Plasma Levels of Secreted Cytokines in Virologically Controlled HIV-Infected Aging Adult Individuals on Long-Term Antiretroviral Therapy

Abstract

HIV-infected (HIV⁺) aging adult individuals who have achieved undetectable viral load and improved CD4 T cell counts due to long-term antiretroviral therapy (ART) may continue to experience inflammation and immunosenescence. Therefore, we evaluated the plasma levels of proinflammatory and anti-inflammatory cytokines in 173 HIV⁺ aging adult individuals with age ranging from 22 to 81 years on long-term ART with viral load mostly <20 HIV RNA copies/mL and compared with 92 HIV-uninfected (HIV^– or healthy controls) aging individuals. We found that the median levels of TNF-α, IFN-γ, IL-1β, IL-6, and IL-10 were higher (p < 0.001 to <0.0001) and IL-17 trended lower in HIV⁺ individuals than healthy controls. Increasing CD4 T cell counts in the HIV⁺ cohort did not significantly change the circulating cytokine levels, although levels of IL-1β increased. However, IL-17 levels significantly decreased with increasing CD4 counts in the healthy controls and yet unchanged in the HIV⁺ cohort. Of note, the levels of circulating IL-17 were significantly reduced comparatively in the healthy controls where the CD4 count was below 500, yet once above 500 the levels of CD4, IL-17 levels were comparable with the HIV⁺ cohort. With increasing CD8 T cell counts, the levels of these cytokines were not significantly altered, although levels of TNF-α, IFN-γ, and IL-6 declined, whereas IL-1β and IL-17 were slightly elevated. Furthermore, increasing age of the HIV⁺ cohort did not significantly impact the cytokine levels although a slight increase in TNF-α, IL-6, IL-10, and IL-17 was observed. Similarly, these cytokines were not significantly modulated with increasing levels of undetectable viral loads, whereas some of the HIV⁺ individuals had higher levels of TNF-α, IFN-γ, and IL-1β. In summary, our findings show that HIV⁺ aging adult individuals with undetectable viral load and restored CD4 T cell counts due to long-term ART still produce higher levels of both proinflammatory and anti-inflammatory cytokines compared with healthy controls, suggesting some level of inflammation.

Introduction

Human immunodeficiency virus type 1 (HIV)-infected adults mount both cellular and humoral immune responses against HIV antigens. Early control of HIV infection is achieved by the innate immunity followed by activation of T and B cells. Cytotoxic CD8 T lymphocytes (CTLs) are generated in most infected individuals that contain HIV infection (da Silva et al., 1999; Mothe et al., 2012), but escape mutants develop during infection (Leviyang and Ganusov, 2015; Marsden and Zack, 2015), and the efficiency of CTLs decrease with disease progression due to the loss of CD4⁺ T cells (Adnan et al., 2006; Kan-Mitchell et al., 2006). In HIV⁺ individuals, if ART is not started early in infection, the vast majority of latent viruses carry CTL escape mutations, which makes CTL ineffective (Deng et al., 2015).

Furthermore, neutralizing antibodies are produced by B cells with the help of CD4 T cells in most HIV⁺ individuals (Caskey et al., 2016; Rusert et al., 2016), but escape mutants develop due to a highly replicating virus. However, HIV infection has a damaging effect on both the innate and adaptive immune cells. HIV infection alters dendritic cell frequencies and innate functions in both acute and chronic infections (Manches et al., 2014), including changes and alterations in functions of macrophages (Howie et al., 2000). In addition, HIV has shown to pathologically change NK cell homeostasis and alter its antiviral functions (Mikulak et al., 2017). The most devastating effects of HIV infection is on CD4 T cells that are directly and indirectly eliminated due to HIV-induced pathology resulting in immunodeficiency and opportunistic infections in untreated HIV⁺ individuals.

As CD4 T cells are reduced in numbers, the efficiency of CD8 T cell functions, B cell responses, CD4 T cell-mediated proliferation, and isotype class switching is diminished. Several studies have shown that HIV infection increases the population of terminally differentiated T cells in HIV+ individuals with uncontrolled viremia causing premature aging, dysregulation of T cell function, and rapid HIV disease progression (Appay and Sauce, 2017; Cao et al., 2009; Pathai et al., 2014). However, HIV⁺ individuals with controlled viremia due to ART exhibit a significant reduction in terminally differentiated T cells and improvement in T cell functions (Behrens et al., 2018).

Due to the success of ART in controlling viremia and improving CD4 T cell counts (Kaplan-Lewis et al., 2017; Lorenzo-Redondo et al., 2016), many HIV⁺ individuals have seen reduced incidence of opportunistic infections and increased life expectancy (Lifson et al., 2017; Lundgren et al., 2015), and attained more than 50 years of age (Effros et al., 2008; United States Senate, 2005). However, HIV⁺ aging individuals are likely to mount a reduced protective immune response because of HIV-mediated immune aging (Effros et al., 2008; United States Senate, 2005) or immunosenescence (Zheng et al., 1997), which causes dysregulation of T cell function and increases the susceptibility of aging individuals to new infections and reactivation of chronic and latent infections, and a reduced response to vaccination (Zheng et al., 1997).

