Impaired Restoration of Plasmacytoid Dendritic Cells in HIV-1-Infected Patients with Poor CD4 T Cell Reconstitution Is Associated with Decrease in Capacity to Produce IFN-α but Not Proinflammatory Cytokines

Naresh Sachdeva, Vishwaratn Asthana, Toye H. Brewer, Deborah Garcia and Deshratn Asthana
J Immunol August 15, 2008, 181 (4) 2887-2897; DOI: https://doi.org/10.4049/jimmunol.181.4.2887

Abstract

We analyzed reconstitution characteristics of plasmacytoid dendritic cells (PDCs) and myeloid DCs-1 in 38 HIV-1-infected patients with impaired restoration of CD4 T cell counts despite prolonged suppression of plasma viremia (discordant) and compared them with 42 patients showing good immunological and virological responses following highly active antiretroviral therapy (HAART). While myeloid DCs showed spontaneous recovery following HAART in both the groups, the discordant patients demonstrated poor peripheral reconstitution of PDCs as compared with concordant patients. The ability of PDCs to produce IFN-α following stimulation with TLR7 ligand imiquimod and TLR9 ligand CpG ODN-2216 was also impaired in discordant patients even after 2 years following initiation of HAART. Lower IFN-α expression in the PDCs following TLR stimulation was further associated with lower expression of transcription factor, IFN regulatory factor-7. In contrast, production of TNF-α and IL-6 following TLR stimulation was comparable in both groups of patients, indicating that impaired reconstitution characteristics do not affect the capacity of PDCs to produce proinflammatory cytokines. The discordant patients had significantly lower baseline CD4 T cell counts and higher baseline viral load at the initiation of HAART implying that lower baseline CD4 T cell counts and higher plasma viral load are associated with impaired restoration of CD4 T cells and PDCs, thus, increasing the susceptibility of discordant patients toward opportunistic infections despite virological control.

Treatment of HIV-1-infected patients with highly active antiretroviral therapy (HAART)3 decreases plasma viral load resulting in increase in whole-blood CD4 T cell counts and improvement in CD4:CD8 T cell ratio to normal levels. However, in some HIV-1-infected patients, there is no appreciable increase in CD4 T cell counts or restoration of CD4:CD8 T cell ratio following HAART, despite suppression of plasma viremia for long periods of time (immunological and virological discordance). Although there is no universal definition of immunological and virological discordance, such responses have been reported in up to 20% of patients 6 mo to 2 years after initiation of HAART (1, 2, 3). Inadequate CD4 T cell restoration specifically in these patients is thought to depend on several virological and host-related factors including previous therapeutic failure, thymic dysfunction, low CD4 T cell counts at the initiation of HAART, advanced stage of disease, increased T cell activation/apoptosis, etc. (1, 4, 5).

Besides CD4 T cells, several studies have reported a reduction in the dendritic cell (DC) numbers, especially plasmacytoid DCs (PDCs), during the onset of HIV-1 infection as well as during the advanced stage of disease (6, 7, 8, 9, 10, 11). The PDCs selectively express CD123, BDCA-2, BDCA-4 Ags, TLR7, and TLR9 and are principal producers of IFN-α (12, 13, 14, 15, 16, 17). Upon stimulation with TLR9 ligands, like unmethylated CpG DNA, herpes simplex virus type 1 and herpes simplex virus type 2 genomic DNA, and TLR7 ligands, like single-stranded RNA viruses, PDCs produce large amounts of IFN-α, besides production of proinflammatory cytokines TNF-α, IL-6, and IL-8 (12, 17, 18, 19, 20). The ability of PDCs to produce high amounts of IFN-α compared with other cell types is mainly attributed to constitutive expression of IFN regulatory factor (IRF)-7, which is required for the stimulation of IFN-α gene expression (21, 22). Numerous studies have demonstrated the critical role of IFN-α in maturation of DCs, and activation of NK and cytotoxic CD8 T cells during antiviral as well as antitumor immune responses (18, 23, 24, 25). IFN-α together with NK cells comprise of major components of innate immune system, in conferring protection from viral infections. Decreases in either PDC count or their ability to produce IFN-α has been linked to increase in plasma HIV-1 RNA levels and has been associated with increased opportunistic infections and AIDS-associated malignancies, such as Kaposi’s sarcoma during HIV-1 infection (6, 26, 27). Another type of DCs, the myeloid DCs (MDC) mainly comprise of MDC-1 population that express CD11c and BDCA-1 and a broad range of TLRs, except TLR-7 and TLR-9 (12, 13). A minor fraction of MDCs, the MDC-2, express high levels of BDCA-3 Ag and their function is largely unknown (13). MDC-1 mainly produce IL-12, besides IL-15 and IL-18, upon antigenic stimulation and are important in generation of Th1 responses (28, 29). MDC-1 have also been reported to decrease during primary HIV-1 infection and later stages of AIDS (30, 31, 32). Both PDCs and MDC-1 exhibit differential restoration following HAART, with PDCs taking longer time in restoration (32, 33).

