. After two washes with HBSS buffer, cells were switched toHBSS containing 1% FBS. After the cells were incubated with vehicle orpoly I:C (50 mg/ml) at 37˚C for 30 min, the fluorescence density was read
2 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY
at 488 nm for excitation and at 525 nm for emission using a SpectraMaxGemini XS fluorescence microplate reader (Molecular Devices).
Nuclear extraction, gel electrophoresis, and Western blottinganalysis
Peritoneal macrophages fromWTand CD11b2/2mice were incubated withpoly I:C (50 mg/ml) or vehicle at 37˚C for 60 or 120 min and washed twicewith cold PBS. Nuclear extraction was performed at 4˚C with a nuclearextraction kit from Affymetrix, following the manufacturer’s instructions.Protein concentrations were determined using the DC protein assay (Bio-Rad). The whole-cell lysates from cultured cells were homogenized inradioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 8], 150 mMNaCl, 5 mM EDTA, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS,and 1:100 protease inhibitor mixture) with phosphatase inhibitors (10 mMNaF, 1 mM Na4P3O7, and 1 mM sodium orthovanadate), sonicated, andboiled for 10 min. Protein concentrations were determined using thebicinchoninic acid assay (Pierce). Protein samples were resolved on 4–12%SDS-PAGE, and immunoblot analysis was performed using Abs against theindicated signaling molecules (Cell Signaling Technology). An Ab againstGAPDH (Sigma-Aldrich) or HDAC2 (Santa Cruz Biotech) was included asan internal standard to monitor loading errors.
Immunofluorescence staining
Macrophages were grown and treated on glass coverslips, fixed in 4%paraformaldehyde, permeabilized with 0.4% Triton X-100, and blocked with5% goat serum. Samples were incubated sequentially with primary Abs andthe corresponding fluorescence probe–conjugated secondary Abs (Invitrogen)and then analyzed using a Zeiss LSM510 Confocal Microscope. To studythe distribution and colocalization of desired proteins, we analyzed imagesfrom $10 random fields from three independent experiments.
Statistical analysis
Data are expressed as mean 6 SEM. Statistical significance was assessedby ANOVA, followed by the Bonferroni t test, using GraphPad Prismsoftware (GraphPad Software, CA). A p value , 0.05 was consideredstatistically significant.
To determine whether Mac-1 is a potential candidate in sensingextracellular dsRNA and regulating the immune response, micedeficient in the CD11b subunit of Mac-1 and age-matched WTmice were injected i.p. with synthetic dsRNA poly I:C (5 mg/kg).
Measurement of serum inflammatory factors 2 h after poly I:Cinjection revealed decreased circulating TNF-a, IFN-b, and IL-12p40 in CD11b2/2 mice compared with WT controls (Fig. 1A).Similarly, the mRNA level of TNF-a, IL-12p40, IFN-b, and IL-6in the liver of CD11b2/2 mice was reduced (Fig. 1B). Collec-tively, Mac-1–deficient (CD11b2/2) mice displayed an impairedimmune response to poly I:C.
Mac-1 deficiency impaired immune responses to poly I:C inmacrophages
Because Mac-1 is expressed dominantly on macrophages that havea central role in the innate immune response, we next comparedinflammatory cytokine induction and investigated inflammatorysignaling cascades after poly I:C stimulation in CD11b2/2 and WTperitoneal macrophages. Consistent with our in vivo results, themRNA level of TNF-a, IL-12p40, IFN-b, and IL-6 was signifi-cantly lower in CD11b2/2 macrophages than in WT controls (Fig.2A). Secreted TNF-a and IL-12p40 in the supernatant showeda significant reduction in CD11b2/2 macrophages (Fig. 2B). IL-12p70 was detected after macrophages were challenged with 50mg/ml poly I:C for 24 h, reaching 2.8 6 0.3 pg/ml in WT mac-rophages and 0.8 6 0.6 pg/ml in CD11b2/2 macrophages. Asimilar pattern was observed for the poly I:C–elicited release ofNO, an inflammatory molecule (Fig. 2C). In contrast, stimulationof cultured peritoneal macrophages with TNF-a (50 ng/ml) orPMA (100 nM) induced indistinguishable immune responsesin CD11b2/2 macrophages and WT macrophages (SupplementalFig. 1). In addition, TLR3 expression in CD11b2/2 and WT mac-rophages was not different (Supplemental Fig. 2). Together, thesedata suggest that the observed impairment in the immune responseof CD11b2/2 macrophages to poly I:C is due to the functionaldeficiency of Mac-1 receptor to poly I:C stimulation and is not dueto a general immune deficit in these cells.
