Andrade, Regis M*; Andrade, Arnaldo F B*; Lazaro, Marta A*; Vieira, Morgana M M†; Barros, Priscila O†; Borner, Alice R S†; Silva-Filho, Renato G†; Santos, Juliana O†; Brindeiro, Rodrigo M‡; Tanuri, Amilcar‡; Bento, Cleonice A M†
Since the beginning of AIDS epidemic, caused by HIV, the proportion of AIDS cases in adults aged more than 50 years has been increasing fast around the world,1 which confirms the need for more studies on this particular population.
Despite the frequencies of HIV-1-specific CD4+ and CD8+ T cells that secrete IL-2 and IFN-γ are good predictors of delayed disease progression and long lasting stable peripheral CD4+ T cells counts, in the great majority of patients viral replication proceeds, and disease progression toward AIDS.2,3 This lack of viral control is mainly accelerated by an early HIV-specific CD4+ T-cell loss and escape mutations in cytolytic T lymphocytes epitopes that commonly develop during infection with HIV-1.4,5
As infection proceeds, the main damage on the immune system is the quantitative and qualitative loss of Th1-like lymphocytes by multiple virus-induced events.6-8 HIV-1 is little cytopathic, and death of target cells by continuous viral budding is not a predominant mechanism of CD4+ T-cell loss.6 Moreover, the destruction of these target cells by HIV-1-specific cytolytic T lymphocytes seems not to contribute significantly to quantitative immune decline9,10 as well. Nowadays, there is a consensus that persistent immune activation after HIV-1 infection is the main cause of immunodeficiency.9,11,12 HIV-1-induced activation is generalized, embracing not only T cells but all components of the immune system, leading to serious disturbances of immune homeostasis.9,11,12
The chronic systemic production of pro-inflammatory cytokines favors cell death by inducing the expression of pro-apoptotic proteins (Bim and Bax), whereas downregulates the levels of the antiapoptotic proteins, such as Bcl-2.10 Furthermore, dysfunctional PD-1+ and Tim-3+ T cells accumulate during the course of infection.13,14 In association with high levels of pro-inflammatory cytokines, impaired IL-2 production and disturbances on its signal transduction pathway have been described as the major deleterious effect on immune homeostasis.15,16 As a consequence of these events, there is an increased T-cell turnover.
Additionally, immune activation directly influences the viral replication process. Many authors have proved the existence of a very close relationship between the synthesis of pro-inflammatory cytokines, such as TNF-α and IL-6, and viral replication.6-8,17-19 Activation of the respective signal transduction pathways by these cytokines induces nuclear factor kB (NF-kB) and nuclear factor of activated T cells (NF-AT), both of them are able to accelerate viral life cycle from HIV-1 proviral DNA.
In chronically HIV-1-infected patients, the production of high levels of pro-inflammatory cytokines can be supported by different immune cells.6-8,20 Some works have shown that macrophages and HIV-specific and nonspecific activated CD8+ T lymphocytes from infected patients are important sources of IL-1β, IL-12, and TNF-α, with impact on clinical disease progression.11,21
As infection proceeds, a preferential loss of the T-helper 1 (Th1)-like responses gradually renders the patients highly susceptible to opportunistic infections and less able to mount protective cellular and humoral immune response after immunizations.22-24 The successful highly active antiretroviral therapy (HAART) helps these patients to restore the immune response by attenuating these immune dysregulation.15,23-27
The marked decrease in the incidence of AIDS-related diseases after the introduction of HAART correlates with the control of plasma viral load (VL) and an increase in peripheral CD4+ cell counts.23 Many studies with HAART-treated HIV-infected young adults have shown that the risk of some opportunistic infections is reduced when patients achieve CD4+ T-cell counts above a certain value.23 However, in addition to increasing CD4+ cell count, the impact of HAART on the degree of functional immune recovery depends on the reduction of the hyperactivation state discussed above.25-29 Normalization of pro-inflammatory cytokines levels and recovery of IFN-γ production have been observed in some patients after successful HAART.15,30-32
Besides the reduction of the degree of immune activation, some host variables, particularly age, have been shown to correlate with the extent of immune restoration after HAART initiation.33-35 Some studies have demonstrated a negative influence of age on CD4+ T-cell numeric recovery after HAART in naive AIDS patients.1,33-35 Nevertheless, older AIDS patients, in general, have good virological response to HAART.35 To our knowledge, no work has evaluated the immune function status in this group of AIDS patients in response to recall antigens after immunization.
