T Lymphocytes + Human CD4 Proinflammatory Myeloid-Related Proteins in Are Both Accentuated by the HIV-1 Transcription and Virus

HIV-1 Transcription and Virus Production Are Both Accentuated by the Proinflammatory Myeloid-Related Proteins in Human CD4 + T Lymphocytes This information is current as of September 28, 2021. Carle Ryckman, Gilles A. Robichaud, Jocelyn Roy, Réjean Cantin, Michel J. Tremblay and Philippe A. Tessier J Immunol 2002; 169:3307-3313; ; doi: 10.4049/jimmunol.169.6.3307

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References This article cites 64 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/169/6/3307.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

HIV-1 Transcription and Virus Production Are Both Accentuated by the Proinﬂammatory Myeloid-Related Proteins in Human CD4؉ T Lymphocytes1

Carle Ryckman,2 Gilles A. Robichaud,2 Jocelyn Roy,2 Re«jean Cantin, Michel J. Tremblay,3 and Philippe A. Tessier3

S100A8, S100A9, and S100A12, collectively known as myeloid-related proteins (MRPs), are highly expressed by the myeloid cell lineage and are found in the extracellular milieu during infections and inflammatory conditions. Recent data showed high levels .of MRPs in the serum of HIV type 1 (HIV-1)-infected patients which correlated with disease progression and low CD4؉ counts Therefore, we set out to investigate the effect of MRPs on HIV-1 replication. We observed a 4- to 5-fold induction of virus production in J1.1, a human T lymphoid cell line latently infected with HIV-1, following treatment with MRPs. Using luciferase- Downloaded from based reporter gene assays, we demonstrated that MRPs induce a dose- and time-dependent activation of the HIV-1 long terminal repeat promoter region that could be blocked by specific anti-MRP polyclonal Abs and by physical denaturation of these proteins. The MRP-mediated induction was acting through the HIV-1 enhancer sequence and was dependent upon NF-␬B activity. These latter results were also confirmed by EMSA experiments conducted in Jurkat cells and freshly isolated PBMCs. In conclusion, we -demonstrate that MRPs induce HIV-1 transcriptional activity and viral replication in infected CD4؉ T-lymphocytes at concen trations similar to those found in the serum of HIV-1-infected patients. The Journal of Immunology, 2002, 169: 3307Ð3313. http://www.jimmunol.org/

