Monocyte-derived immature DCs generated from normal donor PBMCs were treated with plasma from uninfected control donors or plasma from subjects during AHIV (AHIV plasma). DCs were initially pretreated with AHIV plasma obtained during VR to peak viremia (Fiebig stages 1 and 2). We then measured the responsiveness of plasma-treated DCs to selected TLR stimuli. There were no differences between DCs treated with uninfected donor plasma or AHIV plasma, regarding their phenotype (expression of MHC molecules, costimulatory molecules, or markers of maturation, e.g., CD83, CD80, and CD86; data not shown). However, DCs treated with 10% AHIV plasma were substantially compromised in cytokine production (IL-12p70, TNF-α, IL-6) upon TLR stimulation, as shown in one representative experiment depicted in Figure A. Specifically, AHIV plasma–treated DCs produced reduced levels of IL-6, TNF-α, and IL-12p70 relative to uninfected donor plasma–treated DCs (Figure A). Inhibition with 10% AHIV plasma was consistent over multiple experiments; DCs treated with 23 different AHIV plasma samples showed substantially less cytokine production when normalized to average cytokine levels produced by DCs treated with multiple different uninfected control donor plasma samples (Figure B). These 23 different AHIV plasma samples were tested on different donor DCs in multiple experiments normalized against 2 to 5 uninfected control plasma per experiment (the number of experiments ranges from n
= 6 to n
= 50 per individual AHIV plasma samples). This inhibition was also observed irrespective of the source of samples from patients with AHIV, in that AHIV plasma samples purchased from ZeptoMetrix Corp. and those collected through the Center for HIV/AIDS Vaccine Immunology (CHAVI) in North Carolina, USA, and Malawi, Africa elicited comparable DC inhibition (Figure B). For most experiments, DCs were stimulated with the TLR3 agonist poly I:C, because we found that poly I:C is most potent at inducing IL-12p70 production from DCs. Inhibition by AHIV plasma of DC cytokine production was also observed when using other TLR agonists, such as LPS (TLR4 agonist), peptidoglycan (TLR2 agonist), and R848 (TLR7/8 agonist) (Supplemental Figure 1; supplemental material available online with this article; doi:
). Culturing DCs in 10% AHIV plasma overnight inhibited DC cytokine secretion, but treating DCs for as little as 3 hours also induced inhibition (data not shown). Titering plasma indicated that as little as 1% AHIV plasma could be inhibitory (data not shown), although most consistent results were obtained using 10% plasma levels. Furthermore, DCs treated with AHIV plasma had reduced cytokine production relative to that of both DCs treated with normal donor plasma or DCs under plasma-free conditions (Supplemental Figure 2), indicating that AHIV plasma specifically inhibits DCs, rather than being qualitatively poorer for DC stimulation than control donor plasma. Furthermore, DC inhibition by AHIV plasma was not a result of decreased DC viability because AHIV plasma–treated DCs were as viable after treatment as DCs exposed to uninfected donor plasma (data not shown).
AHIV plasma inhibits TLR-stimulated DC function.
DC-mediated antigen presentation is critical for activation of effector CD4+
T cells, and the cytokine milieu produced by DCs influences the subsequent adaptive immune response elicited (10
). The negative effect AHIV plasma has on TLR-stimulated DC cytokine production would be expected to subsequently impact downstream T cell responses. IL-12p70 is critical for polarizing CD4+
T cells toward a Th1 phenotype (9
). Because of the decreased levels of IL-12p70, we hypothesized that AHIV plasma–treated DCs would have a reduced capacity to prime Th1 CD4+
T cells. Accordingly, poly I:C–stimulated DCs treated with plasma from 3 separate donors with AHIV failed to induce Th1 CD4+
T cells (as measured by IFN-γ production from the T cells), whereas poly I:C–stimulated DCs treated with uninfected donor plasma primed potent Th1 responses from naive allogeneic CD4+
T cells (Figure C). NK cells expressing certain receptors have recently been shown to be associated with control of HIV infection (19
), and NK cells are one of the earliest sources of IFN-γ for promoting Th1 responses (20
). DCs also activate and induce IFN-γ secretion by NK cells through DC production of IL-12p70 (12
). Accordingly, NK cells cocultured with AHIV plasma–treated DCs exhibited reduced IFN-γ secretion relative to that of NK cells cocultured with uninfected plasma–treated DCs (Figure D), indicating that the deficiency in DC activity induced by AHIV plasma manifests on both CD4+
T cell and NK cell activation. Inhibited NK cell activation by AHIV plasma–treated DCs results from the defective IL-12p70 and IL-15 production by DCs, as both cytokines can reverse the observed inhibition of NK cells (data not shown). Consistent results were obtained when plasma from multiple additional donors with AHIV was tested on DCs from different normal donors; altogether 12 AHIV plasma samples were tested in T cell priming experiments, and 9 AHIV plasma samples were tested in NK cell activation experiments.
