Plasmacytoid dendritic cell (pdcs), which are found in both

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1 HIV-1 gp120 inhiits TLR9-medited ctivtion nd IFN- secretion in plsmcytoid dendritic cells Elen Mrtinelli*, Cludi Cicl, Donld Vn Ryk, Din J. Goode, Ktilyn Mcleod, Jmes Arthos, nd Anthony S. Fuci Lortory of Immunoregultion, Ntionl Institute of Allergy nd Infectious Disese, Bethesd, MD Contriuted y Anthony S. Fuci, Jnury 3, 2007 (sent for review Novemer 16, 2006) Plsmcytoid dendritic cells (pdcs) ply centrl role in innte nd dptive immune responses ginst virl infections. pdcs secrete type I IFNs nd proinflmmtory cytokines upon stimultion y either TLR7 or TLR9. Throughout the course of HIV infection, the production of type-i IFNs is profoundly impired, nd totl pdc cell counts in peripherl lood correltes inversely with virl lod nd positively with CD4 T cell count. The origin of these defects is uncler. pdcs express CD4, CCR5, nd CXCR4, the primry receptor nd coreceptors, respectively, for the HIV envelope; yet little is known concerning the effects of the virl envelope on these cells. Here, we show tht exposure of pdcs to gp120 results in the suppression of ctivtion of these cells. This suppression is specific for TLR9-medited responses, ecuse TLR7-medited responses re unffected y gp120. gp120 lso suppressed TLR9-medited induction of proinflmmtory cytokines nd expression of CD83, mrker of DC ctivtion. Finlly, gp120 suppressed pdc-induced cytolytic ctivity of nturl killer cells. Tken together, these dt demonstrte tht the direct interction of HIV-1 gp120 with pdcs interferes with TLR9 ctivtion resulting in decresed ility of pdcs to secrete ntivirl nd inflmmtory fctors tht ply centrl role in inititing host immune responses ginst invding pthogens. CpG interferon Plsmcytoid dendritic cell (pdcs), which re found in oth peripherl lood nd in T cell-rich res of secondry lymphoid tissues (1, 2), ply centrl role in immune responses ginst virl infections. pdcs re the principl producers of Type 1 interferons (IFN- / ) (3). These cytokines exhiit potent ntivirl ctivity insofr s they regulte the responses of numerous cell susets involved in oth innte nd dptive immune responses ginst virl pthogens. pdcs respond to viruses nd other pthogens primrily through the recognition of pthogen-ssocited moleculr ptterns (PAMPs), y two intrcellulr Toll-like receptors (TLRs), TLR7 nd TLR9. The former recognizes single strnded RNA (4), wheres the ltter recognizes unmethylted DNA motifs (5). The enggement of TLR7 nd TLR9 y PAMPS ctivtes pdcs to rpidly produce high levels of type 1 IFNs nd moderte mounts of inflmmtory cytokines, including TNF- nd IL-6 (6). Through TLR-ctivtion, humn pdcs promote B cell-, T cell- nd nturl killer (NK) cell-medited immune responses. The interction etween pdcs nd NK cells hs prticulrly profound effect on innte immune responses. Activtion of pdcs, fter enggement of TLR-9, ctivtes utologous NK cells nd enhnces their cytolytic ctivity (7, 8). In this regrd, pdcs hve een identified s the non-t, non-b, nonmonocytic cell type required for NK cellmedited killing of virus-infected cells or tumor cell lines (9), nd pdc-derived IFN- exerts dominnt role in this interction (10). The role of pdcs in HIV disese is suject of gret interest. HIV infection results in decrese in the numer of circulting pdcs (11, 12). Levels of circulting pdcs correlte inversely with plsm virl lod nd directly with CD4 T cell counts (12, 13). These effects cn e prtilly reversed y ntiretrovirl therpy (14). Additionlly, pdcs isolted from individuls cutely infected with HIV exhiit n impired ility to secrete type 1 IFNs (15, 16). The underlying cuse(s) of these ltertions in pdc numers nd ctivity, s result of HIV infection, re unknown. However, given