The present study aimed to evaluate the impact of age in reconstituting the humoral and cellular immune response to tetanus vaccination in young and aged AIDS patients with virological and immunological success after HAART, using healthy age-matched groups as controls.
Patients and Tetanus Immunization
The study examined a group of 20 HIV-1-infected individuals more than 55 years old (mean: 58.9 years; range: 55-65) who were successfully treated with antiretroviral therapy. A second group included 20 young HIV-1-infected patients (mean: 31.1 years; range: 22-38), with similar infection characteristics. All individuals were recruited from the Centre of Epidemiology at the University Hospital of the State University of Rio de Janeiro, Brazil. As controls, gender matched healthy young (n = 20; mean: 27.6 years; range: 20-40, 50% male) and healthy aged individuals (n = 20; mean: 61 years; range: 60-65, 45% male) were enrolled in the study. The written consent for participation was obtained from all, and the study was approved by the Ethics Committee of the same University Hospital.
All characteristics of the HIV-infected individuals, obtained from medical records, are presented in Table 1, including the HAART scheme and the CD4+ T-cell count at baseline and 24 months after the beginning of antiretroviral therapy. The CD4+ cell counts are expressed as absolute values, and the VL was transformed to log10-scale to normalize distribution. As shown in the Table 1, despite the difference in baseline VL, all patients were successfully treated with a 3-drug combination. All of them had achieved undetectable plasma HIV-1 RNA levels, defined here as less than 80 copies per milliliter, within 3 months after therapy, and have remained below this level for at least 2 years (data not shown). Importantly, the patients started therapy with similar baseline CD4+ T-cell counts and responded immunologically by increasing these counts to comparable values of CD4+ cells after therapy. To avoid problems concerning multidrug failure, all the AIDS patients were in their first antiretroviral scheme.
As all these individuals had received tetanus toxoid (TT) vaccine during their childhood, and not during adolescence or adult life, a single boost of TT (Sanofi Pasteur, SA, Lyon, France) in the deltoid muscle was given to all individuals, and the blood samples were collected by venipuncture on day 30 after vaccination for all experiments. Specifically for the evaluation of humoral response, blood samples were also collected immediately before TT immunization. Among the HIV-1-infected patients, the clinical and virological status remained stable 2 months after TT immunization.
Quantification of TT-Specific Immunoglobulin G in the Serum
Immediately before and 30 days after TT immunization, the blood samples were collected and the serum was obtained for TT-specific immunoglobulin G (IgG) quantification by SERION ELISA Classic kit (Immunomat TWIN System, Würzburg, Germany). Values lower than 0.01 IU/mL were considered not protectors, values between 0.11 and 0.5 IU/mL indicated sufficient protection, and values >0.5 IU/mL indicated long lasting protection.
Cell Culture and Stimulation
Thirty days after TT immunization, blood samples were collected from both control individuals and successfully HAART-treated HIV-1-infected patients. Peripheral blood mononuclear cells (PBMC) were obtained by centrifugation on Ficoll-Hypaque gradients as previously described.18 For some experiments, CD8+ cell-depleted PBMC were obtained by negative selection using magnetic beads coated with anti-CD8 monoclonal antibody (Dynal Biotech, Great Neck, NY). The efficacy of this procedure was approximately 96% as evaluated by flow cytometry (data not shown). The number of viable cells of each preparation was measured by Trypan blue exclusion in a hemocytometer. Viable cells were adjusted to a concentration of 1 × 105 cells per well and cultured in a 96-well round bottom microtitre plates with 200 μL RPMI 1640 added with 2 μmol/L L-glutamine (GIBCO, Carlsbad, CA), 10% fetal calf serum, 20 U/mL penicillin, 20 μg/mL streptomycin, and 20 μmol/l HEPES buffer. The cultures were stimulated with TT at 1 μg/mL (SBL Vaccin, Stockholm, Sweden) in the presence or absence of recombinant human IL-2 (rhIL-2) at 20U/mL (BD Systems, Minneapolis, MN). In some experiments for evaluation of TT-induced cytokine production, saturating doses of anti-IL-10 mAb (22 μg/mL; B&D System) or isotype control (IgG2a) were added at the beginning of the cultures and 3 days later. The cells were maintained for 7 days at 37° C in a humidified 5% CO2 incubator for proliferation and cytokine assays.