Poly I:C bound to Mac-1 receptor on the cell surface ofmacrophages
A dsRNA-binding assay using cell extracts from peritoneal mac-rophages or RAW 264.7 cells (a mouse macrophage–like cellline) 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 6SEM 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
Poly I:C–conjugated agarose beads pulled down the CD11b subunitof Mac-1, whereas unconjugated beads or beads conjugated withsingle-stranded poly C failed to do so (Fig. 3A). These resultsindicate that Mac-1 can specifically recognize double-strandedpoly I:C. To further confirm the interaction between poly I:Cand Mac-1, a surface binding assay was performed using flowcytometry and confocal imaging. FITC-labeled poly I:C boundto the surface of WT macrophages, whereas such surface bindingwas dramatically decreased in CD11b2/2 macrophages (Fig. 3B,3C). Furthermore, the surface binding was also significantly re-duced in WT macrophages or RAW 264.7 cells pretreated withfibrinogen, an endogenous ligand of Mac-1, whereas such an in-hibitory effect of fibrinogen was not observed in CD11b2/2
macrophages (Fig. 3B, 3C). Consistent with this result, confocalimaging (Fig. 3D) showed that CD11b deletion or fibrinogenpretreatment reduced the surface binding of poly I:C in macro-phages. In addition, immunofluorescence staining of CD11b re-vealed colocalization of CD11b with surface-bound Cy3–poly I:Cin WT macrophages (Fig. 3E). Further overlapping analysisshowed .75% overlap of Mac-1 with Cy3–poly I:C on the cellsurface (Fig. 3E). Overall, these data provide strong evidencethat Mac-1 can recognize and bind double-stranded poly I:Con the surface of the macrophage, indicating that Mac-1 may
mediate immune responses to poly I:C through outside-in sig-naling.
Mac-1 facilitated the internalization of poly I:C
It is well known that internalization of extracellular dsRNA is acrucial step to subsequent antiviral responses induced by endo-somal TLR3 activation (6, 11, 24). Our confocal imaging analysisdetected intracellular poly I:C within 15 min after Cy3-labeledpoly I:C was added to macrophages, and further accumulation ofintracellular poly I:C was observed with extended time (30 min).However, the uptake of poly I:C was significantly attenuated inCD11b2/2 macrophages compared with WT macrophages (Fig.4A). In contrast to the differential internalization of poly I:C,the uptake of Cy3-labeled poly C (ssRNA) was indistinguish-able in WT and CD11b2/2 peritoneal macrophages, indicating thespecificity of CD11b on dsRNA poly I:C (Fig. 4A). The flowcytometric analysis revealed ∼40% reduction in the uptake ofFITC-labeled poly I:C into CD11b2/2 macrophages comparedwith WT macrophages (Fig. 4B). These results indicate a criticalrole for Mac-1 in the internalization of poly I:C into macrophages.Such a conclusion was confirmed by the finding that Mac-1–blocking Ab significantly attenuated the uptake of poly I:C intoWT macrophages but not CD11b2/2 macrophages (Fig. 4C).
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 RNAwas 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.
4 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY
As a common cellular signaling pathway of integrins (27, 28),the activation of PI3K was specifically described in previousstudies on Mac-1–signaling transduction (29, 30). PI3K was alsoimplicated in the regulation of endocytosis and intracellular mem-brane trafficking in macrophages (31, 32). In this study, we foundthat wortmannin, a PI3K inhibitor, blocked the uptake of poly I:Cinto the macrophage (Fig. 4D). Importantly, when challenged withpoly I:C, the phosphorylation of AKT, a key kinase downstream ofPI3K, was significantly impaired in CD11b2/2 macrophages (Fig.4E). Taken together, the reduced uptake of poly I:C in the settingof PI3K inhibition in WT macrophages and the diminished PI3K
activation in CD11b2/2 macrophages suggest that Mac-1 pro-motes the internalization of poly I:C through activating the PI3Kpathway. Thus, Mac-1 may facilitate immune responses in mac-rophages through enhancing the uptake of extracellular poly I:Cinto the endosome, where TLR3 resides.