pportunistic infections and inflammatory situations fre- activated endothelial cells (13), and keratinocytes (14, 15). quently result in an enhanced viral load in individuals S100A8 and S100A9 associate noncovalently to form homodimers O infected with HIV type 1 (HIV-1).4 Various cytokines and the heterodimer S100A8/A9 (16Ð18). MRPs have been shown and soluble factors are released in the serum of these patients dur- to exert several extracellular proinflammatory activities. For ex- ing opportunistic pathologies, some of which may directly con- ample, S100A9 stimulates neutrophil adhesion to fibrinogen (19), tribute to the virus replicative cycle by positively affecting the while S100A8 has been reported to be an extremely potent che- regulatory elements of HIV-1 (i.e., the long terminal repeat (LTR)) motactic factor for murine myeloid cells (20). In addition, by guest on September 28, 2021 (1Ð3). Virus production is thus influenced by the microenviron- S100A8/A9 enhances migration of monocytes across endothelial ment that is surrounding virus-infected cells. cells (21). Endothelial cells incubated with S100A12 express in- The myeloid-related proteins (MRPs) S100A8, S100A9, and creased levels of ICAM-1 and VCAM-1 due to an induction of S100A12 are calcium-binding proteins that belong to the S100 NF-␬B, resulting in the adhesion of lymphocytes to endothelial protein family (4, 5). Expression of the MRPs is mostly confined cells (22). In addition, S100A12 mediates expression of TNF-␣ to neutrophils, monocytes (5, 6), and activated macrophages (7Ð and IL-1␤ when incubated with a murine macrophage cell line 10). They are also expressed by certain epithelial cells (11, 12), (22). Local secretion of MRPs has been detected in chronic periodon- Centre de Recherche en Infectiologie, Pavillon Centre Hospitalier de l’Universite« tal infections (23, 24) and particularly high concentrations were Laval, Centre Hospitalier Universitaire de Que«bec, and De«partement de Biologie Me«dicale, Faculte«deMe«decine, Universite«Laval, Que«bec, Canada found in the serum of patients suffering from chronic bronchitis, cystic fibrosis, and tuberculosis (7, 16, 25, 26). High levels of Received for publication May 20, 2002. Accepted for publication July 18, 2002. serum S100A8/A9 have also been found in HIV-1-seropositive The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance patients with advanced immune deficiency (Centers for Disease with 18 U.S.C. Section 1734 solely to indicate this fact. Control and Prevention stages II and III) (27Ð29). Elevated 1 This study was supported by an Arthritis Society of Canada Grant (to P.A.T.) and S100A8/A9 levels in AIDS patients correlated with the onset of Canadian Institutes of Health Research (CIHR) HIV/AIDS Research Program Grants HOP-14438, MOP-37781, and HOP-15575 (to M.J.T.). C.R. is supported by a stu- opportunistic infections. Other studies demonstrated high levels of dentship from the K.H. Hunter Charitable Foundation and the CIHR. J.R. is the S100A8/A9 in cerebrospinal fluids, saliva, and inflamed gingiva recipient of a CIHR Doctoral Award, while G.A.R. holds a PhD Fellowship from the from AIDS patients during ongoing opportunistic infections (30Ð Fonds de la Recherche en Sante«du Que«bec-Fonds pour la Formation de Chercheurs et l’Aide a`la Recherche Health Program. P.A.T. holds a Scholarship Award from the 32). Some of these studies revealed that MRP levels were inversely Arthritis Society of Canada and M.J.T. has been allocated a Tier 1 Canada Research proportional to CD4ϩ T cell counts and in linear correlation with Chair in Human Immunoretrovirology. viral load in patients with advanced HIV-1 disease (i.e., AIDS) 2 C.R., G.A.R., and J.R. contributed equally to this work. (28, 29). More recently, S100A8 derived from cervico-vaginal se- 3 Address correspondence and reprint requests to Dr. Philippe A. Tessier and Dr. cretions was shown to induce virus production in a latently HIV- Michel J. Tremblay, Centre de Recherche en Infectiologie, Local RC709, Pavillon Centre Hospitalier de l’Universite«Laval, Centre Hospitalier Universitaire de Que«bec, 1-infected monocytoid cell line (33). Despite the numerous data 2705 Boulevard Laurier, Ste-Foy, Que«bec, Canada G1V 4G2. E-mail addresses: presenting an association between MRPs and HIV-1 pathogenesis, [email protected] and [email protected] the understanding of this correlation remains elusive. 4 Abbreviations used in this paper: HIV-1, HIV type 1; LTR, long terminal repeat; MRP, myeloid-related protein; iono, ionomycin; RAGE, receptor for advanced gly- Regulation of HIV-1 is intimately linked to the activity of its cation end-product. promoter positioned in the 5Ј LTR sequence which is, in turn,