the centrl role tht these cells ply in oth innte nd dptive ntivirl immune responses, understnding the mechnisms wherey pdcs interct with nd respond to HIV my provide fundmentl insights into HIV pthogenesis. pdcs cn e infected with HIV in vitro, ut productive infection is chieved only y using high-titer stocks (17, 18), nd it is unlikely tht in vivo direct infection ccounts for the ltered pdc ctivity descried ove. Incution of pdcs with HIV-1 virions induces their mturtion nd the production of IFN. This response is driven y TLR7 recognition of HIV ssrna (19). However, dditionl interctions etween pdcs nd virion components, including the virl envelope, my lso impct pdc mturtion nd function. In this regrd, pdcs express CD4, CCR5, nd CXCR4, ll of which re signltrnsducing lignds for the virl envelope protein gp120. gp120 hs een shown to trnsduce intrcellulr signls in CD4 T cells nd mcrophges (20, 21) nd cn hve profound effect on the function nd viility of these cells (22, 23). Yet, little is known out gp120-medited effects on pdcs, lthough one report indictes tht HIV envelope protein promotes IFN- secretion (24). In this report, we exmined the effect of gp120 on pdcs function. We found tht gp120 disrupts TLR9-medited ctivtion in reltively specific mnner. gp120 tretment suppressed TLR-9-induced secretion of type-1 IFNs nd of other inflmmtory cytokines. Functionlly, pdcs exposed to gp120 exhiited reduced cpcity to induce cytotoxic ctivity in NK cells. As possile mechnism of gp120-medited interference with the IFN pthwy, we descrie its inding to BDCA-2, C-type lectin receptor expressed on the surfce of pdcs. Results gp120 Inhiits TLR9-, ut Not TLR7-. Medited Secretion of IFN- from pdcs. Upon endocytosis of HIV-1 virions, pdcs re ctivted nd secrete IFN- (19). Although this response requires virus cpture y CD4 receptors on the surfce of pdcs, severl reports suggest tht ctivtion nd IFN- secretion occur s consequence of intrcellulr TLR7 recognition of virl ssrna (19). However, one study reports tht gp120 lone, in the sence of RNA, cn trigger pdc ctivtion nd IFN- secretion (24). To etter understnd the effect of exposure of pdcs to HIV gp120, we cultured freshly isolted pdcs for 18 h in the presence or sence of either n R5 or n X4 gp120 recominnt protein. Using sensitive multisutype INF- ELISA, we were unle to detect gp120-induced IFN- (Fig. 1). In contrst, cultures treted with CpGs induced high levels of IFN- (Fig. 1). Considering tht gp120 disrupts the mturtion of monocyte-derived DC (25), we sought to determine whether gp120 would lso disrupt TLR-medited ctivtion nd mturtion of Author contriutions: E.M., C.C., J.A., nd A.S.F. designed reserch; E.M., D.V.R., D.J.G., nd K.M. performed reserch; E.M., C.C., D.V.R., nd J.A. nlyzed dt; nd E.M., J.A., nd A.S.F. wrote the pper. The uthors declre no conflict of interest. Arevitions: pdc, plsmcytoid dendritic cell; gp120tr, gp120 trimer. *To whom correspondence should e ddressed t: Ntionl Institutes of Helth, 10-11N210, 9000 Rockville Pike, Bethesd MD, E-mil: mrtinee1@niid.nih.gov. This rticle contins supporting informtion online t /DC PNAS Ferury 27, 2007 vol. 104 no. 9 cgi doi pns

2 c d e Fig. 1. IFN- production induced y TLR9-lignds, ut not y TLR7-lignd is inhiited y oth monomeric nd trimeric gp120. () pdcs were treted for with CpG (5 g/ml), R5-gp120 (100 nm), or mock-treted with PBS. IFN- in the superntnts ws detected y using humn IFN- multisutype ELISA. () IFN- produced from freshly isolted pdc treted with CpGs (500 ng/ml) in presence or sence of R5-gp120 monomer or trimer (10 nm), SIV monomer (10 nm), or control recominnt protein (10 nm). (c) IFN- produced from pdcs treted with the TLR 7 lignd Imiquimod (1 g/ml) in the presence or sence of R5-gp120 monomer or trimer (gp120tr) (10 nm). (d) IFN- produced from pdcs exposed to HSV-2 for 18 h in the presence or sence of HIV gp120. (e) IFN- produced from pdcs exposed to HSV-2, or treted with CpG, in the presence or sence of gp120. Error rs represent SDs clculted from replictes. Results shown re representtive of t lest five independent experiments IMMUNOLOGY pdcs. As descried ove, freshly isolted pdcs were treted with CpGs, which ctivte pdcs through TLR9 (26), or lterntively with Imiquimod, n imidzoquinoline compound tht ctivtes pdcs through TLR7 (27), in the presence or sence of gp120. As expected, overnight exposure to CpG lone induced significnt levels of IFN- ; however, concomitnt tretment with n R5 gp120 reduced the CpG-induced-IFN- levels y 50% (Fig. 1). Similr effects were oserved with n X4 gp120 (dt not shown). This reduction ws significnt reltive to CpG-treted cells in five experiments (P 0.006). gp120 is presented s trimer on HIV virions (28). To determine whether this form of gp120 induced similr effects, pdcs were exposed to gp120 trimer in the presence of CpG, nd n even greter suppression of IFN- secretion ws oserved (Fig. 1). Additionlly, we evluted recominnt simin immunodeficiency virus (SIV) gp120 derived from the sooty mngey virus SIVsmm/PBj (29). This envelope protein suppressed IFN- levels to greter degree thn ny of the HIV-1 gp120s tested (Fig. 1). Finlly, s control, we treted cells with CpG nd recominnt control protein, in this instnce scd4, to demonstrte tht the suppression we oserved ws not generl phenomenon resulting from the exposure to ny recominnt protein; s expected, we oserved no significnt effect on IFN- secretion (Fig. 1). Susequently we evluted severl concentrtions of gp120 rnging from 200 nm to 1 nm, nd determined tht its effect on IFN- secretion ws concentrtion-dependent nd tht, in the mjority of the experiments, 10 nm gp120 consistently reduced IFN- y t lest 50% (dt not shown). In mrked contrst, gp120 exerted no effect on TLR7-medited induction of IFN- (Fig. 1c). pdcs treted overnight with Imiquimod induced significnt levels of IFN-, nd neither monomeric nor trimeric gp120 suppressed this induction [the differences in the mount of the IFN- secreted were not significnt (P 0.05)]. Thus, the gp120-medited suppression of TLR9-IFN- secretion ws not the result of generlized effect on pdcs. Indeed, ecuse there is sustntil overlp in TLR7 nd TLR9 signl trnsduction pthwys (30), gp120 disruption of TLR9 stimultion would pper to reflect reltively specific effect. In ddition to CpG, we tested the effect of gp120 on the stimultion of pdcs y HSV-2, nturlly occurring TLR9 gonist (5). Although virus lone induced INF-, inclusion of gp120 in cultures together with HSV-2 reduced IFN- Fig. 2. gp120 suppresses TLR9-medited up-regultion of CD83, ut not CD86, on pdcs. pdcs were treted with CpGs in the presence or sence of R5-gp120 monomer or trimer nd stined with PE nti-cd83 () or CD86 () mas nd nlyzed y flow-cytometry. Men fluorescence intensities (MFI) were verged from three different experiments. Error rs represent SDs clculted on them. Mrtinelli et l. PNAS Ferury 27, 2007 vol. 104 no

3 c Fig. 3. Secretion of inflmmtory cytokines induced y TLR9 stimultion in pdcs is inhiited y gp120. Concentrtions of TNF-, IL6, nd IP10 in the superntnts of pdc cultures with CpGs in the presence or sence of R5-gp120 monomer or trimer were detected y using BD Cytometric Bed Arry (CBA) cell signling flex sets. SDs were clculted from replictes. Results shown re representtive of t lest five independent experiments. secretion to n undetectle level (Fig. 1d). Finlly, we determined whether IFN- secretion ws lso suppressed y gp120. Both CpG nd HSV-2 induced IFN-, nd the ddition of gp120 to cultures suppressed IFN- secretion y oth of these TLR9 