It is well known that poly I:C activates IRF3 signaling via TLR3,leading to the induction of IFN and IFN-inducible genes. Theattenuation of poly I:C–elicited IFN-b induction in CD11b2/2
macrophages and mice (Fig. 1) suggests that Mac-1 might par-
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
ticipate in TLR3-dependent IRF3 activation. IRF3 is present inan inactive form in the cytoplasm in unstimulated innate immunecells; upon the phosphorylation of its serine, the active IRF3translocates to the nucleus, leading to the transcription of IFN andIFN-inducible genes. We next examined the phosphorylation andthe nuclear translocation of IRF3 protein by using the nuclearfraction of WT and CD11b2/2 macrophages. To rule out possibleinterference from any contaminated cytoplasmic IRF3 in thenuclear fraction, we used phospho-IRF3 (Ser396) Ab to determineIRF3 activation. As expected, we observed decreases in poly I:C–induced phosphorylation and nuclear translocation of IRF3 inCD11b2/2 macrophages compared with WT macrophages (Fig.5A), indicating that Mac-1 enhanced TLR3-dependent IRF3 ac-tivation.To further determine the downstream-signaling cascades of
poly I:C–elicited Mac-1 activation, the MAPK and NF-kB path-ways, two major signaling pathways responsible for proinflam-matory cytokine induction, were assayed in WT and CD11b2/2
macrophages. WT macrophages exhibited time-dependent phos-phorylation in the MAPK and NF-kB pathways (Fig. 5B, 5C) afterpoly I:C (50 mg/ml) stimulation. CD11b2/2 macrophages displayeda significant reduction in the phosphorylation of JNK1/2, the p65subunit of NF-kB, and IkB, as well as the degradation of IkB-a(Fig. 5B, 5C). The phosphorylation of p38 was also impaired inCD11b2/2 macrophages, although its reduction was less prom-inent than the reduction in JNK phosphorylation (Fig. 5B). Theseresults indicate an important role for Mac-1 in the activation ofJNK and NF-kB by poly I:C. We next treated WT macrophageswith either a JNK inhibitor (SP600125; 5 mM) or an NF-kB in-
hibitor (Compound A; 1 mM) (33) for 30 min and then challengedthe cells with poly I:C. Both inhibitors significantly suppressed therelease of TNF-a and IL-12p40, which further demonstrated theimportance of the activation of JNK and NF-kB in the poly I:C–induced immune response (Fig. 5D). Together, these results sug-gest that Mac-1 contributes to the poly I:C–induced immune re-sponse in macrophages through promoting the activation of theMAPK and NF-kB pathways.In response to extracellular dsRNA, NF-kB activation is thought
to be involved in the downstream signaling of TLR3 activation (7),but the engagement of TLR3 does not seem to be required for theactivation of MAPK signaling in macrophages (34). Thus, ourfindings described above (Fig. 5) suggest that, in addition to thecontribution to the TLR3–NF-kB pathway by facilitating dsRNAinternalization, Mac-1 may induce TLR3-independent signalingpathways, such as the MAPK pathway. Bafilomycin A (BFA), aninhibitor of vacuolar-type H+-ATPase, blocks the acidification ofthe endosome and, therefore, inhibits TLR3 activation (35). In thepresence of BFA, considerable induction of the proinflammatorycytokines TNF-a and IL-6 persisted after poly I:C treatment,whereas the induction of IFN-b was abolished (Supplemental Fig.3). These data support the premise that type I IFN induction byextracellular poly I:C is TLR3 dependent (36, 37), but they alsosuggest that other proinflammatory cytokines can be induced byextracellular poly I:C in a TLR3-independent manner. Interest-ingly, the persisting levels of TNF-a and IL-6 in the setting ofBFA were significantly lower in CD11b2/2 macrophages than inWT controls, further supporting the involvement of Mac-1 in aTLR3-independent response to extracellular poly I:C.
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.
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.