dependent upon an enhancer region (Ϫ104/Ϫ81) necessary for a for 16 h at room temperature. rMRPs were eluted with PBS. The digestion productive HIV-1 infection cycle in infected cells (34Ð37). The and elution process was repeated once to cleave the remaining undigested enhancer region is characterized by two tandemly positioned recombinant proteins. Contaminating thrombin was extracted from the elu- ␬ ates using streptavidin-agarose and contaminating LPS was removed by NF- B elements and, as demonstrated recently, a NFAT binding polymyxin B-agarose (Pierce, Rockford, IL). Eluted proteins were ana- site (35, 38Ð40). Since then, it has been shown that the NF-␬B and lyzed by immunoblot and SDS-PAGE. Control for bacterial contamination NFAT transcriptional elements act synergistically upon the viral of the recombinant proteins consisted of untransformed HMS174 stimu- enhancer region to positively modulate HIV-1 transcriptional ac- lated, lysed, and processed for purification like the recombinant proteins. Stimulation experiments with this control were conducted using the same tivity and replication (40Ð44). Therefore, HIV-1 replication is dilutions as for MRPs. Proteins were inactivated by heating at 90¡C for 15 tightly linked to T cell activation due to overlapping signal trans- min or by incubation with polyclonal Abs (anti-MRPs) for 30 min at room duction requirements between cellular gene expression and acti- temperature before incubation with T lymphocytes. Polyclonal antisera vation of viral regulatory elements. against recombinant human S100A8, S100A9, and S100A12 were gener- We set out to assess the possible modulatory effect of recom- ated after repeated injections in New Zealand White rabbits at 4-wk intervals. binant S100A8, S100A9, S100A12, and S100A8/A9 proteins on the life cycle of HIV-1 in a cell type recognized as a major cellular reservoir of this retrovirus, i.e., the CD4ϩ T cell. Using Jurkat- ELISA derived cell lines and freshly isolated PBMCs, we show in this The measurement of virus-encoded p24 protein was determined by an in- study that MRPs induce HIV-1 transcriptional activity and virus house enzymatic assay and was described previously (50). gene expression through a NF-␬B-dependent signal transduction pathway. These results suggest that MRPs should be considered as Downloaded from secreted factors contributing to the pathogenesis of HIV-1 Transfections infection. Transient transfections of T cell lines using the DEAE-dextran method were performed as previously described (51). Briefly, cells (5 ϫ 106) were first washed once in transfection solution buffer (137 mM NaCl, 25 mM Materials and Methods Tris-HCl (pH 7.4), 5 mM KCl, 0.6 mM Na HPO , 0.5 mM MgCl , and 0.7 Cells used in the current study 2 4 2 mM CaCl2) and incubated in 0.5 ml of transfection solution containing 15 ␮ ␮ http://www.jimmunol.org/ The lymphoid Jurkat T cell line (45) was obtained from the American Type g of DNA from the indicated plasmid(s) and 500 g/ml of DEAE-dextran Culture Collection (Manassas, VA). The 1G5 T cell line is a Jurkat deriv- (final concentration; Pharmacia, Piscataway, NJ) for 25 min at room tem- ϫ 6 ative that harbors two stably integrated constructs constituted of the lucif- perature. Thereafter, cells were diluted at a concentration of 1 10 /ml using complete culture medium supplemented with 100 ␮M of chloroquine erase gene under the control of the HIV-1SF2 LTR (46). 1G5 cells were obtained through the National Institutes of Health AIDS Repository Re- (Sigma-Aldrich) and transferred into 6-well plates. After 45 min of incu- agent Program. We also made use of J1.1, a Jurkat E6.1-derived T cell line bation at 37¡C, cells were centrifuged, resuspended in complete culture that is latently infected with HIV-1 (47). All of these cell lines were main- medium, and incubated at 37¡C for 24 h. To minimize variations in plasmid tained in complete culture medium made of RPMI 1640 supplemented with transfection efficiencies, cells were transfected in bulk and were next sep- 10% FBS (HyClone Laboratories, Logan, UT), glutamine (2 mM), peni- arated into various treatment groups. cillin G (100 U/ml), and streptomycin (100 ␮g/ml). Primary PBMCs from