Recently, we observed a group of discordant patients that failed to show any increase in their CD4 T cell counts despite maintaining low to undetectable plasma viral load for more than 1 year (34). Though, there are various reports on the recovery of immune cells following HAART, mainly focused on CD4, CD8 T cell subsets, and NK and B cells, there is very little information on the restoration of DC subsets during discordant responses in HIV-1-infected patients, particularly during impaired CD4 T cell reconstitution. In the present study, we investigated longitudinal variations in the reconstitution of peripheral DC subsets in relation to changes in CD4 T cell counts and plasma viral load following HAART in the discordant and concordant HIV-1-infected patients. In addition, we assessed the capacity of PDCs to release IFN-α and proinflammatory cytokines TNF-α and IL-6 following stimulation with TLR7 and TLR9 ligands to understand whether poor reconstitution of CD4 T cells influences any of the functional characteristics of PDCs. Our results demonstrate that while MDCs show spontaneous recovery following HAART in both the groups of patients, recovery of PDC numbers and their ability to produce IFN-α is impaired in the discordant patients even after 2 years following HAART, despite prolonged virological control. We also observed that defective IFN-α production by the PDCs is associated with minor variations in the expression of IRF-7. Further, we observed that poor reconstitution characteristics of PDCs did not influence their capacity to produce proinflammatory cytokines.

Materials and Methods

Study subjects and specimens

Peripheral whole-blood specimens were collected in Sodium heparin and EDTA tubes from 80 HIV-1-infected patients and 12 age- and sex-matched HIV-negative subjects (normal healthy controls) from the University of Miami-Miller School of Medicine outreach clinic, Project Outreach (Florida City, FL) and Borinquen Health Care Center (Miami, FL). We observed the pattern of CD4, CD8, B, and NK cell counts and plasma HIV-1 RNA levels (recorded every 4–6 mo for over 2 years) of 1100 patients to recruit study subjects that demonstrated a strict discordant or concordant profile and divided them into two groups: discordant (n = 38) and concordant (n = 42). The study was duly approved by the Institutional Review Board of the University of Miami-Miller School of Medicine. The criteria for selection of discordant patients was: undetectable plasma HIV-1 RNA (<50 copies/ml) for at least 1 year with a) whole-blood CD4 T cell count less than 200/mm3 or b) CD4:CD8 ratio less than 1.0 with CD4 T cell counts less than 400/mm3 and CD4 T cell increase not greater than 100/mm3 following HAART (Fig. 1⇓) (1, 5). Patients showing positive immunological and virological response following HAART having CD4 T cell increase greater than 100/mm3 following HAART with virological control were placed in the concordant group. Patients were excluded for non-adherence to HAART, any acute illness, opportunistic infections, lymphomas, or psychiatric illness. The whole-blood samples of the recruited subjects were submitted to the Laboratory for Clinical and Biological Studies where all investigations were performed.

FIGURE 1.
FIGURE 1.

Pattern of median CD4 T cell counts and plasma viral load of (A) discordant (n = 38) and (B) concordant (n = 42) HIV-1-infected patients observed over 2 years before analysis. CD4 T cell counts and plasma viral load of each patient were recorded every 6 mo. The concordant patients showed an overall improvement in CD4 T cell counts and decrease in plasma viral load with inverse relation between the two parameters throughout the period. The discordant patients showed a continuous discordant response between CD4 T cell counts and plasma viral load during 12–24 mo. The arrow indicates the time point when the discordant patients demonstrated no appreciable increase in their CD4 T cell counts despite maintaining low to undetectable viral loads for more than a year.

Flow cytometry

Immunophenotyping of the DC subsets was performed on fresh EDTA whole-blood specimens of the recruited subjects using the dendritic cell enumeration kit (Miltenyi Biotec) as per manufacturer’s instructions. Flowcytometric acquisition and analysis was performed on the 4- color flowcytometer, FACSCalibur (BD Biosciences) and the percentage of PDC (CD14−, CD19−, BDCA1−, BDCA2+), MDC-1 (CD14−, CD19−, BDCA1+, BDCA2−), and MDC-2 (CD14−, CD19−, BDCA1−, BDCA2−, BDCA3+) populations were analyzed using the Cell Quest Pro software (v 5.2). In all the analyses, a minimum of 1,000,000 events were analyzed and dead cells were excluded after reaction with dead cell discriminator dye. Relative DC frequencies were calculated after subtraction of the gated events determined for the isotype control sample ran in parallel with the test sample. Absolute numbers of DC subsets were determined from the white blood counts obtained on Coulter AcT5 5-part differential hematology analyzer (Beckman Coulter).

Stimulation of PDCs with TLR7 and TLR9 ligands

PBMCs were isolated from heparin-preserved fresh whole blood by density gradient centrifugation with Ficoll-paque Plus lymphocyte separation medium (Amersham Biosciences). The PDCs were purified from the PBMCs using the blood dendritic cell Ag 4 (BDCA4) dendritic cell isolation kit (Miltenyi Biotec) as per manufacturer’s instructions. Briefly, the PDCs were labeled with anti-BDCA4 Ab coupled colloidal paramagnetic microbeads and passed twice through minimagnetic separation columns using the OctoMACS separator supplied by the same manufacturer. Purity of the separated PDCs was measured by flow cytometry following staining with anti-BDCA2-PE Ab (Miltenyi Biotec). PDCs were counted and adjusted to 5 × 103 cells/well and cocultured with 5 × 105 PBMCs/well of the same patient/control in 24-well flat-bottom plates in 1 ml of RPMI 1640 medium supplemented with l-glutamine, 10% heat-inactivated human AB serum (Mediatech), and 1% penicillin and streptomycin (Invitrogen) (Costar). The cells were stimulated for 20 h with 5 μg/ml TLR7 ligand imiquimod (R837) or TLR9 ligand, 3 μM CpG-A (ODN 2216) or control CpG-A (Invivogen), while negative control wells contained cells without any stimulant. Following stimulation, cell-free culture supernatants were harvested and frozen once at −80°C. In some experiments, PDCs were purified from the cocultures, counted, assessed for viability, and used for real-time RT-PCR analysis.