Of note, the TT dose (1 μg/mL) was previously established by our group by evaluating PBMC proliferative response to different TT concentrations (0,1-10 μg/mL) in a group of healthy immunized individuals (median 39.7 years old; range 22-65). Higher doses of TT did not modify the maximal [3H]-thymidine uptake (data not shown). Similarly, the IL-2 dose was also previously established by a dose-response study in continuous T-cell line proliferation by IL-2-dependent cell line (CTLL) assay (data not shown), and IL-10 mAb doses were established in our previous work.18
Approximately 1 × 105 cells per well of PBMC were activated or not with TT (1 μg/mL), with or without rhIL-2 (20 U/mL) for 7 days. The cellular proliferation was measured after addition of [3H]-thymidine (0.51 μCi/well) for the last 12 hours of incubation. The cells were harvested in glass fiber filters in an automatic cell harvester, and radioactive incorporation was measured using a liquid scintillation counter.
The supernatants from PBMC or CD8+ cell-depleted PBMC cultures activated or not with TT plus IL-2 were collected after 7 days and cytokines were measured using OptEIA enzyme-linked immunosorbent assays (BD Pharmigen, San Diego, CA), according to manufacturer's protocol. Briefly, each pair of monoclonal antibodies was used to detect human IL-1β, IL-10, IL-4, TNF-α, and IFN-γ. The reaction was revealed with streptavidin-horseradish peroxidase, using 3,30,5,50 tetramethylbenzidine as substrate. Recombinant human IL-1β, IL-10, IL-4, TNF-α, and IFN-γ at concentrations ranging from 10 to 500 pg/mL were used to construct standard curves.
The Effect of TT on In Vitro HIV-1 Replication
To evaluate the impact of TT addition on in vitro viral replication, the supernatants of TT-activated cell cultures were collected 7 days after the beginning of the cultures and stored at −70°C. This time was chosen because, in previous experiments, the peak of in vitro HIV-1 replication occurred at this point.18 HIV-1 RNA was measured in the supernatants with a commercial reverse transcriptase-polymerase chain reaction kit (Amplicor HIV Monitor Test, Roche Molecular System, Branchburg, NJ), with a detection threshold of 80 copies per milliliter.
The nonparametric Mann-Whitney U test was applied to determine whether the groups were statistically different for each given variable. The impact of TT vaccination on both IgG titers and in vitro HIV-1 replication for the young and aged AIDS patients was analyzed using paired Student t test. The significance in all experiments was defined as P < 0.05.
In Vivo IgG Concentration and In Vitro Lymphoproliferative Response to TT was Lower in Successful HAART-Treated Aged HIV-1-Infected Patients
The first immune event studied was the lymphoproliferative response induced by TT in cell cultures obtained from previously TT-immunized young and aged individuals. As demonstrated in Figure 1, lymphoproliferative response to TT in younger HIV-1-infected patients was similar to age-matched control group (uninfected young) and it was higher compared with aged groups. On the other hand, among the older individuals, the level of counts per minute (cpm) was lower in the HIV-1-infected patients (Fig. 1). As in both aging and AIDS, the reduction in lymphocyte proliferation is related to impairment of IL-2 production,16,36 we added to each cell cultures optimal doses of human recombinant IL-2. Although the addition of IL-2 had no significant effect on TT-specific cell proliferation among cultures from young individuals, it elevated, but not to normal levels, the response to TT in cell cultures from the aged ones. More important, even in the presence of IL-2, the TT-specific proliferative response remained lower in aged HIV-1-infected group. Of note, without previous TT vaccination, the in vitro lymphoproliferation to TT with or without IL-2 was undetectable in PBMC cultures from young and aged AIDS patients (dada not shown). As IL-2 alone does not induce cell proliferation (data not shown), but it improved the proliferation performance in response to TT, we decided to do the other in vitro immunological assays in presence of TT plus IL-2.
Concerning humoral response, the dosage of TT-specific IgG in the serum revealed lower antibody concentrations in samples from aged patients (Fig. 2). Four weeks after vaccination, all healthy elderly had raised IgG titers, but antibody concentrations were still lower than in young groups. No statistical difference was observed postvaccination in young patient groups. Of note, despite 100% of young AIDS patients had acquired high levels of anti-TT IgG (long lasting protection), only 30% of the aged AIDS patients raised IgG titers higher than 1.1 IU/mL after vaccination.