Since TLR-stimulated DC cytokine production was inhibited by AHIV plasma, we also tested whether plasma from subjects with HIV-1 at other stages of infection inhibits DCs. Plasma from patients with chronic HIV who were not on antiretroviral therapy or long-term nonprogressors (LTNPs; patients with HIV who have maintained stable CD4+ T cell counts) also inhibited DC cytokine production, although the inhibition was not as potent as with AHIV plasma (Supplemental Figure 3A). We also tested the capacity of DCs treated with different plasma to prime Th1 responses, with AHIV plasma–treated DCs promoting very poor Th1 responses, whereas untreated chronic HIV- and LTNP plasma-treated DCs elicited Th1 responses more comparable to those of DCs treated with uninfected donor plasma (Supplemental Figure 3B). Because of the higher potency of inhibition by AHIV plasma, we focused our studies on elements of AHIV plasma that inhibit DCs.
In order to determine at which time point plasma from patients with AHIV becomes inhibitory to DCs, we tested plasma isolated from patients with AHIV over sequential time points during the acute phase of infection, including the eclipse phase and VR, spanning approximately 45 days before and after viremia (8 panels of AHIV plasma). As shown in Figure , the time point when viremia was first detected is indicated as day 0 and the following phase is noted as VR. Plasma collected from uninfected donors over sequential time points did not mediate any significant inhibitory effects on DCs, therefore cytokine levels from DCs treated with plasma from infected donors were normalized to levels produced by uninfected donor plasma–treated DCs. As is shown in Figure , the inhibitory effect of AHIV plasma on TLR-stimulated DC production of IL-12p70 occurs concurrently with VR, and, for some AHIV samples, the inhibitory effect is transient, whereas in others it is maintained during high viral load. Plasma collected from donors with acute HCV or acute HBV did not mediate inhibition of DC IL-12 production, even at time of VR, indicating that the inhibition mediated by AHIV plasma is not a generalized feature of plasma from the acute phase of all virus infections (Figure ). The same kinetics of inhibition was observed on poly I:C–stimulated DC production of IL-6 and TNF-α upon AHIV but not acute HCV plasma treatment of DCs (data not shown).
AHIV plasma–mediated DC inhibition occurs concurrently with VR.
Because AHIV plasma inhibits TLR-stimulated DC cytokine production at time points in infection corresponding to VR, it is possible that the elevated levels of HIV virions in the plasma may be responsible for these effects. However, treating DCs with live HIV-1, a laboratory strain (ADA) or a founder strain, added together with uninfected donor plasma did not inhibit cytokine production (Figure A). This indicates that the inhibitory effects of AHIV plasma are mediated by factors resulting from acute infection and not by the virus itself. It should be noted that HIV-1 by itself, when added to uninfected donor plasma, was not inhibitory to DCs across a wide range of doses, including supraphysiological doses corresponding to 300 ng/ml of p24 protein (data not shown).
AHIV plasma–mediated DC inhibition is independent of virus and occurs in primary myeloid DCs.
Because of the effects observed with acute HIV plasma, we evaluated the function of primary mDCs from patients with acute HIV at different Fiebig stages for their capacity to produce inflammatory cytokines upon TLR stimulation. Whole PBMCs were stimulated with CLO97, a TLR7/8 agonist, and cytokine production by mDCs was assessed. In agreement with our in vitro data, mDCs from patients with AHIV at Fiebig stages 1 and 2 exhibited an impaired capacity to produce cytokines, with significantly fewer cells producing TNF-α (P = 0.003) and results for IL-12 production that approached significance (P = 0.08) (Figure B). As with the inhibitory effects of AHIV plasma on monocyte-derived DCs, this phenomenon seemed transient in that the inhibition manifested at the earliest Fiebig stages (Fiebig stages 1 and 2) of AHIV when VR occurs (Figure B). Therefore, mDCs are inhibited by factors elicited early on in AHIV.