Cytokine analysis

Cell-free culture supernatants were tested for IFN-α using a human multi-species IFN-α ELISA kit as per protocol provided by the manufacturer (PBL Biomedical Laboratories). IFN-α released per PDC in each sample was calculated as described earlier (10). Levels of proinflammatory cytokines TNF-α and IL-6 were measured using a protein biochip array system, Evidence Investigator (Randox laboratories), as described previously (35). Supernatants from cultures without TLR ligands were used as controls.

Real-time RT-PCR analysis

The PDCs obtained from the coculture experiments were used for extraction of mRNA using the μMACS mRNA isolation kit (Miltenyi Biotec). cDNA was synthesized from the mRNA before the final elution step of the mRNA isolation procedure, on the magnetic columns placed on the thermoMACS separator using the μMACS one-step cDNA kit (Miltenyi Biotec). Real-time PCR assays were conducted on the ABI PRISM 7000 Sequence Detector System using primers and TaqMan probes specific for the conserved sequences in the target genes; IFN-α (Forward; 5′-CCACAAAAGATTCATCTGCTGCTT-3′, Reverse; 5′-TCATTCAGCTGCTGGTAGAGTTC-3′, FAM-labeled probe, 5′-CCTCCTAGACAAATTC-3′) (designed to amplify the major human IFN-α subtypes), IRF-7 (Forward; 5′-CCTGTGGACACCTGTGACA-3′, Reverse; 5′-TCCTGTCGCAGCAGACG-3′, FAM-labeled probe, 5′-CTGGCCACACGACCTG-3′), and IL-6 (Assay ID Hs00174131_m1) and TNF-α (Assay ID Hs00174128_m1) with β-actin as endogenous control (Assay ID 4310881E) (Applied Biosystems). A duplex PCR involving amplification of target with endogenous control was performed for each sample in 96-well clear plates. Amplification was conducted as follows: 95°C, 10 min, 42 cycles of 95°C, 15 s and 60°C, 1 min. Relative quantity of the target genes was calculated using the normalized amount of the target with reference to β-actin, determined by the standard curve constructed from pooled cDNA dilutions obtained from the normal healthy controls.

Statistical analysis

Comparisons between the different groups for all of the parameters tested were performed using the Mann-Whitney U test. The correlations between variables were calculated using the Spearman’s rank correlation test and linear regression analysis were plotted using the Graph Pad Prism software (v 4.0).

Results

Discordant responses persist for long periods of time despite virological suppression

The discordant patients had similar duration of HIV-1 infection and HAART regimens as compared with the concordant patients (Table I⇓). However, the discordant patients had significantly higher plasma viral load and lower CD4 T cell counts as compared with the concordant group before initiation of HAART (p < 0.01). The trend of CD4 T cell counts and plasma viral load of all the patients in the cohort was observed for a period of over 24 mo before the current analysis (Fig. 1⇑). The discordant patients demonstrated moderate increase in their CD4 T cell counts from 0 to 12 mo. However, 12 mo onward, these patients failed to show any increase in their CD4 T cells despite maintaining undetectable plasma viral load. In contrast, the concordant patients demonstrated constant increase in their CD4 T cell counts coupled with decline in plasma viremia after initiation of HAART, with an inverse relation between plasma viral load and CD4 T cell counts during the entire period of observation. There were no significant differences in the median absolute B cell counts between the two groups, although the median absolute NK cell counts were higher in discordant patients (p = 0.054). Other demographic and clinical characteristics of patients and controls are described in Table I⇓.

View this table:
Table I.

Characteristics of the recruited subjects and their treatment regimens at the time of analysisa

Poor restoration of CD4 T cell counts in discordant patients impairs restoration of PDCs but not MDCs

In HIV-1-infected patients, PDCs have been reported to bear a direct correlation with CD4 T cell counts and indirect correlation with plasma viral load (8). Consistent with the earlier reports, the HIV-1-infected subjects demonstrated lower absolute counts of PDCs (median, 6560/ml; range, 600–33,320/ml) and MDC-1 (median, 16,590/ml; range, 1830–117,040/ml) as compared with normal healthy controls (PDC, median, 15,125/ml; range, 6760–35,340/ml and MDC-1, median, 23,400/ml; range, 2050–64,080/ml) with absolute counts of PDCs being significantly lower in HIV-1-infected subjects (p = 0.0005).