healthy donors were isolated by Ficoll-Hypaque density gradient centrif- by guest on September 28, 2021 ugation. Before being used for preparation of nuclear extracts, these cells Stimulations and reporter gene assays were first cultured in complete RPMI medium containing 20% FBS in the Cells were seeded at a density of 105 cells per well (100 ␮l) in 96-well ␮ presence of 3 g/ml of PHA-P (Sigma-Aldrich, St. Louis, MO) and 30 flat-bottom plates. Cells were either left unstimulated or treated with PMA U/ml of recombinant human IL-2 for 3 days at 37¡C under a 5% CO2. (20 ng/ml; Sigma-Aldrich)/ionomycin (iono, 1 ␮M; Calbiochem, La Jolla, ␮ Plasmids CA) or the indicated concentrations of MRPs in a final volume of 200 l for8hat37¡C unless otherwise specified. Luciferase activity was then The p␬B-TATA-LUC plasmid containing the HIV-1 ␬B enhancer region determined with a Dynex 96-well plate luminometer device (Chantilly, (Ϫ105/Ϫ70) and a TATA box placed upstream of the luciferase reporter VA) following a previously described protocol (51). gene was used in this study (48). This plasmid was a generous gift from Dr. W. C. Greene (The J. Gladstone Institutes, San Francisco, CA). pNF-␬B- LUC contains five consensus NF-␬B-binding sequences placed upstream to Preparation of nuclear extracts and EMSA the luciferase gene along with a minimal promoter (Stratagene, La Jolla, ϫ 6 CA). The dominant negative I␬B␣-expressing vector pCMV-I␬B␣ Cells (5 10 ) were either left untreated or incubated for1hat37¡C with S32A/36A PMA/iono or MRPs (10 ␮ has been described previously (48) (a kind gift from Dr. W.C. Greene). The g/ml). Incubation with the stimulating agents DNA filler pCMV-EcoRV/SmaI was constructed from the expressing vec- was terminated by the addition of ice-cold PBS and nuclear extracts were tor pCMV-I␬B␣ S32A/36A with EcoRV/SmaI digestion. pm␬B-LTR-Luc prepared according to the described microscale preparation protocol (52). Protein concentrations were determined by the bicinchoninic assay with a was kindly provided by Dr. K. Calame (Columbia University, New York, ␮ NY). It contains the luciferase reporter gene under the control of mutated commercial protein reagent kit (Pierce). EMSA was performed with 10 g ␬ Ϫ ϩ of nuclear extracts incubated for 20 min at room temperature in 20 ␮lof NF- B (CTCACTTTCC) HIV-1 LTR ( 453 to 80) (49). pNFAT-Luc, ϫ which contains the minimal IL-2 promoter with three tandem copies of the 1 binding buffer (100 mM HEPES (pH 7.9), 40% glycerol, 10% Ficoll, 250 mM KCl, 10 mM DTT, 5 mM EDTA, 250 mM NaCl, 2 ␮g poly(dI- NFAT-binding site, was a kind gift from Dr. G. Crabtree (Howard Hughes ␮ ␥ 32 Medical Institute, Stanford, CA). dC), 10 g nuclease-free BSA fraction V) containing 0.8 ng of [ - P]- labeled dsDNA oligonucleotide. The following consensus binding site rMRPs and polyclonal anti-MRP antiserum dsDNA oligonucleotides were synthesized inhouse and used as probes and/or competitors: NF-␬B(5Ј-ATGTGAGGGGACTTTCCCAG-GC-3Ј); Human S100A8, S100A9, and S100A12 cDNAs were synthesized by RT- Oct-2A (5Ј-GGAGTATCCAGCTCCGTAGCATGCAAATCCTCTGG- PCR from neutrophil RNA isolated using TRIzol reagent according to 3Ј); and enhancer region (Ϫ104/Ϫ82) from the LTR of HIV-1 NL4-3 strain the manufacturer’s instructions (Life Technologies, Grand Island, NY). (5Ј-CAAGGGACTTTCCGCTGGGGACTTTCCAGGG-3Ј). DNA-protein cDNAs were cloned into the pET28 expression vector (Novagen, Madison, complexes were resolved from free-labeled DNA by electrophoresis in WI) and transformed in Escherichia coli HMS174. Expression of rMRPs native 4% (w/v) polyacrylamide gels. The gels were subsequently dried was induced with 1 mM isopropyl ␤-D-thiogalactoside for 16 h at 16¡C. and autoradiographed. Cold competition assays were conducted by adding After incubation, cultures were centrifuged at 5,000 ϫ g for 10 min. The a 100-fold molar excess of unlabeled dsDNA oligonucleotide simulta- pellet was resuspended in PBS/0.5 M NACl/1 mM imidazole and lysed by neously with the labeled probe. Supershift assays were performed by pre- sonication. Lysates were then centrifuged at 55,000 ϫ g for 25 min and the incubation of nuclear extracts with 1 ␮g of Ab (anti-p50 subunit from supernatants were collected. Recombinant His-tagged MRPs were purified NF-␬B; Santa Cruz Biotechnology, Santa Cruz, CA) in the presence of all using a nickel column. His-tagged proteins bound to the column were the components of the binding reaction for 30 min on ice before the ad- cleaved from their His-tag by adding 10 U of thrombin and were incubated dition of the labeled probe. The Journal of Immunology 3309

Results MRPs promote virus production in a latently HIV-1-infected T cell line To decipher the possible modulatory role of MRPs on virus production, a latently HIV-1-infected established T lymphoid cell line (i.e., J1.1) was treated with S100A8, S100A9, S100A12, or S100A8/A9. As depicted in Fig. 1, the level of virus production was strongly up-regulated in J1.1 cells by all MRPs tested by an ϳ4-fold induction when compared with untreated cells. No such induction of HIV-1 production could be seen when J1.1 cells were instead treated with mock protein puriﬁcation solution, therefore demonstrating that the noticed enhancing effect was not due to a bacterial product contaminant. These results indicate that MRPs are potent inducers of HIV-1 production in human CD4ϩ T cells. These observations prompted us to investigate at the molecular level the different intracellular events leading to MRP-mediated activation of HIV-1 gene expression in CD4-expressing T cells. Downloaded from MRPs mediate activation of HIV-1 transcription In an attempt to study the effect of MRPs on HIV-1 LTR-driven transcriptional activity, both a dose response and kinetic analyses were conducted using the Jurkat-derived 1G5 T cell line, which carries two stably integrated constructs made of the luciferase re-