Higher Pro-Inflammatory Profile Associated With Lower Levels of Th1-Cytokines Observed in Aged HIV-1-Infected Patients
Concerning the investigation of cytokine network disturbances, we observed that TT-activated PBMC cultures from HIV-infected patients produce high levels of IL-1β and TNF-α, mainly in aged patients (Fig. 3). Despite their high tendency to produce pro-inflammatory cytokines, the production of IFN-γ was lower in aged HIV-infected patients (Fig. 3). Furthermore, this low in vitro IFN-γ production correlated with low in vivo anti-TT IgG titers (data not shown).
The levels of IL-10 have been described to increase with age, and this cytokine is known to impair Th1-mediated response.10,37 Among the aged patients, the neutralization of endogenous IL-10 increased IFN-γ production only in TT-activated PBMC from the healthy group (Fig. 4). Finally, concerning IL-4 release, no statistical difference was observed between the groups.
Unlike the healthy young individuals, both aged and AIDS patients have high counts of abnormal peripheral CD8+ T cells,21,37-39 and to analyze the contribution of this T-cell subset to the deficient Th1 response observed in older group, we cultured CD8+ cell-depleted PBMC on the same previously mentioned conditions. As observed in Figure 5, despite the depletion of CD8+ cells has significantly reduced the IL-1β and, mainly, TNF-α release from aged HIV-infected patients, no modification was observed concerning IFN-γ production. In healthy aged group, the depletion of this T-cell subset reduced all cytokines evaluated but did not change the cytokine profile of the cultures (Fig. 5).
To evaluate whether the lower ability to perform Th1 response to TT in the aged group was related or not to the level of in vitro HIV-1 replication, supernatants from TT-activated cell cultures were collected 7 days after the beginning of the cultures and the concentration of HIV-1 RNA was quantified by reverse transcriptase-polymerase chain reaction. Interestingly, the level of viral replication was significantly lower in the TT-activated cell cultures from successfully HAART-treated aged patients (811.5 ± 1028) when compared with the younger group (2488 ± 2046) (P = 0.0075).
The pharmacological treatment of AIDS patients by HAART leads to the suppression or reduction in plasma VL and to an increase in CD4+ T-cell count.15 However, the success of immunological reconstitution after HAART can be less efficient is some patients, particularly in aged ones. Recent studies have described a negative influence of age on CD4+ cell recovery after HAART initiation in HIV-infected patients, despite a better viral control in the older ones.33-35,40 Nevertheless, in our study, even working with aged HIV-1-infected patients who had augmented the CD4+ T-cell counts to similar levels to those observed in younger patients, the functional humoral and cellular immune recovery to the recall antigen TT was significantly lower. Similar results were obtained by our group when T-cell polyclonal activators were used.18 Many studies have found a direct correlation between HAART-induced functional immune reconstitution to different antigens and good rise in peripheral CD4+ T-cell count.25-27 Our results, however, suggest that, in aged patients, who represent an increasing group of HIV-1 victims, the elevation in CD4+ T-cell counts after HAART may be merely a mathematical event, rather than a true functional immune recovery.
In our study, among AIDS patients, the lymphoproliferation to TT was higher in the younger group. Some authors have previously shown that reduction in lymphoproliferation in both young and elderly HIV-1-infected adults is due, at least in part, to their diminished ability to produce IL-2.16,36 In our system, although exogenous IL-2 had augmented the cell proliferation to TT in the aged group, it remained much lower than in younger patients. The incomplete ability of IL-2 to restore cell proliferation could be related to a damage in CD25 signal transduction pathway described in elderly individuals by some authors.39,41 The absence of effect of IL-2 on cultures of young patients may represent a saturation of in vitro cellular proliferative potential to TT.