Among the HIV-1-infected subjects, the discordant and concordant groups had similar absolute counts of MDC-1 (discordant, median, 17,350/ml; range, 1830–117,040/ml and concordant, median, 15,060/ml; range, 4000–66,220/ml). However, the discordant patients had significantly lower absolute counts of PDCs (median, 4875/ml; range, 600–24,960/ml) as compared with the concordant patients (median, 9535/ml; range, 960–33,320/ml) (p = 0.0008) at the time of analysis (Figs. 2⇓ and 3⇓). The MDC-2 population was rare in the HIV-1-infected patients vs the normal healthy controls (data not shown).

FIGURE 2.
FIGURE 2.

Flowcytometric analysis of PBMCs isolated from a representative patient specimen for enumeration of DCs. After exclusion of (A) cell debris and platelets, (B) B cells, monocytes, granulocytes, and dead cells were excluded by drawing a region R2 from where (C) PDC, MDC-1, and (D) MDC-2 subsets were separated on the basis of the expression of different blood dendritic cell Ags with respect to (E and F) isotype control Abs.

FIGURE 3.
FIGURE 3.

Distribution of peripheral PDC and MDC-1 cell counts in discordant (n = 38) and concordant (n = 42) HIV-1-infected patients and normal healthy controls (n = 12). A, The discordant patients had significantly lower PDC counts as compared with concordant patients (p = 0.008, Mann-Whiney U test). B, Both the groups had similar peripheral MDC-1 counts. For each subset, boxes represent 25th and 75th percentile with the median value (solid line) between boxes, while the whiskers represent the minimum and maximum values.

The discordant patients had controlled plasma viremia but failed to show recovery of CD4 T cell numbers. Therefore, we investigated the kinetics of DC subsets in discordant and concordant subjects in relation to restoration of CD4 T cell counts and plasma viral load in those patients (11 discordant and 16 concordant), where the data on DC subsets before initiation of HAART for up to 2 years was available. Before initiation of HAART, absolute peripheral PDC counts were lower in discordant patients (median, 6190/ml; range, 690–13,220/ml) as compared with concordant patients (median, 7130/ml; range, 1275–19,880/ml), although the differences were statistically insignificant. However, after initiation of HAART, discordant and concordant groups showed different patterns of reconstitution of peripheral PDC and MDC-1 population in relation to CD4 T cell counts. As observed in the entire group of discordant patients, there was poor reconstitution of CD4 T cell numbers despite virological control following HAART in the selected discordant group (Fig. 4⇓). Interestingly, these discordant patients showed no increase in peripheral PDC counts following HAART for up to 12 mo (Fig. 4⇓A). There was a moderate increase in the PDC numbers between months 12 and 24, which was significantly lower than the concordant group at the end of 24 mo (median, 7130/ml; range, 1510–14,770/ml vs 13820/ml; range, 1690–28430/ml, p = 0.022). In contrast the concordant group showed consistent increase in peripheral PDC counts following HAART, with maximum increase between 0 and 6 mo similar to the trend observed with CD4 T cell counts (Fig. 4⇓B). Peripheral MDC-1 counts were also low in discordant (median, 11,030/ml; range, 2130–27,410/ml) and concordant groups (median, 14,100/ml; range, 3430–26,850/ml) before initiation of HAART. However, in contrast to PDC population, the MDC-1 population demonstrated a consistent increase in both the groups with stabilized peripheral counts at the end of 24 mo following HAART (Fig. 4⇓, C and D).

FIGURE 4.
FIGURE 4.

Reconstitution of peripheral PDC and MDC-1 counts observed in selected group of discordant (n = 11) and concordant (n = 16) patients in relation to CD4 T cell counts following initiation of HAART. A, The discordant patients showed no increase in median PDC counts following HAART for up to 12 mo. There was a moderate increase in the PDC numbers between months 12 and 24, which was significantly lower (p = 0.022) than the concordant group at the end of 24 mo. The discordant patients also demonstrated poor reconstitution of CD4 T cells during the observation period. B, In contrast the concordant group showed consistent increase in median PDC counts following HAART, with maximum increase between 0 and 6 mo similar to the trend observed with CD4 T cell counts. C and D, The MDC-1 population demonstrated a consistent increase in both the groups with stabilized counts at the end of 24 mo following HAART.

Production of IFN-α by the PDCs following TLR stimulation is impaired in the discordant patients

The PDCs possess the capacity to secrete huge amounts of IFN-α when stimulated with TLR7 or TLR9 ligands. We used the same approach to assess the function of PDCs in discordant and concordant HIV-1-infected patients. Purity of the PDCs isolated from all the specimens was at least 85% (ranging from 85 to 93%). Time line analysis of IFN-α production in PDC-enriched PBMC cocultures showed maximum amount of IFN-α in culture supernatants until 20 h following stimulation with TLR ligands, after which there was no increase in IFN-α production. Overall, stimulation with TLR9 ligand CpG-2216 yielded ∼8-fold higher amounts of IFN-α/cell from PDC-enriched PBMC cocultures as compared with stimulation with TLR7 ligand imiquimod (p = 0.003) (Fig. 5⇓A). IFN-α production on per-cell basis ranged between 0.02–2.95 pg and 0.02–0.46 pg following TLR9 and TLR7 stimulation, respectively. The normal controls produced significantly higher amounts of IFN-α as compared with HIV-1-infected patients upon stimulation with TLR9 ligand (median, 1.62 pg/cell; range, 1.06–2.95 pg/cell vs median, 0.745 pg/cell; range, 0.05–2.28 pg/cell) and TLR7 ligand (median, 0.23 pg/cell; range, 0.1–0.4 pg/cell vs median, 0.095 pg/cell; range, 0.02–0.46 pg/cell) (p < 0.01). Notably, in comparison of HIV-1-infected patients, the concordant group demonstrated significantly higher amounts of IFN-α production by the PDCs isolated following stimulation with TLR9 ligand (median, 1.04 pg/cell; range, 0.25–2.28 pg/cell vs median, 0.36 pg/cell; range, 0.05–1.5 pg/cell) (p = 0.036) as well as TLR7 ligand (median, 0.115 pg/cell; range, 0.02–0.46 pg/cell vs median, 0.04 pg/cell; range, 0.02–0.28 pg/cell) (p = 0.029) as compared with the discordant group (Fig. 5⇓, B and C).