gonists (Fig. 1e). gp120 Inhiits TLR9-Medited Up-Regultion of CD83 nd the Secretion of Inflmmtory Cytokines. Both CD83 nd CD86 re memrne receptors tht re expressed t very low (CD86) or undetectle (CD83) levels on immture pdcs ut re up-regulted upon ctivtion of these cells y TLR lignds (6). CD86 is one of the principl costimultory receptors on APCs (31), wheres the function of CD83 is lrgely unknown (32). Of note, CD83 upregultion fter culture of DC isolted from HIV-infected individuls is reduced reltive to DCs isolted from helthy donors (33). We tested the effect of gp120 on TLR-medited induction of oth CD83 nd CD86. R5 gp120 suppressed TLR9-medited induction of CD83 (Fig. 2) (P 0.007). A recominnt R5 trimer exerted stronger suppressive effect, wheres X4 gp120 suppressed CD83 in mnner comprle with n R5 gp120 (dt not shown). In contrst, none of the recominnt envelopes tested suppressed TLR9-medited induction of CD86 (Fig. 2) (P 0.05). TLR7- medited induction of CD83 nd CD86 were unffected y exposure of pdcs to gp120 (dt not shown), consistent with the inhiitory ctivity of gp120 on TLR9-, ut not TLR7-, medited induction of IFN-. Along with IFN-, ctivted pdcs secrete moderte mounts of other inflmmtory cytokines, including TNF-, IL1, IL4, IL6, IL12p70, nd IP10. Using flow cytometrysed ed rry ssy (detection limit 3.9 pg/ml), we were le to detect only TNF-, IL6, nd the chemokine IP10 fter TLR9- medited stimultion. As with IFN-, gp120 significntly suppressed the production of ll three cytokines (Fig. 3) (P 0.05). The gp120 trimer ws notly more potent, suppressing TNF- secretion y 70%. gp120 Interferes with pdc-driven NK Cell Cytotoxicity. NK cells respond to virl infections y producing IFN- nd lysing infected cells (10). This ntivirl response requires NK cell ctivtion, nd pdcs ply n importnt role in this ctivtion. Viruses stimulte pdcs through either TLR7 or TLR9, nd they respond y secreting IFN- nd TNF-, which, in turn, contriute to the ctivtion of NK cells (7, 8). In light of our oservtions tht gp120 cn suppress TLR9-medited secretion of IFN- nd TNF- (Figs. 1 nd 3), we sought to determine whether these effects were of sufficient mgnitude to impct pdc-driven NK cell cytolytic ctivity. We ctivted pdcs overnight with CpG in the presence or sence of gp120. Cells were then wshed to remove gp120 from the culture medium. Freshly isolted utologous NK cells were then dded for n dditionl 24 h, fter which n NK sensitive cell line, K562, ws dded to the culture. Cell killing ws mesured fter 2 hours y using flow cytometry-sed ssy. In good greement with previous reports, cytolytic ctivity ws clerly enhnced when NK cells were cocultured with CpG-stimulted pdcs reltive to control cultures lcking pdcs (Fig. 4). Of note, we oserved significnt reduction in the cytolytic ctivity of NK cells ctivted with pdcs tht were treted with CpG in the presence of gp120 reltive to pdcs tht were treted with CpG in the sence of gp120 (P 0.05). Both monomeric (Fig. 4) nd trimeric (Fig. 4) gp120 Fig. 4. NK cell cytotoxicity induced y TLR9-stimulted pdcs is inhiited y gp120. pdcs were treted with CpGs in the presence or sence of n R5 gp120 monomer () or trimer (). Stimulted pdcs were dded to homologous NK cell cultures. NK cells cultured in the sence of pdcs were included s control. PKH67-K562 trget cells were dded to cultures t rtios: 1:2, 1:1, nd 2:1 (NK:K562). Frequency of cell killing ws determined y flow cytometric mesurement of the numer of K562 cells stining positively for PKH67-green nd propidium iodide. NK cytotoxicity is reported s the percentge of ded K562 cells clculted y sutrcting in ech condition the percentge of ded cells of the untreted control (K562 cultured without NK cells). SDs were clculted on the replictes cgi doi pns Mrtinelli et l.