6 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY
Mac-1–dependent, TLR3-independent activation of NOX2enhanced immune responses to poly I:C
Previous studies showed that Mac-1 ligands can induce the pro-duction of superoxide free radicals in macrophages and neu-trophils where NOX2 acts as a major source of superoxide duringinflammation (20, 30). Therefore, we examined the effect of theinteraction of poly I:C and Mac-1 on NOX2 activation in mac-rophages. As shown in Fig. 6A, poly I:C (50 mg/ml) inducedsignificant production of extracellular superoxide in WT macro-phages. On the contrary, poly I:C failed to do so in macrophagesdeficient in CD11b or gp91 (the catalytic subunit of NOX2).However, PMA (a commonly used NOX2 stimulator) triggeredrobust superoxide production in both WT and CD11b2/2 mac-rophages; as expected, PMA was inactive in gp912/2 macro-phages. Although superoxide radical is membrane impermeable,its downstream products, H2O2 and peroxynitrite (the reactionproduct of superoxide and NO), are membrane permeable. Con-sistent with extracellular superoxide production, iROS were alsoelevated after poly I:C treatment in WT macrophages, whereasdeficiency in either Mac-1 or NOX2 abolished such iROS pro-duction (Fig. 6B). These results indicate that poly I:C acti-vates NOX2 to secrete superoxide anion in a Mac-1–dependentmanner.
Next, we demonstrated the contribution of NOX2 activationto poly I:C–elicited induction of proinflammatory cytokines. The
production of TNF-a and IL-12p40 in poly I:C–treated macro-
phages was significantly attenuated by the genetic deletion of
gp91 (Fig. 6C) or by the pharmacological inhibition of NOX2
activity by apocynin, a widely used NOX2 inhibitor (Fig. 6D). The
downstream signaling of NOX2 activation was determined in WT
and gp912/2 macrophages. The phosphorylation of p38, JNK, and
p65 was attenuated in gp912/2 macrophages compared with WT
macrophages (Fig. 6E). Such reduced phosphorylation of p38,
JNK, and p65 in gp912/2 macrophages was also observed in
Mac-1–deficient macrophages (Fig. 5B, 5C). These results, com-
bined with the finding that Mac-1 is required for poly I:C–induced
NOX2 activation (Fig. 6A, 6B), indicated that poly I:C–elicited
activation of Mac-1 activated NOX2 and, thereby, stimulated the
MAPK and NF-kB pathways to promote proinflammatory cy-
tokine production.Interestingly, poly I:C–induced superoxide production was not
altered in TLR32/2 macrophages compared with WT cells (Fig.
6F). In addition, the inhibition of NOX2 activity by apocynin led
to a similar reduction in the induction of TNF-a and IL-12p40
in TLR32/2 and WT peritoneal macrophages (Fig. 6G). These
results indicate that TLR3 was not involved in the activation of
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
60 min) in the whole-cell lysates. (C) The activation of NF-kB was indicated by the phosphorylation of p65 and Ikba (an inhibitory cofactor of NF-kB) and
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 mean6 SEM from three independent experiments in triplicate. *p, 0.05 versus WT controls, #p, 0.05 versus poly I:C–treated WT
NOX2. Altogether, our findings demonstrated that poly I:C acti-vates NOX2 to release superoxide anion and to participate in theinduction of proinflammatory cytokines in a TLR3-independent,but Mac-1–dependent, manner (Fig. 7). In addition, through fa-cilitating the internalization of poly I:C, Mac-1 activation mayamplify TLR3-dependent proinflammatory responses to dsRNA(Fig. 7).