During AHIV, the apoptosis of CD4+
T cells results in elevated levels of apoptotic MPs, which are released from the apoptotic cells into the plasma (15
). Elevated MPs occur concurrently with VR (15
). Accordingly, we detected elevated apoptotic MPs in AHIV plasma relative to those in uninfected control plasma (Supplemental Figure 4A). Based on our data indicating that AHIV plasma becomes inhibitory at the time of VR (Figures and ), we surmised that apoptotic MPs elevated during VR impede DC function. Apoptotic MPs can also be experimentally generated from apoptotic PBMCs and isolated by ultracentrifugation. Isolated MPs were clearly apoptotic, as determined by interaction with annexin V, which binds phosphatidylserine (ref. 21
and data not shown). Electron microscopy of isolated MPs indicated small membrane-bound fragments, ranging in size from 0.1 to 1 μm (Supplemental Figure 5), as would be expected for plasma MPs (20
). Mass spectrometric analysis of apoptotic MPs indicated that they were enriched in ER-derived proteins, such as Erp44, as well as stress-related proteins, such as hypoxia upregulated protein 1α (HIP-1α) and various heat shock proteins (HSPs) (Supplemental Figure 6 and Supplemental Table 1, A and B). Using apoptotic MPs isolated from the supernatant of UV-irradiated PBMCs (to induce apoptosis), we tested the effects of experimentally derived apoptotic MPs on DC function. As controls, DCs were treated with MPs from the supernatant of non-UV-irradiated PBMCs or no MPs. Apoptotic MPs inhibited cytokine production from poly I:C–stimulated DCs (Figure A), although upregulation of DC activation markers and viability was unaffected (data not shown). Apoptotic MPs also inhibited DC cytokine production stimulated by TLR agonists, such as flagellin (Figure B), LPS, and R848 (data not shown). The dose of experimental MPs used was comparable to the number of MPs observed in AHIV plasma preparations by FACS analysis (Supplemental Figure 4A). As with AHIV plasma, apoptotic MP-treated DCs also exhibited reduced priming of Th1 CD4+
T cells, as indicated by reduced levels of IFN-γ and elevated IL-5 production (indicative of Th2 skewing) from cocultured CD4+
T cells (Figure C). In accordance with reduced Th1 priming, apoptotic MP-treated DCs did not prime naive CD8+
T cells, as indicated by inhibited levels of IFN-γ production from CD8+
T cells cocultured with apoptotic MP-treated DCs (Figure D). Moreover, apoptotic MP-treated DCs did not activate NK cells (Figure E). We next isolated MPs from 9 different AHIV plasma samples (at time points when plasma is inhibitory to DCs) and confirmed that MPs derived from AHIV plasma inhibit TLR-stimulated DC cytokine production (Figure F). DCs were treated with an equal number of AHIV- and control plasma-derived MPs, and yet only the MPs derived from AHIV plasma were inhibitory (Figure F). Therefore, in addition to exhibiting elevated levels, MPs from AHIV plasma are also qualitatively different than MPs derived from control plasma. Relative to uninfected MPs derived from control plasma, MPs derived from AHIV plasma are CD41–
, indicating that they are predominantly nonplatelet derived (Supplemental Figure 4, B and C). MPs derived from AHIV plasma and uninfected plasma were analyzed for specific lineage markers (CD3, CD19, CD14, and CD16), and overall the expression for any of these markers was low. CD41 is highly expressed on MPs derived from uninfected plasma, indicating that control MPs are primarily platelet derived, whereas MPs in AHIV plasma are probably derived from many different PBMC subsets that undergo apoptosis during AHIV (Supplemental Figure 4, B and C).
Apoptotic MPs inhibit TLR-stimulated DC function.
Because MPs are elevated in AHIV plasma during VR, we tested whether there is a synergy between virus and MPs that elicits DC inhibition. Plasma from patients with acute HCV or HBV was not inhibitory (Figure ), and accordingly, MPs derived from acute HCV plasma did not inhibit DC cytokine production even when DCs were treated with MPs derived from acute HCV plasma combined with live HIV-1 (Supplemental Figure 7A). Additionally, to further rule out the role of virus in MP-mediated DC dysregulation, apoptotic MPs were generated by UV irradiation of HIV-infected PBMCs. Apoptotic MPs from HIV-infected PBMCs did not elicit greater DC inhibition than apoptotic MPs from uninfected PBMCs (Supplemental Figure 7B). Finally, filtering out most MPs from supernatant of HIV-infected, irradiated PBMCs by passage through a 0.2-μM filter (which allows HIV-1 virus to pass through) showed that MPs and not virus are responsible for DC inhibition because removal of most MPs ameliorates DC inhibition (Supplemental Figure 7B).