porter gene driven by the HIV-1SF2 LTR. As shown in Fig. 2A,a http://www.jimmunol.org/ signiﬁcant up-regulation of HIV-1 LTR activity was observed upon treatment of 1G5 cells with concentrations as low as 1 ␮g/ml of S100A8, S100A9, S100A12, or S100A8/A9. Maximal luciferase activity was observed when using MRPs at a concentration of 10 ␮g/ml. This protein concentration (10 ␮g/ml) was thus used throughout the following sets of experiments unless speciﬁed otherwise. It should be noted that all MRPs tested were able to activate HIV-1 LTR-dependent reporter gene activity.

Kinetic analysis of HIV-1 LTR activity was also performed us- by guest on September 28, 2021 ing 1G5 cells stimulated with MRPs for increasing periods of time. FIGURE 2. Dose response and kinetic analysis of MRP-mediated in- As shown in Fig. 2B, a significant increase in HIV-1 LTR activity duction of HIV-1 LTR activity. A, 1G5 cells were either left untreated of was detected as early as 2 h following stimulation whereas max- were treated with the mock protein purification solution (using the same imal activation was observed ϳ8-h posttreatment. PMA/iono stim- dilution as for MRPs), PMA/iono (20 ng/ml and 1 ␮M, respectively), or ulation, used as a positive control, resulted in a 69-fold induction increasing doses of MRPs (S100A8, S100A9, S100A12, and S100A8/A9) over untreated control. These results demonstrate that the effect of ranging from 0.01 to 50 ␮g/ml for8hat37¡C. B, 1G5 cells were either left untreated or were treated with the mock protein purification solution, PMA/ iono, or MRPs (S100A8, S100A9, S100A12, and S100A8/A9) (10 ␮g/ml) for the indicated time periods. Cells were then lysed and luciferase activity was monitored with a microplate luminometer. Results are presented as fold induction in luciferase activity either over untreated samples for PMA/ iono-treated samples or over mock protein purification solution-treated samples from MRP-treated samples. These data are from the calculated means Ϯ SEM of three different lysed cell samples in the same experimental setting and are representative of three different experiments.

MRPs on HIV-1 LTR activity is dose-dependent and transient in human CD4ϩ T cells. To demonstrate the specificity of MRP-mediated HIV-1 LTR activity, we performed a set of experiments in which MRPs were either denaturated by heat or inhibited using MRP-specific neutralizing antisera. As shown in Fig. 3A, physical denaturation of FIGURE 1. Treatment of latently HIV-1-infected T cells with MRPs these proteins completely abolished their ability to induce LTR lead to virus production. J1.1 cells were either left untreated or were treated activation in 1G5 cells. To further address and confirm the spec- with the mock protein purification solution (using the same dilution as for ificity of the MRP-mediated effect on HIV-1 LTR activity, we MRPs) or 10 ␮g/ml of MRPs (i.e., S100A8, S100A9, S100A12, and S100A8/A9) for 24 h at 37¡C. Supernatants were harvested and virus pro- made use of neutralizing polyclonal antisera directed against duction was evaluated using a p24 enzymatic assay. Results shown are the MRPs. As shown in Fig. 3B, the addition of specific Abs to the means Ϯ SEM of triplicate samples from one experiment that is represen- MRPs significantly diminished MRP-mediated luciferase activity tative of two others. in 1G5 cells when compared with preimmune sera. In addition, the 3310 MRPs INDUCE HIV-1 REPLICATION IN T LYMPHOCYTES Downloaded from http://www.jimmunol.org/