FIGURE 5.
FIGURE 5.

Secretion of IFN-α by the PDC-enriched PBMC cocultures following stimulation with TLR ligands. A, Stimulation with TLR9 ligand CpG-2216 yielded ∼8-fold higher amounts of IFN-α per PDC as compared with stimulation with TLR7 ligand imiquimod (p = 0.003*). The concordant patients (n = 42) demonstrated significantly higher amounts of IFN-α production per PDC following stimulation with (B) TLR9 ligand (p = 0.036*) and (C) TLR7 ligand (p = 0.029*) as compared with discordant patients (n = 38). The normal healthy controls demonstrated significantly higher amounts of IFN-α production per PDC following stimulation with either of the TLR ligands (p < 0.01*) in comparison to HIV-1-infected subjects. Boxes represent 25th and 75th percentile with the median value (solid line) between boxes, while the whiskers represent the minimum and maximum values. ∗, Mann-Whiney U test.

Next, we addressed the issue whether the capacity of PDCs to secrete IFN-α (following stimulation with TLR ligands) is associated with peripheral CD4 and PDC counts. In all the HIV-1-infected patients recruited in the study, CD4 T cell counts showed a significant positive correlation with PDC counts (r = 0.28, p = 0.011) and their capacity to release IFN-α following stimulation with TLR9 ligand (r = 0.61, p < 0.0001) and TLR7 ligand (r = 0.29, p = 0.008) (Fig. 6⇓, A and B). Further, we correlated the capacity of PDCs to release IFN-α with post-HAART increase in CD4 T cell counts in all the HIV-1-infected patients. We observed that increase in CD4 T cell counts correlated significantly with the function of PDCs (TLR9 stimulation; r = 0.69, p < 0.0001; TLR7 stimulation; r = 0.43, p = 0.014). In group-wise comparisons as well, the discordant patients showed a highly significant positive correlation of CD4 T cell counts with PDC counts (r = 0.57, p = 0.001) and their capacity to release IFN-α following TLR stimulation (TLR9, r = 0.71, p < 0.0001; TLR7, r = 0.53, p = 0.002). The concordant patients also showed similar correlation of CD4 T cell counts with PDCs (r = 0.25, p = 0.023) and their capacity to release IFN-α following TLR stimulation (TLR9, r = 0.49, p = 0.011; TLR7, r = 0.22, p = 0.038). Thus, it appears that poor reconstitution of CD4 T cell counts also impairs functional capacity of PDCs, besides affecting their numbers particularly in the discordant HIV-1-infected patients.

FIGURE 6.
FIGURE 6.

Correlation of CD4 T cell counts with PDC counts (right y-axis) and release of IFN-α (left y-axis) following stimulation with (A) TLR9 ligand CpG-2216 and (B) TLR7 ligand imiquimod in all of the 80 HIV-1-infected patients recruited in the study. Calculations were performed using the Spearman’s rank correlation test. Solid linear regression lines represent relationship of CD4 T cell counts vs PDC counts, whereas dotted lines represent relationship of IFN-α/PDC vs CD4 T cell counts. CD4 T cell counts showed a significant positive correlation with PDC counts (r = 0.28, p = 0.011) and their capacity to release IFN-α following stimulation with TLR9 ligand (r = 0.61, p < 0.0001) and TLR7 ligand (r = 0.29, p = 0.008).

Lower IFN-α expression in PDCs of discordant patients is associated with low expression of IRF-7