4 % of Mx CXCR4 CD4 CCR5 ISOT C gp120 CD4A gp120 CD4A w-o C ++ gp120 ISOT C presence of Leu3A, significnt level of residul, non CD4- medited inding remined (Fig. 5), indicting tht gp120 inds other unidentified receptors on pdcs. Becuse inding to C-type lectin receptors is clcium-dependent (35), we then compred CD4-independent inding in the presence or sence of C 2 (Fig. 5) nd noted significnt reduction in CD4-independent inding in the sence of C 2. We conclude tht gp120 inds pdcs in oth CD4-dependent nd -independent mechnisms FL2-H medited this suppression, consistent with the cpcity of oth proteins to suppress the secretion of IFN- nd TNF-. We cnnot rule out the possiility tht dditionl pdc ctivtion fctors were dysregulted y gp120 tretment nd lso plyed role in the reduced ctivtion of NK cells. Nevertheless the reduced cpcity of NK cells to kill trgets when CpG-treted pdc re exposed to gp120 underscores the potentil of gp120 to disrupt the network of ntivirl immune responses in which pdcs re involved. gp120 Intercts with pdcs Through CD4-Dependent nd -Independent Mechnisms. To etter understnd the mechnisms underlying gp120-medited disruption of pdc function, we investigted the interctions etween gp120 nd pdc surfce receptors. Most DCs sutypes express CD4 nd the HIV coreceptors CXCR4 nd CCR5. In ddition, gp120 is lso susceptile to cpture y severl ntigen-cpturing C-type lectin receptors tht re expressed on vrious DCs nd APCs. These include DC-SIGN, expressed on myeloid DCs, lngerin on Lngerhns cells, nd the mcrophge mnnose receptor expressed on mcrophges (34). All of these receptors ind to gp120 crohydrte residues, ut none of the ove-mentioned receptors is present on pdcs. We mesured oth CD4-dependent nd -independent inding on pdcs. Consistent with previous reports, flow-cytometric nlysis of these cells demonstrted expression of CD4 s well s CCR5 nd CXCR4 (Fig. 5). pdcs were lso stined with iotin-conjugted R5-tropic gp120, followed y fluoresceine conjugte. High levels of gp120 inding were detected on pdcs (Fig. 5). To determine how much of this inding ws CD4-medited, cells were preincuted with locking nti-cd4 ma, Leu3. Although inding ws reduced in the FL2-H Fig. 5. gp120 inds pdcs through oth CD4-dependent nd C 2 - dependent interctions. () Freshly isolted pdc were stined with nti-cd4, nti-ccr5, nd nti-cxcr4 mas or n isotype control ma. () Freshly isolted pdc were preincuted with n unleled nti-cd4, gp120-locking ma (Leu3), nd stined with iotin-gp120- streptvidin-pe in the presence or sence of C 2. gp120 Binds to the C-Type Lectin Receptor BDCA-2. BDCA-2 is C-type lectin receptor expressed on pdcs, which functions primrily s n ntigen-cpturing receptor (36). It hs een reported tht BDCA-2 ligtion nd cross-linking results in the inhiition of CpG-medited induction of IFN- / (36, 37). Given the results presented ove, we investigted the possiility tht HIV-1 gp120 inds this C-type lectin receptor. In preliminry experiments, we consistently oserved prtil reduction of the gp120 inding to the surfce of pdcs in the presence of n nti-bdca-2 ma (dt not shown). To determine directly whether gp120 inds BDCA-2, we trnsiently trnsfected COS-7 cells with BDCA-2 expression vector. After 48 h, high level of BDCA-2 expression ws chieved (Fig. 6). We reproducily oserved low ut significnt levels of gp120 inding to the BDCA-2-trnsfected cells (P 0.03), ut not mock-trnsfected cells, nd this inding ws inhiited y the removl of C 2 from the stining uffer (Fig. 6). Trimeric gp120 ound to BDCA-2 trnsfected cells to greter degree (Fig. 6), suggesting tht the ffinity of gp120 BDCA-2 interctions re fcilitted y vidity effects ssocited with envelope trimers. BDCA-2-medited inhiition of IFN- / secretion hs een linked to tyrosine phosphoryltion (36). We sked whether gp120 tretment of pdcs lso promoted tyrosine phosphoryltion of intrcellulr sustrtes. Overnight-cultured pdcs were treted with either gp120 or n irrelevnt control protein (scd4) for 30 min, fter which cells were lysed, nd the tyrosine phosphoryltion of two sustrtes, p38 nd ERK1/2, ws monitored with phosphospecific ntiodies y flow cytometry. CpG DNA, which is known to induce the phosphoryltion of these two sustrtes, ws used s positive