Many immune disturbances described in both healthy elderly individuals and chronically HIV-1-infected patients are related to dysregulation in cytokine network,38,42 and in AIDS patients, the magnitude of functional immune recovery by HAART is directly proportional to the degree at which immune hyperactivation is controlled.15,43 Similarly to what happens throughout several decades of a normal person's life, it has been proposed that, in HIV-1 infection, the persistent systemic immune activation by chronic antigen exposure is the main reason of accelerated immunosenescence.9,11,12,37-39 In our work, IL-1β and TNF-α releases were higher in TT-activated cell cultures from healthy aged individuals and from AIDS patients. Systemic pro-inflammatory cytokines suppress both B-cell formation in bone marrow and thymopoiesis, resulting in progressive reduction of naive B-cell and T-cell counts.37 In this scenario, an accumulation of abnormal terminally differentiated effector T cells occurs.38,39 These abnormal T cells have short telomeres, a highly restricted T-cell receptor repertoire, an impaired capacity to migrate to lymph nodes, and a decreased ability to be stimulated by antigen-presenting cells, a result of the loss of the costimulatory molecules CD28 and CD27.37-39,21 Among these cells, Th1 lymphocytes are the most susceptible to the deleterious effects of oxidative stress related to chronic antigen stimulation and pro-inflammatory cytokines.44 Furthermore, the defective IL-2 production jeopardizes the long living cells by not inducing anti-apoptotic proteins, like bcl-2.21,37-39
IL-2-secreting and IFN-γ-secreting Th1 cells are not only the main coordinators of protective immune response against infectious diseases, but they are pivotal in responding to T-dependent antigens vaccination, such as TT.45,46 In our study, the stronger pro-inflammatory profile observed in aged HIV-1-infected patients is paradoxically associated with a dramatic damage on IFN-γ production by TT-activated cell cultures, even after IL-2 addition.
Authors have demonstrated that functional damage in Th1 cell subset in healthy elderly individuals impairs Ig isotype switching and somatic mutation, which are essential for the production of high-affinity antibodies and decreases titers of IgG in the serum.47-49 In these individuals, the antitetanus antibody levels before booster TT dose did not reach protective titers in 50% of them. In our cohort, no aged AIDS patient had protective levels of anti-TT IgG at baseline. These levels efficiently raised in only 30% of them postvaccination. Altogether, these observations suggest that the prevalence and intensity of the humoral immune response to tetanus decay in aging, and this phenomenon is amplified by HIV-1 infection. Furthermore, our results also demonstrate that HAART does not efficiently restore, in older patients, the ability to adequately mount a humoral and cellular immune response.
In our previous study, the peripheral CD8+ T cells were the main sources of pro-inflammatory cytokines in CD3-activated cell cultures from HIV-1-infected patients.18 It is known that, as disease progresses, specific and nonspecific chronically HIV-activated CD8+ T cells contribute to a generalized state of immune activation by secreting high levels of IL-1β and TNF-α. Successfully HAART-treated patients have a tendency to reduce this immune dysregulation.21,50 In our study, depletion of CD8+ cells diminished significantly the levels of these pro-inflammatory cytokines, but did not improve the production of IFN-γ by TT-stimulated cultures from aged AIDS patients, indicating that the Th1-mediated response to TT could not be functionally restored in this patient group.
Advanced age has been associated with an expanded peripheral regulatory T-cell pool, such as IL-10-secreting T cells, probably to control the hyperactivated state.37 In HIV-infected patients, excessive production of IL-10 has been suggested to cause deleterious effects by inhibiting the production of Th1 cytokines that are implicated in promoting resistance against several pathogens.51 In the present study, although elevated IL-10 production was detected in the TT-activated cell cultures from both HIV-infected and HIV-uninfected aged subjects, the blockade of this cytokine did not improve the IFN-γ production in aged AIDS patients, in contrast to the healthy aged group. This finding is in agreement with the theory that, during the course of HIV infection, IFN-γ-producing cells are the most destroyed cells.6,24,52
Pro-inflammatory cytokines, such as IL-1β, IL-6, and TNF-α, leads not only to immunologic exhaustion, but also renders CD4+ T cells more susceptible to direct HIV infection by enhancing the expression of the HIV coreceptor CCR5.53 Despite the pro-inflammatory cytokines detected in cultures from the aged groups, IL-10 was the highest produced cytokine in these cultures in response to TT. In our previous work, the ability of CD4+ T cells from elderly AIDS patients to produce high levels IL-10 was directly associated to low in vitro HIV-1 replication.18 In aged patients, although anti-IL-10 mAb significantly elevated the number of RNA copies in polyclonaly activated CD4+ T-cell cultures, it did not elevate the IFN-γ release.18 Therefore, despite the low IFN-γ production, the good virological response to HAART in aged patients observed by physicians and described by some authors54 could be explained, at least in part, by the higher IL-10 levels in this age group.18
In conclusion, our work reveals a complex immune dysfunction in aged HIV-1-infected patients, even successfully treated with HAART. The results reported here indicate a dramatic loss of TT-specific IFN-γ-secreting T cells associated with persistent immune hyperactivation that could possibly be extended to other recall antigens. A better characterization of all immune disorders in aged AIDS patients to recall antigens can provide valuable information that might help to design better immunoprophylatic and perhaps immunotherapeutic strategies for this particular group.
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