DiscussionExtracellular dsRNA, as a potent stimulator of the innate immuneresponse, induces inflammatory cytokines and chemokines in manycell types. However, the precise mechanism underlying its extra-cellular recognition and intracellular signaling remains largelyunknown. In this study, we demonstrate that Mac-1 functions asa novel surface receptor for dsRNA in macrophages, as summa-rized in Fig. 7. Specifically, Mac-1 binds extracellular dsRNA onthe surface of macrophages to mediate cellular immune responses.Further mechanistic studies indicate that Mac-1 activation facili-tates the internalization of dsRNA and also activates NOX2, whichenhances TLR3-dependent proinflammatory responses and trig-gers TLR3-independent oxidative immune responses. Thus, Mac-1 plays a distinct role in dsRNA-induced immune responses. Our
findings suggest that Mac-1 acts as a bona fide PRR for extra-cellular dsRNA to signal downstream inflammatory responses.As a member of b2 integrins, Mac-1 has long been recognized
to participate in immune responses to infection and was sug-gested to be a therapeutic target (18, 38). For instance, Mac-1 (alsoknown as CR3) can bind complement components (such as iC3b)induced by invading pathogens to activate innate and adaptiveimmune responses (39, 40). CD11b-deficient mice were used touncover the specific contribution of Mac-1 to many immune re-sponses, such as FcR-triggered inflammation (41), phagocytosis ofcomplement-opsonized particles (42), and immune reactions tobacteria or LPS (13, 21). Recent studies of Ross River virus(RRV)–associated arthritis/myositis indicated a role for Mac-1 invirus-induced inflammation (19). RRV causes severe leukocyte-mediated inflammatory responses in joint and skeletal muscletissues, leading to a chronic inflammatory manifestation similarto arthritis/myositis. CD11b2/2 mice exhibit decreased proin-flammatory and cytotoxic effectors (e.g., S100A9/S100A8 andIL-6) and less severe tissue damage compared with WT mice,indicating the contribution of Mac-1 to RRV-induced chronic in-flammation. Although the investigators considered the activatedcomplement components to be the stimulus for Mac-1, a direct
FIGURE 6. Mac-1–dependent activation of NOX2 enhanced poly I:C–elicited immune response. Macrophages from WT, CD11b2/2, and gp912/2 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
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 gp912/2
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.
8 dsRNA-ELICITED MAC-1 ACTIVATION ON THE INNATE IMMUNITY
link between complement and Mac-1 was not demonstrated (19).Viral-produced dsRNA is a powerful viral PAMP, and it stimulatesboth innate and adaptive antiviral immune responses (1). Previousstudies showed that Mac-1 acts as a membrane-bound PRR andbinds a number of PAMPs and damage-associated molecularpatterns (22, 42, 43). Further studies to define the role of viraldsRNA and Mac-1 activation in virus infection and host antiviralinflammatory response will identify novel disease mechanisms re-lated to virus pathogens.CD11b2/2mice displayed impaired immune responses to poly I:C,
suggesting that Mac-1 may contribute to viral dsRNA-inducedinflammation (Fig. 1). A dsRNA-binding assay and a cell sur-face–binding assay revealed that Mac-1 can recognize poly I:Con the surface of macrophages, indicating that it is a novel surfacePRR for dsRNA (Fig. 3). The ligand-binding ability of Mac-1 maybe due to intrinsic properties of the I-domain (the major bindingsite for Mac-1 ligands) in the CD11b subunit (44). The I-domainis known to interact with a large array of ligands. However, thebinding profile of the I-domain is still unclear. The blockage ofpoly I:C binding by the I-domain–binding ligand fibrinogen (Fig.3B–D) and the attenuation in poly I:C internalization by the anti-CD11b Ab specific for residues 250–350 within the I-domain (Fig.4C) suggest that poly I:C may bind to the I-domain of CD11b.Structural analysis of the I-domain reveals a metal ion–dependentadhesion site (44); this site is thought to prefer negative-chargedgroups, such as aspartate or glutamate residues of ligands (e.g.,ICAM-1) (18). Such coordination may also be important forthe binding of the negative-charged phosphate group of poly I:Cto this domain. However, our dsRNA-binding assay indicatedthat the recognition was specific for dsRNA (poly I:C), but notfor ssRNA (poly C), which showed that the negative-chargedgroup is not a determining factor for the binding of poly I:C toMac-1.We next demonstrated that Mac-1 promotes endocytosis of
extracellular poly I:C. In fact, several reports revealed the in-
volvement of Mac-1 in the phagocytosis of its ligands (20, 42), butlittle 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 thesefindings, inhibitors of SR-As (fucoidan or dextran sulfate) blockedthe uptake of poly I:C into macrophages (Supplemental Fig. 4).The reduction in poly I:C internalization in macrophages deficientin 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 theuptake of dsRNA. A recent study reported that Mac-1 and SR-Asmediated the phagocytosis of degenerated myelin by macrophagesthrough a PI3K-dependent pathway (45). Blockage of poly I:C entryinto macrophages by a PI3K inhibitor (Fig. 4D) and attenuation ofPI3K activation in CD11b2/2 macrophages (Fig. 4E) imply thatMac-1 facilitates the endocytosis of poly I:C through activationof the PI3K pathway. The endocytosis of extracellular dsRNA isa critical step for antiviral TLR3 activation to induce type I IFNproduction via IRF3 (36, 37, 46). Although TLR3 partially lo-cates to the surface in some cell types (47–49), it only bindsdsRNA in the low-pH endosome (11). The reduction in poly I:Cinternalization (Fig. 4A, 4B), IRF3 activation (Fig. 5A), and pro-duction of type I IFN (IFN-b) in CD11b2/2 macrophages (Fig.2A) indicates that Mac-1–mediated endocytosis of poly I:C par-ticipates in the TLR3-dependent antiviral response.TLR3 is a well-established receptor for extracellular dsRNA.