FIGURE 3. MRP-mediated induction of HIV-1 LTR-driven activity is abolished by heat denaturation and MRP-specific Abs. A, 1G5 cells were treated with the mock protein purification solution, PMA/iono combination, or with the native or heat inactivated MRPs (i.e. S100A8, S100A9, S100A12, and S100A8/A9) (10 ␮g/ml). B, 1G5 cells were treated with the mock protein purification solution, PMA/iono, or MRPs (5 ␮g/ml) and by guest on September 28, 2021 incubated with either MRP-specific polyclonal Abs or preimmunized control serum. Cells were then lysed and luciferase activity was monitored FIGURE 4. MRPs mediate activation of HIV-1 enhancer and lead to the using a microplate luminometer. Results are presented as fold induction in binding of cellular factors onto the enhancer domain. A, Jurkat cells were luciferase activity either over untreated samples for PMA/iono-treated transiently transfected with an HIV-1 enhancer-driven luciferase reporter samples or over mock protein purification solution-treated samples form gene construct, i.e., p␬B-TATA-Luc. Cells were treated with the mock MRP-treated samples. These data are from the calculated means Ϯ SEM of protein purification solution, PMA/iono, or MRPs (10 ␮g/ml) for8hat three different lysed cell samples in the same experimental setting and are 37¡C, then lysed and luciferase activity was monitored using a microplate representative of three different experiments. luminometer. Results are presented as fold induction in luciferase activity either over untreated samples for PMA/iono-treated samples or over mock protein purification solution-treated samples form MRP-treated samples. antisera were ineffective at inhibiting PMA/iono-induced LTR ac- These data are from the calculated means Ϯ SEM of three different lysed tivity, thus confirming that the effect detected was caused by cell samples in the same experimental setting and are representative of the MRPs. three different experiments. B, Labeled HIV-1 enhancer oligonucleotide was incubated with nuclear extracts from Jurkat cells or PBMCs that were MRPs are acting on HIV-1 enhancer region either left untreated (lane 1) or were treated with PMA/iono combination (lane 2), S100A8 (lane 3), S100A9 (lane 4), or the mock protein purifi- Because it is well-established that HIV-1 transcriptional activity cation solution (lane 5). Binding specificity was tested by adding a 100- and expression is predominantly driven through the LTR enhancer fold molar excess of a probe composed of either the cognate HIV-1 en- domain (Ϫ104/Ϫ82), we set out to evaluate whether MRP-induced hancer oligonucleotide (lane 6) or a nonspecific probe (i.e., Oct-2A) (lane HIV-1 activity acts through the HIV-1 enhancer using a Jurkat cell 7). These results are from one experiment that is representative of two line transiently transfected with the luciferase gene under the con- others. trol of the LTR enhancer region (i.e., p␬B-TATA-Luc). These cells were either left untreated or stimulated with PMA/iono or MRPs. As shown in Fig. 4A, all MRPs significantly up-regulated the LTR enhancer-driven luciferase activity in Jurkat cells. In an 4) demonstrated the presence of intense enhancer/protein com- attempt to confirm these observations, EMSA were performed in plexes when compared with complexes obtained from untreated both Jurkat cells and freshly isolated human PBMCs. Nuclear ex- (lane 1) or mock protein purification solution-treated cells (lane tracts from untreated, PMA/iono-, S100A8-, S100A9-, or mock 5). Specific and nonspecific competitions using 100-fold concen- protein purification solution-treated Jurkat cells or PBMCs were trations of nonlabeled probes confirmed the specificity of the sig- incubated with radiolabeled probes corresponding to the HIV-1 nal (lanes 6 and 7). Altogether, these results strongly suggest that LTR enhancer sequence. As illustrated in Fig. 4B, nuclear extracts the enhancer domain is playing a key role in the MRP-mediated from cells treated with PMA/iono (lane 2) or MRPs (lanes 3 and effect on HIV-1 LTR activity. The Journal of Immunology 3311