Based on our data, we surmised that elevated apoptotic MPs in AHIV plasma contribute to the inhibition of DCs that accounts for dysfunctional Th1 priming as well as NK cell activation. We strove to elucidate the mechanism by which apoptotic MPs inhibit human DCs. In order to determine which receptors on DCs could be mediating the inhibitory effects of apoptotic MPs, we isolated MP-binding proteins from surface-biotinylated DCs and performed mass spectrometry (MS). Cell surface proteins that specifically bind apoptotic MPs were identified by comparing mass spectrometric analysis of apoptotic MP pull-down preparations and excluding proteins that appeared in control MP preparations. A list of DC surface proteins (or associative signaling molecules) that specifically bound apoptotic MPs is shown in Table . Several candidate proteins were identified, including CD35 (complement receptor 1), CD44 (phagocytic glycoprotein 1), and FcγRIIb (CD32b; inhibitory Fc receptor). All of these were sequentially tested, but only CD44 was determined to play a role in apoptotic MP-mediated DC inhibition.
MP-derived proteins bound to DCs identified by MS/MS
CD44 is upregulated on circulating mDCs from patients with AHIV and early HIV (Supplemental Figure 8). It is possible that the upregulation results from inflammation associated with early HIV infection (7
). Binding of CD44 selectively to apoptotic MPs was confirmed by staining apoptotic MPs with CD44-Fc chimeric protein (Figure A). Blockade of CD44 on DCs with anti-CD44 monoclonal antibody (data not shown) or competing for MP interaction with CD44 on DCs using soluble CD44-Fc chimeric protein (Figure B) relieved apoptotic MP-mediated inhibition of poly I:C–stimulated DC cytokine production. Blocking CD35 or FcγRIIb did not have any effect on apoptotic MP-mediated DC inhibition of cytokine production (data not shown). Blocking CD44 with anti-CD44 monoclonal antibody, or pretreating MPs derived from AHIV plasma with CD44-Fc, ameliorated the inhibitory effects of MPs derived from AHIV plasma on DCs, as indicated by restoration of IL-12p70 production from DCs stimulated with poly I:C (Figure C). In order to test whether CD44 ligation of DCs is sufficient to inhibit DCs, we used receptor-targeted antibodies crosslinked on beads (22
). As shown in Figure D, crosslinking CD44 on DCs inhibits IL-12p70 production in response to poly I:C stimulation and IL-6 production in response to LPS. Thus, signaling through CD44 on DCs is sufficient to inhibit TLR-stimulated DC cytokine secretion. Despite binding to annexin V, treatment of apoptotic MPs with annexin V failed to alleviate their inhibition of DCs (Supplemental Figure 9A); moreover, neither did treatment of apoptotic MPs with another phosphatidylserine binding protein, MFG-E8 (Supplemental Figure 9B) alleviate inhibition of DCs, indicating that apoptotic MP-mediated DC inhibition is not due to any classical phosphatidylserine receptor, such as Tim-4, BAI, or Stabilin-2 (23
Inhibition of DCs by AHIV plasma is dependent on CD44.
Interestingly, CD44 also mediates endocytic uptake by DCs (24
), which led us to examine whether uptake of apoptotic MPs is required for their MP-mediated DC inhibition. Receptor-mediated uptake by DCs is commonly regulated by the GTPase Rac1, which controls actin filament nucleation (29
). Blocking Rac1 signaling abrogates DC uptake capacity (29
). Rac1 and associated molecules such as Rap-1b also appeared in our mass spectrometric analysis (though for both control and apoptotic MPs; Table ). We hypothesized that the inhibitory effect of apoptotic MPs is linked to their uptake by DCs. Supporting this, inhibition of Rac1 signaling via a specific chemical inhibitor almost completely reversed the inhibitory effects of apoptotic MPs on DC cytokine production (Figure A). Using CFSE-labeled MPs and confocal microscopy, we determined that Rac1 inhibition abrogates engulfment of MPs by DCs but still allows DCs to bind MPs (Figure B). This is in agreement with the role of Rac1 in regulating actin polymerization during phagocytosis/endocytosis. Thus, blocking MP uptake with cytochalasin D treatment (which disrupts actin filament formation) of DCs completely abrogated the inhibitory effects of apoptotic MPs on DC cytokine production (Figure C). Although these data suggest that engulfment of MPs is required for DC inhibition, it should be kept in mind that downstream signaling events could also be affected by Rac1 inhibition and actin filament disruption, irrespective of MP uptake by DCs.
Mechanisms of apoptotic MP-mediated DC inhibition.