However, extracellular dsRNA is still able to induce a significantamount of multiple proinflammatory cytokines in TLR32/2 mac-rophages or microglia (7, 12). The TLR3-independent inflamma-tory responses triggered by dsRNAwere confirmed by our similarfindings (Fig. 6G). Most importantly, the current study delineateda TLR3-independent, but Mac-1–dependent, mechanism that un-derlines NOX2-associated oxidative immune responses to extra-cellular dsRNA. First, poly I:C stimulated WT macrophages butnot macrophages deficient in Mac-1 or gp91 (the catalytic subunitof NOX2) to generate extracellular superoxide and iROS (Fig. 6A,6B). Second, TLR32/2 and WT macrophages released the sameamount of extracellular superoxide upon poly I:C challenge (Fig.6F). Third, the genetic deletion or the pharmacological inhibitionof NOX2 reduced poly I:C–elicited proinflammatory cytokineproduction (Fig. 6C, 6D). Fourth, poly I:C–mediated productionof proinflammatory cytokines in TLR32/2 macrophages was de-creased by NOX2 inhibition (Fig. 6G). Last, the impaired acti-vation of MAPKs and NF-kB in macrophages deficient in Mac-1(Fig. 5B, 5C) or gp91 (Fig. 6E) further implies that Mac-1 caninduce inflammatory signaling through NOX2-induced iROS. In-deed, growing evidence has indicated an important role for iROSin inducing cellular immune responses to viral infection (50–52).Two independent groups showed recently that, in airway epithelialcells treated with either poly I:C or respiratory syncytial virus,NADPH oxidase is the major source of iROS and plays a crucialrole in mediating downstream cellular immune responses (53, 54).In this article, we reported an important role for NOX2-associatedROS in dsRNA-mediated innate immune responses in macro-phages.In summary, the current study demonstrated that Mac-1 acts as
a novel signaling PRR on the cell surface, sensing extracellulardsRNA. We further elucidate that poly I:C activates inflammatoryoxidative enzyme NOX2 to produce ROS and to participate in theinduction of proinflammatory cytokines in a TLR3-independent,but Mac-1–dependent manner. Through facilitating the internali-zation of poly I:C, Mac-1 activation also amplifies TLR3-depen-dent proinflammatory responses to dsRNA. Our results providenew insight into how macrophages recognize extracellular signals
FIGURE 7. The role of Mac-1 in dsRNA-mediated signaling in the
macrophage. The membrane receptor TLR3 recognizes and binds inter-
nalized extracellular dsRNA only in acidified endosomes and then acti-
vated TLR3 induces type I IFN production via IRF3 and proinflammatory
cytokine generation via the NF-kB pathway. The present study identifies
Mac-1 (CD11b/CD18 or CR3) as a novel PRR on the surface of macro-
phages. Mac-1 senses and binds extracellular dsRNA to facilitate the en-
docytosis of extracellular dsRNA, thereby amplifying the TLR3-dependent
signaling, or to activate oxidative enzyme NOX2 to produce ROS, thereby
activating the MAPK and NF-kB pathways to induce proinflammatory
associated with lytic virus infections and identify a potentialtherapeutic target for virus-related inflammatory diseases.
AcknowledgmentsWe thank Anthony Lockhart for assistance with animal colony manage-
ment and maintenance. We also thank Dr. Monte S. Willis (University of
North Carolina, Chapel Hill, NC) for reviewing this manuscript.
DisclosuresThe authors have no financial conflicts of interest.
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