HIV-1 activation induced by MRPs is mediated through the NF-␬B factor Previous findings demonstrated that NF-␬B and NFAT can act in a concerted manner to up-regulate the HIV-1 enhancer region (43, 53). To delineate the involvement of these two transcription factors in MRP-mediated HIV-1 activation, we transiently transfected Ju- rkat cells with a luciferase-based reporter vector that is regulated by several NF-␬B consensus binding sites. Data from Fig. 5A demonstrate that all MRPs tested act as strong inducers of NF-␬B. This observation suggests that the transcriptional factor NF-␬Bisre- sponsible for the MRP-dependent up-regulation of HIV-1 gene expression. In contrast, MRPs were unable to mediate activation of NFAT as assessed by transient transfection of Jurkat cells with a NFAT-driven reporter gene construct (data not shown). To sub- stantiate the involvement of NF-␬B in the observed MRP-mediated induction of HIV-1 LTR activity, Jurkat cells were next transiently transfected with an HIV-1 LTR-driven reporter gene vector that carries mutations in the two NF-␬B binding sites. When using Downloaded from this molecular construct, MRPs were no longer able to mediate activation of the regulating elements of HIV-1 (Fig. 5B), thereby confirming the pivotal role played by NF-␬B in this phenomenon. Our next series of investigations were aimed at defining the nature of the protein complex that binds to the HIV-1 enhancer following treatment with MRPs. To this end, mobility shift assays were conducted using again as a probe the HIV-1 enhancer region http://www.jimmunol.org/ that was incubated along with nuclear extracts from S100A8- treated Jurkat cells and human PBMCs. A more intense retarded FIGURE 6. NF-␬B is the predominant factor binding to the LTR en- ϩ complex was detected following treatment of Jurkat cells and hancer sequence in CD4 T cells upon MRP-induced activation. Labeled PBMCs with either PMA/iono (positive control) or S100A8 (Fig. HIV-1 enhancer oligonucleotides were incubated with nuclear extracts 6, compare lanes 2 and 3 with lane 1). The formation of the com- from Jurkat cells or PBMCs that were either left untreated (lane 1) or were treated with PMA/iono combination (lanes 2 and 8), or S100A8 (lane 3–7 and 9)for1hat37¡C. Binding specificity was tested by adding a 100-fold molar excess of a probe composed of either the consensus NF-␬B binding

site (lane 4), the cognate HIV-1 enhancer oligonucleotide (lanes 7 and 8), by guest on September 28, 2021 or a nonspeciﬁc probe (i.e., Oct-2A) (lane 9). For gel supershift assays, nuclear extracts were also incubated with an Ab speciﬁc for the p50 subunit of NF-␬B(lane 5). These results are from one experiment that is representative of two others. NA, not applicable.

plex induced by S100A8 treatment was totally competed away by an excess of unlabeled consensus NF-␬B probe (lane 4) but not NFAT and Oct-2A probes (lanes 6 and 9). Finally, the S100A8- induced complex was supershifted when anti-p50 polyclonal Abs were included in the EMSA reaction mixture (lane 5) demonstrating that the p50 subunit of NF-␬B is a major component of the retarded complex that binds to the HIV-1 enhancer region.

Discussion Opportunistic infections frequently result in an increase in viral load in HIV-1-infected individuals and a decrease in CD4ϩ T cell count. A number of inflammatory mediators released in the serum of the patients during these pathologies contribute to HIV-1 replication (1Ð3). Recently, cytokine-like activities have been as- cribed to MRPs (19, 21, 22), which are released during inflamma- FIGURE 5. MRPs mediate NF-␬B-dependent up-regulation of HIV-1 tory episodes by neutrophils and monocytes (54Ð57). High levels LTR activity. Jurkat cells were first transiently transfected with pNF-␬B- of MRPs have been detected in the serum of patients suffering Luc (A), pLTR-Luc (B), or pm␬B-TATA-Luc (B). Cells were next treated from bronchitis and tuberculosis (7, 16, 26), as well as in patients with either the mock protein purification solution, PMA/iono, or MRPs (10 suffering from AIDS (27Ð32). The presence of these proteins in ␮g/ml) for8hat37¡C. Cells were then lysed and monitored for luciferase HIV-1 seropositive patients was correlated with an increased viral activity. Results are presented as fold induction in luciferase activity over either untreated samples for PMA/iono-treated samples or over mock pro- load and a decrease in the number of circulating CD4-expressing tein purification solution-treated samples from MRP-treated samples. T lymphocytes. Because MRPs have been recently demonstrated These data are from the calculated means Ϯ SEM of three different lysed to activate monocytes as well as lymphocytes, it was tempting to cell samples in the same experimental setting and are representative of speculate that the release of MRPs during opportunistic infections three different experiments. could affect the replicative cycle of HIV-1 in T lymphocytes. 3312 MRPs INDUCE HIV-1 REPLICATION IN T LYMPHOCYTES