control. Both gp120 nd CpG DNA medited p38 nd ERK1/2 tyrosine phosphoryltion, wheres the control protein did not [supporting informtion (SI) Fig. 7). This oservtion is consistent with BDCA-2-medited signling. Although we cnnot rule out the possiility tht gp120 is lso signling through CD4, under similr conditions, CD4 ligtion does not induce tyrosine phosphoryltion of intrcellulr sustrtes on pdcs (38). Discussion As the principl producers of IFN-, pdcs ply centrl role in ntivirl immune responses (3). Type I interferons regulte rod rnge of responses involving T, B, nd NK cells. In HIV disese, these cell popultions exhiit profound functionl defects (39). The IMMUNOLOGY Fig. 6. gp120 inds BDCA-2 COS-7-trnsfected cells. () BDCA-2 expression on BDCA-2-trnsfected COS-7 cells vs. mock-trnsfected cells. gp120 inding to mock-trnsfected nd BDCA-2-trnsfected cells in the presence or sence of C 2. () gp120-trimer inding to BDCA-2-trnsfected cells. Results re representtive of three independent experiments. Mrtinelli et l. PNAS Ferury 27, 2007 vol. 104 no

5 potentil role of pdcs in immune dysfunction in HIV hs therefore generted sustntil interest. As Bhrdwj nd collegues hve elegntly shown, pdcs respond to HIV RNA through TLR7 (19). To etter understnd the specific effects of gp120 ligtion, in the sence of other virl components, including virl RNA, we treted freshly isolted pdcs with severl recominnt R5 nd X4 gp120 proteins, including trimeric R5 envelope. Under the conditions we used, none of these gp120s induced IFN- (Fig. 1), consistent with the oservtions of Beignon et l. (19). These results differ from report y Gessni nd collegues (24) who did oserve gp120- induced IFN-. This discrepncy my reflect the fct tht those experiments we crried out in the presence of IL3, which cn predispose pdcs to secrete IFN- (40). gp120 tretment did however produce specific ltertions in pdc function. When pdcs were treted simultneously with gp120 nd either TLR9 gonist (CpG) or HSV-2, TLR9-medited responses were lunted. We oserved reduced secretion of IFNs, TNF-, nd IL6 nd reduced up-regultion of CD83. We further demonstrted tht NK cells, which re ctivted y pdcs to kill trget cells, exhiited reduced cytolytic ctivity fter exposure of CpG-treted pdcs to gp120. These effects did not result from reduced pdc viility, s evidenced y our oservtion tht CD86 up-regultion in untreted vs. gp120-treted pdcs ws unchnged. Moreover, gp120 tretment hd no discernile effect on pdc responses to TLR7-induced secretion of cytokines, or the up-regultion of cell surfce mrkers of ctivtion. These dichotomous effects on TLR9- vs. TLR7- medited ctivtion re surprising insofr s the downstrem signling pthwys ssocited with these two types of stimultion re lrgely overlpping. Of note, TLR7 nd TLR9 pthwys differ in their sensitivity to chloroquine (41). Additionl studies re required to understnd the mechnism y which gp120 disrupts TLR9-, ut not TLR7-, medited ctivtion of pdcs. Becuse pdcs express high levels of CD4 nd significnt levels of CCR5 nd CXCR4, gp120-medited signl trnsduction through these receptors my contriute to the responses descried in this report. Although gp120 signling through these receptors is well documented in lymphocytes (20, 21), little is know out gp120- medited signling through these receptors on pdcs. In ddition, in this report, we demonstrte tht BDCA-2, C-type lectin, which is expressed on pdcs, inds HIV-1 gp120 in C 2 -dependent mnner, rising the possiility tht this interction contriutes to suppression of IFN- production. Two lines of investigtion suggest this to e the cse. First, two recent reports show tht BDCA-2 ligtion with n nti-bdca-2 ntiody inhiits CpG DNA-induced IFN- production (36, 37). Second, we oserved gp120-medited tyrosine phosphoryltion of intrcellulr sustrtes, which occurs s consequence of BDCA-2 ligtion, ut not CD4 ligtion (38). Thus, gp120-medited suppression of CpG-induced IFN- secretion ppers to result directly from ligtion of BDCA-2. Interestingly, signling through BDCA-2 interferes with the nucler trnsloction of IRF-7, one of the principl trnscription fctors involved in the regultion of IFN- production (42). Although we could not identify CD4-medited signling in pdcs, cross-linking