To confirm whether the observed differences in IFN-α production are due to differences in the expression of IFN-α in the PDCs isolated from different groups, we quantitated the expression of IFN-α and IRF-7 from the PDCs isolated from the cocultures by real-time RT-PCR analysis. The transcription factor IRF-7 is essential for the induction of IFN-α genes via the virus-activated, MyD88-independent pathway and the TLR-activated, MyD88-dependent pathway (22). A total of 2.0 × 103 PDCs were isolated and analyzed from cocultures of 15 subjects each from the discordant and concordant groups, and 5 normal healthy controls. The discordant and concordant patients did not show significant differences in the expression of IFN-α and IRF-7 before stimulation (Fig. 7⇓, A and D). Following stimulation, the normal healthy controls showed higher relative expression of IFN-α mRNA (∼3-fold with TLR9 stimulation and 2-fold with TLR7 stimulation) in the PDCs, significantly higher than the HIV-1-infected patients (p = 0.002 and 0.046, respectively). Consistent with the results of IFN-α ELISA, the concordant patients showed significantly higher relative expression of IFN-α mRNA as compared with discordant patients following TLR9 stimulation (p = 0.04) (Fig. 7⇓B). TLR7 stimulation also resulted in higher relative IFN-α mRNA levels in PDCs of concordant patients, although the difference was not statistically significant (p = 0.262) (Fig. 7⇓C). Expression of IFN-α mRNA correlated significantly with the IRF-7 mRNA levels (p = 0.004). Expression of IRF7 was also higher in the PDCs isolated from normal healthy controls, especially after stimulation with TLR9 ligand (p = 0.018) (Fig. 7⇓E). Expression of IRF-7 did not show much difference between discordant and concordant subjects as seen with IFN-α, with concordant patients showing slightly higher levels of IRF-7 following stimulation with TLR9 ligand (p = 0.135) and TLR7 ligand (p = 0.191) (Fig. 7⇓, E and F). Overall, in all of the subjects expression of IRF-7 correlated significantly with expression of IFN-α (TLR9 stimulation, r = 0.76, p < 0.0001; TLR7 stimulation, r = 0.62, p < 0.001). Although these observations confirmed our results on differences in the expression levels of IFN-α between various groups, they also indicated that small changes in the concentration of IRF-7 might be associated with regulation of TLR-mediated expression of IFN-α.

FIGURE 7.
FIGURE 7.

Relative expression of IFN-α and IRF-7 in PDCs isolated from the cocultures as quantitated by real-time RT-PCR analysis. The purity of the isolated PDCs ranged from 85 to 93%. A and D, The discordant and concordant patients did not show significant differences in expression of IFN-α and IRF-7 before stimulation. B, The concordant patients (n = 15) showed significantly higher IFN-α mRNA levels as compared with discordant patients (n = 15) following stimulation with TLR9 ligand CpG-2216 (p = 0.04*). C, Stimulation with TLR7 ligand imiquimod also resulted in higher relative IFN-α mRNA levels in PDCs of concordant patients, although the difference was not statistically significant (p = 0.262*). E, The expression of IRF7 was also higher in the PDCs isolated from concordant group, after stimulation with TLR9 ligand, although the difference was statistically insignificant (p = 0.135*). F, Expression of IRF-7 did not show any significant difference between discordant and concordant subjects following stimulation with TLR7 ligand (p = 0.191*). The normal healthy controls (n = 5) showed significantly higher relative expression of IFN-α mRNA after stimulation with either of TLR ligands and IRF-7 expression following stimulation with TLR9 ligand (p = 0.018*) compared with HIV-1-infected patients. Solid lines represent median values. ∗, Mann-Whiney U test.

Discordant and concordant patients release comparable levels of IL-6 and TNF-α following stimulation with TLR ligands

In addition to IFN-α, TLR stimulation in PDCs results in secretion of proinflammatory cytokines TNF-α and IL-6 in an IRF-independent pathway (36). We also investigated production of proinflammatory cytokines IL-6 and TNF-α in the PDC-enriched cocultures compared with non-PDC-enriched cocultures. In accordance with previous reports, production of proinflammatory cytokines was significantly higher following TLR9 stimulation (p < 0.01), as compared with TLR7 stimulation (37). In comparison to HIV-1-infected subjects, PDCs isolated from normal healthy controls produced similar amounts of IL-6 and TNF-α following stimulation with TLR9 ligand CpG-2216 or TLR7 ligand imiquimod (Fig. 8⇓, A–D). Among the HIV-1-infected subjects, PDCs isolated from the concordant subjects released similar levels of IL-6 following stimulation with TLR9 ligand (p = 0.384) and TLR7 ligand (p = 0.617) as compared with discordant subjects (Fig. 8⇓, A and B). As observed with IL-6, PDCs isolated from discordant and concordant patients also produced comparable levels of TNF-α following stimulation with TLR9 and TLR7 ligands (p = 0.304 and p = 0.413, respectively) (Fig. 8⇓, C and D). The production of proinflammatory cytokines by the PDCs following stimulation with TLR ligands was also assessed by real-time RT-PCR analysis in 15 discordant and 15 concordant subjects, and 5 normal healthy controls. Again, the PDCs isolated from the discordant subjects expressed similar levels of IL-6 and TNF-α mRNA following stimulation with either of the TLR ligands in the cocultures as compared with PDCs isolated from the concordant subjects (Fig. 8⇓, E–H). Thus, unlike IFN-α, impaired reconstitution did not appear to influence the capacity of PDCs to produce proinflammatory cytokines.

FIGURE 8.
FIGURE 8.