The data presented in this study demonstrate that MRPs up- might act synergistically with MRPs to induce HIV-1 transcrip- regulate HIV-1 transcriptional activity and virus production in tion. Further studies are required to solve this issue. CD4ϩ T lymphocytes. We show that recombinant S100A8, The results presented in this study reveal for the first time that S100A9, S100A12, and S100A8/A9 proteins induce HIV-1 repli- MRPs such as S100A8, S100A9, S100A12, and S100A8/A9 can cation in a chronically infected Jurkat cell line (J1.1). More spe- up-regulate HIV-1 transcription and virus production in human cifically, MRP treatment resulted in a dose- and time-dependent CD4-expressing T cells. Although we have been able to define that activation of the HIV-1 LTR promoter sequence that is exerted MRPs mediate their positive effect on HIV-1 LTR via an NF-␬B through the enhancer region. Maximal stimulation with MRPs oc- signaling pathway, the cellular receptor(s) that bind MRPs on hu- curred at 10 ␮g/ml, a concentration previously demonstrated to be man CD4ϩ T cells has not been investigated. The receptor for in the range of those found in the serum of HIV-1-infected patients advanced glycation end-products (RAGE) was recently reported to (14 ␮g/ml) (27), thereby providing a physiological relevance to the be a natural receptor for S100A12; to date, RAGE remains the sole present findings. Although S100A8 and S100A12 are chemotactic receptor known to bind MRPs. However, S100A8 and S100A9 do at much lower concentrations (10 ng/ml), similar concentrations not bind to RAGE-transfected Chinese hamster ovary cells, sug- had been reported to induce VCAM-1 and ICAM-1 expression by gesting that they interact with a distinct receptor (64). Although we endothelial cells, and IL-2 production by PBMCs (21, 22). Mech- observed RAGE gene expression in PBMCs using RT-PCR (data anistic analysis revealed that MRP-induced HIV-1 activation was not shown), we suspect that a distinct receptor is responsible for dependent upon the NF-␬B binding sites located within the HIV-1 the MRP-mediated induction of NF-␬BinCD4ϩ lymphocytes. LTR enhancer region. Inhibition of RAGE with specific Abs or by competition with sol- Downloaded from Several known inducers of HIV-1 transcription such as PGE2, uble RAGE could confirm this hypothesis. TNF-␣, anti-CD3 Abs, and phorbol ester agents (e.g., PMA) pro- Previous studies have indicated that high levels of S100A8/A9 mote HIV-1 replication through an induction of the transcription are found in sera from HIV-1-infected patients, which correlated factor NF-␬B (39, 58). NF-␬B is also important for expression of with the evolution of immune deficiency and opportunistic infec- several cytokines (e.g., IL-1, IL-6, and TNF-␣) and adhesion mol- tions (27Ð29). Whether the presence of MRPs in the serum of these ecules (e.g., ICAM-1 and VCAM-1) (59Ð61). The fact that in- patients is a consequence or a cause of HIV-1 replication remains

flammatory proteins such as MRPs activate HIV-1 replication in unknown. Interestingly, Hashemi et al. (33) demonstrated recently http://www.jimmunol.org/ CD4ϩ T cells in a NF-␬B-dependent manner is in agreement with that S100A8 protein derived from cervico-vaginal secretions acti- the previous observations indicating that S100A12 mediates nu- vates HIV-1 replication in a latently infected monocytoid cell line. clear translocation and activation of NF-␬B (22). In this work, This observation, along with the data presented in this study, could activation of NF-␬B by S100A12 resulted in expression of help to explain the correlation between the increased levels of ICAM-1 and VCAM-1 by endothelial cells. Therefore, interaction serum MRPs and the reduction of CD4ϩ cell counts and enhance- between MRPs and T cells is likely to result not only in HIV-1 ment of HIV-1 load (28, 29). Globally, our results suggest that gene expression and virus production, as shown in the present MRPs could represent significant contributors to HIV-1 disease study, but also in an increased surface expression of ICAM-1 on progression in seropositive patients with ongoing opportunistic in- such a cellular subpopulation. Given that ICAM-1 has been shown fections and/or inflammatory conditions. by guest on September 28, 2021 to be inserted within the HIV-1 envelope and to result in an en- hancement of virus infectivity (50, 62), production of MRPs due to Acknowledgments some specific opportunistic infections could also result in produc- We thank Pascal Rouleau for his technical help in the purification of the tion of HIV-1 particles that would be more infectious. recombinant MRPs. In addition to ICAM-1, S100A12 also induces IL-2 production and secretion from PBMCs, as well as the release of TNF-␣ and References IL-1␤ from mononuclear phagocytes (22). It can thus be proposed 1. 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