CD4 hs lso een reported to suppress CpG-medited IFN- secretion (38). Thus, the unique cpcity of gp120 to ind oth CD4 nd BDCA-2 my contriute to the suppression of IFN- secretion in response to TLR9, ut not TLR7, stimultion. In conclusion, pdcs respond to HIV in complex mnner tht includes effects medited y oth virl RNA nd the HIV envelope. HIV virions induce pdcs to secrete IFN- through TLR7 recognition of virl RNA (19). In this report, we show tht, in ddition to virl RNA, pdcs lso respond to the virl envelope protein in mnner tht renders them less le to respond to DNA viruses nd other TLR9-stimulting pthogens, including cteri. Thus, direct interctions etween HIV nd pdcs my contriute to chronic ctivtion of the immune system nd simultneously suppress responses to specific opportunistic infections. In light of the centrl role tht these cells ply in regulting the immune system, the results reported herein my provide insight into the role of HIV pdc interctions in HIV-driven dysfunction of the immune system. Mterils nd Methods Cells nd Regents. pdcs were otined from PBMCs derived from helthy donors y using BDCA-4 Miltenyi selection kit (Miltenyi Biotec, Auurn CA). Cell purities routinely exceeded 95% y flow cytometric nlysis using BDCA-2 ma. Cells were used immeditely fter selection, without preculture. Homologous NK cells were isolted from elutrited lymphocytes y using negtive selection enrichment kit (Stemcell Technologies, Vncouver BC, Cnd). NK purity ws 98%. The K-562 cell line ws otined from the Americn Type Culture Collection (Mnsss, VA) (43). The TLR9- lignds (TLR9L), CpG A (ODN2216), nd CpG C (ODN2395) were otined from Invivogen (Sn Diego, CA) nd were used interchngely (unless otherwise specified) t 500 ng/ml. The TLR7-lignd (TLR7L) Imiquimod (Invivogen) ws used t 1 g/ml. Recominnt gp120 proteins (JR-FL, 92Ug037, 92Ug021, nd SIV PBj1.9) were produced nd purified s descried (44). The trimeric recominnt gp120 92Ug037 ws constructed y using the firitin domin of phge T7 (45). Purified envelope proteins underwent three successive Triton X-114 extrctions to remove trce endotoxins (46) nd were tested for endotoxin with the Limulus moeocyte lyste (LAL) ssy (BioWhittker, Wlkersville, MD) ( 0.1 unit/ g) gp120 ws iotinylted y using NHS-EZ link Biotin Regents (Pierce, Rockford IL). Anti-CD4 PE, nti-ccr5 PE, nti- CXCR4 PE, nti-cd83 PE, nti-cd86 PE, or Streptvidin FITC nd PE were from BD Biosciences (Sn Diego, CA), nti-bdca-2 PE nd APC were from Miltenyi Biotec. CD4 ma nd Leu3A were from BD Biosciences (Sn Jose, CA). FACS stining uffer: 10 mm Hepes, 150 mm NCl, 10 mm CCl, 0.09% N Azide, 2% Fetl Bovine Serum (FBS). Fluorescence ws mesured on BD FACSCliur (BD Biosciences, Sn Jose). Cytokine Anlysis. Superntnts were collected 18 h fter stimultion. IFN- nd IFN- were determined y ELISA using multisutype IFN- nd IFN- ELISAs (PBL Biomedicl, Pisctwy NJ) per the mnufcturers instructions. TNF-, IL1, IL4, IL6, IL10, IL12p70 nd IP10 were mesured vi Cytometric Bed Arry (CBA) cell signling flex sets (BD Biosciences Sn Jose CA). NK Cytotoxicity Assy. NK cell cytotoxicity ssy ws crried out s previously descried with minor modifictions (47, 48). Briefly, freshly isolted pdcs were stimulted with TLR gonists with or without gp120 s descried ove. Autologous NK cells were kept overnight t 4C. After 18 h, pdcs were wshed to remove gp120, nd NK cells were dded t rtio of 1 pdc per 2 NK cells for n dditionl 18 h. Cells were then wshed, nd PKH72-green leled K562 cells were dded to the culture for n dditionl 2 h. Cells were wshed nd resuspended in stining uffer contining propidium iodide (PI). The frequency of killed K562 cells ws clculted s the numer of (PI /PKH7 2 cells)/(pi /PKH7 2 cells in the sence of NK cells). Trnsfection of Cells with BDCA-2 Plsmid. COS-7 cells were trnsiently trnsfected y using Profectin (Qigen, Vlenci CA) following the mnufcturer s instructions. The humn BDCA-2 coding sequence ws synthesized (DNA 2.0, Menlo Prk CA) nd inserted into pcmv 3.0 (Promeg, Mdison, WI). Surfce expression of BDCA-2 ws checked with BDCA-2-PE (Miltenyi Biotech) nd gp120 inding ws ssessed with iotinylted gp cgi doi pns Mrtinelli et l.

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