Box and whisker plots showing secretion of proinflammatory cytokines IL-6 and TNF-α by the PDC-enriched PBMC cocultures following stimulation with TLR ligands. PDCs isolated from the concordant subjects (n = 42) released slightly higher levels of IL-6 following stimulation with TLR9 ligand CpG-2216 (A) and TLR7 ligand imiquimod (B) as compared with discordant subjects (n = 38), though the differences were statistically insignificant. C and D, Levels of TNF-α released by the PDCs following stimulation with either of the TLR ligands were also comparable between the two groups. Boxes represent 25th and 75th percentile with the median value (solid line) between boxes, while the whiskers represent the minimum and maximum values. E–H, Scatter plots (with median values) showing expression of IL-6 and TNF-α, analyzed from isolated PDCs (purity, 85–93%) following TLR stimulation by real-time RT-PCR analysis in 15 discordant and 15 concordant subjects, and 5 normal healthy controls. No significant differences were observed in the mRNA levels of IL-6 and TNF-α in the PDCs following stimulation with either of the TLR ligands between the discordant and the concordant patients.

Discussion

The principal objective of HAART is to inhibit HIV-1 replication, ultimately leading to immune reconstitution in infected individuals, traditionally measured by improvement in CD4 T cell numbers. There are no standardized or universally accepted criteria for discordant responses in HIV-1 infection and little is known about the pathogenesis of discordant responses, which seems to depend on the interaction of a multitude of viral-, host-, and treatment-related factors. The discordant patients recruited in this study did not show expected rise in their CD4 T cell counts and CD4:CD8 ratios despite maintaining low to undetectable viral loads for long periods of time, leaving the ultimate goal of HAART unachieved (Fig. 1⇑). We investigated reconstitution characteristics of DC subsets for a period of more than 2 years in a large cohort of discordant and concordant HIV-1-infected subjects and assessed the capacity of PDCs to produce IFN-α following TLR stimulation, conventionally recognized as a functional marker of PDCs. The discordant patients showed poor numerical and functional restoration of PDCs as compared with the concordant group. Generally, PDCs decrease in their numbers following HIV-1 infection; however, they gradually recover following HAART in ∼8–12 mo, though taking longer time in restoration than the MDC-1 counts (10, 31, 32). It is also known that PDC numbers and function are inversely related to plasma viral load and directly related to CD4 T cell counts with PDCs responding more readily to variations in viral load (26, 31, 38). In contrast, we observed that PDCs, like CD4 T cells, do not show restoration to normal levels in the discordant patients even after 2 years, despite prolonged viral suppression, indicating that plasma viremia does not entirely influence PDC numbers or functions in some HIV-1-infected patients. In contrast, restoration of MDC-1 population was unaffected by defective immune reconstitution. Our results, thus, show that PDCs from HIV-1-infected individuals with different CD4 T cell reconstitution characteristics behave differently in terms of expansion and effector functions, suggesting that PDC counts and IFN-α production in vitro could also serve as alternate markers of defective immune reconstitution in HIV-1 patients.

The PDCs express higher levels of CD4 on their surface as compared with MDCs, besides expressing CCR5 and CXCR4, required for HIV-1 infection (39). Therefore, it is highly probable that factors affecting CD4 T cell reconstitution, such as immune activation, damage to GALT during primary HIV-1 infection, and loss of CD4 progenitor cells, also influence numerical and functional restoration of PDCs. To understand whether the loss of PDCs is linked to initial CD4 T cell counts or viral load, we investigated the baseline CD4 T cell counts and plasma viral load of discordant and concordant patients at the initiation of HAART. We observed that the discordant patients indeed had significantly lower baseline CD4 T cell counts and higher baseline viral load at the initiation of HAART, as compared with concordant patients (Table I⇑) indicating that lower baseline CD4 T cell counts and higher plasma viral load may be associated with impaired restoration of CD4 T cells and PDCs in these patients. In a similar longitudinal study involving 21 HIV-1-infected subjects, high baseline viral load was associated with poor recovery of PDCs (11). Though there are differences in disease progression in both the groups investigated, it may be likely that the discordant patients perhaps had delayed initiation of HAART, as early treatment is reported to effectively restore PDC and CD4 T cell counts (40, 41). Earlier, we had shown that the plasma cytokine milieu of discordant patients is associated with elevated levels of cytokines, such as vascular endothelial growth factor, epidermal growth factor, IL-1α, and IL-1β, which are reflective of the primary immune damage inflicted by high levels of HIV-1 replication in these individuals before the initiation of HAART (35). Therefore, it is likely that initial immune damage caused to the lymphoid tissue during primary HIV-1 infection affects recovery of CD4 T cells and PDCs, although other factors, such as bone marrow precursor or thymic damage, drug associated cytotoxicity, and suboptimal adherence to HAART, cannot be excluded in some patients.

The PDCs isolated from discordant patients also showed lower expression of IRF-7, especially after TLR9 stimulation. Constitutive expression of IRF-7 and the ability to produce considerable amounts of IFN-α seem to represent characteristic features of PDCs (21). Engagement of TLRs in PDCs triggers a signaling cascade that rapidly activates IRF-7 and transcription of IFN-α genes (42). Though there may be other reasons associated with loss of PDC function, like nuclear translocation of IRF-7 (43), our results here demonstrate that decrease in expression of IFN-α is linked to decreased expression of IRF-7 and small changes in IRF-7 expression could influence production of IFN-α in the PDCs following TLR stimulation. It may also be noted that IRF-5 is also suggested to participate in TLR7-mediated induction of IFN-α secretion, although the mechanism and extent of this participation is still unclear (44). In contrast to IFN-α, the capacity of PDCs to release proinflammatory cytokines was independent of their reconstitution characteristics. Following stimulation with TLR7, TLR8, and TLR9, proinflammatory cytokines IL-6, IL-1β, TNF-α, and IL-12p40 are produced in a MyD88-dependent but IRF-7-independent pathway involving activation of transcription factors NF-κB and ATF2-c-Jun via IκB kinase-mediated IκB degradation and MAPK activation (36). In another report, CpG-mediated activation of B cells and PDCs as well as TNF-α production of PDCs remained largely unchanged (45). In a more recent report, An et al. (46) have shown that low concentrations of phosphatase SHP-1 leads to preferential production of proinflammatory cytokines, over IRF-7-mediated IFN-α production. Guiducci et al. (47) have highlighted a critical involvement of PI3K in IRF-7-mediated production of IFN-α, but not in proinflammatory responses of PDCs including TNF-α and IL-6 production. Therefore, in context of our observations, it seems likely that impaired reconstitution characteristics of PDCs affect IRF-7-mediated IFN-α synthesis to a greater extent as compared with synthesis of proinflammatory cytokines.

The observation that discordant patients have impaired PDC numbers and function despite persistent undetectable plasma viral load has important clinical implications. IFN-α is not only important in antiviral responses but also enhances the survival of PDCs in an autocrine fashion and up-regulates IL-12 receptor-α expression on immature CD4 T cells that colocalize to the same paracortical areas of lymphoid tissue as do the PDCs (48, 49, 50). Therefore, loss of IFN-α generation during HIV-1 infection leads to loss of adaptive immune responses in lymphoid tissues, contributing mainly toward the Th-1 functional deficits that characterize AIDS. In situations of viral rebound following drug-resistance or incompliance of therapy, the PDCs can get infected with HIV-1 in peripheral sites or in the bloodstream and join other immature DCs in bringing HIV-1 into contact with naive CD4 T cells. This could lead to a further selective demise of HIV-1 reactive nascent CD4 Th-1 cells that might otherwise have been destined to defend against HIV-1 or opportunistic pathogens (27). Soumelis et al. (6) have confirmed that it is only when both CD4 T cell and PDC counts (<2/mm3) fall below critical levels that opportunistic infections develop. Besides DC maturation, IFN-α also drives differentiation of virus-specific B cells into mature plasma cells that might be diminished in the discordant patients affecting their humoral immune responses (51). However, in our study, the discordant patients did not show any active opportunistic infection at the time of analysis. The discordant patients had slightly higher NK cell counts as compared with the concordant patients possibly compensating for the loss of PDC-mediated innate immune effector functions. It appears that pre-HAART viral load levels and immune damage do not affect recovery of NK cells over a longer period of time (>2 years) in the discordant patients following viral suppression as observed in our study. In contrast, delayed restoration of NK cell numbers has been reported during the first year of HAART in previous studies (11, 41). Taken together, these observations warrant additional studies to determine the kinetics of recovery of NK cell subsets and their functions for longer periods of time following HAART. Though we did not examine the functions of NK cells, it is also possible that IFN-α-mediated activation of NK cells might be affected in the discordant subjects. Also, TNF-α-mediated cytotoxicity might be contributing to the protective responses toward opportunistic infections in these patients, as TNF-α can enhance NK cell-mediated lysis of infected cells and maturation of PDC and MDC-1 cells, counterbalancing IFN-α deficiency (52). Nevertheless, poor reconstitution of CD4 T cells and loss of PDC number and function always keep the discordant patients at higher risk of contracting opportunistic infections regardless of their plasma viral load.

To conclude, there is increasing evidence on the role of PDCs and IFN-α production in HIV-1 infection and other diseases. We have demonstrated that inadequate CD4 T cell restoration influences restoration of PDC numbers and functions that further affects innate immune effector functions in HIV-1-infected patients with discordant immunological and virological responses. Our results, thus, delineate the importance of restoration of key immune cells like PDCs, besides CD4 T cells, and warrant investigating use of immune intervention strategies like polyethyleneglycol-IFN-α- or IFN-α-inducing TLR ligands in diseases associated with loss of PDC-mediated protective immune responses.

Acknowledgments

We thank all of the study participants, technical staff of the Laboratory for Clinical and Biological Studies, and Jessica Weinstein, Mihay Gonzalez, Margarita Ashman, and Leonardo Davila for assistance with the experiments. We also thank Evril Antoine, Nurse Coordinator (Project Outreach), and Vincent Delgado, Deputy Director Administration (Borinquen Health Care Center) in recruitment of participants.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • 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 with 18 U.S.C. Section 1734 solely to indicate this fact.

  • ↵1 This work was supported by the University of Miami-Laboratory for Clinical and Biological Studies educational and training funds.

  • ↵2 Address correspondence and reprint requests to Dr. Deshratn Asthana, Laboratory for Clinical and Biological Studies, University of Miami-Miller School of Medicine, 1550 Northwest 10th Avenue, Fox Building, Suite 118, Miami, FL 33136. E-mail address: desh{at}miami.edu

  • ↵3 Abbreviations used in this paper: HAART, highly active antiretroviral therapy; DC, dendritic cell; PDC, plasmacytoid DC; IRF, IFN regulatory factor; MDC, myeloid DC.

  • Received February 29, 2008.
  • Accepted June 6, 2008.

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