Both aging and HIV⁺ individuals have less protective immunity against infections likely due to dysregulation of cytokine expression (Haynes et al., 1999; Heeney, 2002), defective CD4⁺ T cell function, reduced IL-2 production (Kedzierska and Crowe et al., 2001; Watanabe et al., 2010), and weakened proliferation and differentiation of CD4 T cells to antigens (Haynes et al., 1999; Heeney, 2002; Zheng et al., 1997). More importantly, both aging and HIV infection are associated with inflammation that increases the expression of proinflammatory cytokines such as IL-1β, IL-6, TNF-α, and chemokines (French et al., 2015; Reuter et al., 2012; Stacey et al., 2009; Tudela et al., 2014). HIV⁺ individuals who are virally suppressed with ART still have residual viral replication that causes immune activation and inflammation and release of proinflammatory cytokines (Guo et al., 2014; Okay et al., 2020).

We have shown that CD4 T cells of HIV⁺ aging individuals with controlled viremia and improved levels of CD4 T cell counts due to ART produced IL-2, IL-10, and IFN-γ comparable with healthy controls (Behrens et al., 2018) and both CD4 and CD8 T cells produced higher levels HIV-specific IFN-γ and TNF-α (Behrens et al., 2020). However, evaluating the secreted levels of proinflammatory and anti-inflammatory cytokines in the plasma of these HIV⁺ aging adult individuals with controlled viremia on ART could further provide some important insights into the modulation of these cytokines.

Cytokines are both pleomorphic and pleiotropic and can be grouped as proinflammatory or anti-inflammatory and serve the function of initiating or mediating inflammation by regulating immune function and affecting viral replication. In addition, cytokines can be further grouped as Th1, Th2, or Th17 cytokines based on type of infections encountered. As HIV infection progresses, cytokines shift from a Th1 proinflammatory response to a Th2 anti-inflammatory response contributing to HIV disease progression (Kedzierska and Crowe et al., 2001). However, HIV⁺ individuals successfully treated with ART still have increased levels of plasma IFN-γ¹⁰ (Watanabe et al., 2010) and decreased levels of IL-10 (Brockman et al., 2009). The proinflammatory cytokines such as TNF-α, IFN-γ, and IL-1β, are involved in inflammatory, cell-mediated, and cytotoxic reactions as part of the Th1 and cell-mediated immunity.

Furthermore, IL-6, a pleomorphic cytokine, made by T cells, macrophages, and endothelial cells, performs several functions, including acute-phase response and inflammation (Tanaka et al., 2014). We have evaluated the plasma levels of several of these cytokines, including IL-1β, IL-6, IL-10, IL17, IFN-γ, and TNF-α in HIV⁺ aging adult individuals on ART with undetectable viral load and improved CD4 T cell counts.

Here we show that HIV⁺ aging individuals with controlled viremia and improved CD4 T cell counts on long-term ART still make proinflammatory cytokines, including TNF-α, IFN-γ, IL-1β, IL-6, and anti-inflammatory cytokine IL-10, higher than healthy controls, but not IL-17. These cytokines were differentially modulated with increasing CD4 and CD8 T cell counts, age, and viral load of HIV⁺ individuals on ART.

Materials and Methods

Human subjects and cell preparation

This study was approved by the Institutional Review Board of the University of Arizona, Tucson. Informed and signed consents were obtained from all participants in the study. The HIV⁺ individuals' cohort consisted of 173 clinically diagnosed HIV⁺ individuals with age ranging from 22 to 81 years (median; age: 55 years, CD4 counts: 614 cells/mL, CD8 counts: 663 cells/mL) receiving health care and antiretroviral therapy (ART) at the Petersen HIV Clinic, Division of Infectious Disease, Banner University Medical Center, Tucson, AZ, USA. The uninfected control cohort (HIV^– or healthy controls) included 92 healthy age-matched (age ranging from 23 to 75 years, median age: 53 years, CD4 counts: 696 cells/mL, CD8 counts: 338 cells/mL) HIV^– uninfected (healthy controls).

Approximately 60% of the individuals from both HIV⁺ and healthy control (HIV–) cohorts were seropositive from cytomegalovirus (CMV) antibodies, which could be either active or past infection, as individuals retain the antibodies for the entirety of their life after CMV infection. While several HIV clinical laboratories consider <50 HIV RNA copies/mL as undetectable viral load, Department of Health and Human Services (DHHS)/Center for Disease Control and Prevention (CDC) considers <200 HIV copies/mL as undetectable viral load.

The demographics and clinical and laboratory findings for these cohorts are summarized in Table 1. Blood samples were collected into 10 mL sodium heparin Vacutainer tubes (BD, Sunnyvale, CA, USA). Blood samples were processed at the University of Arizona Biorepository Laboratory as per their protocols, and plasma was aliquoted and cryopreserved for future analysis. One K2 ethylenediaminetetraacetic acid tube was collected to determine complete blood counts, using an A^c-T 5diff CP machine (Beckman Coulter, Pasadena, CA, USA).

Table 1.

Demographics and Laboratory Parameters of HIV⁺ and HIV^– (Healthy Controls) Individuals

	Number of subjects (n)	Age (years)	Median age (years)	Ethnicity	Lymphocyte count (cells/mL)	Median lymphocyte count (cells/mL)	CD4 count (cells/mL)	Median CD4 count (cells/mL)	CD8 T cell count (cells/mL)	Median CD4 count (cells/mL)	Viral load (copies/mL)
HIV+ Cohort	173	22–81	55	A, AA, MA, O, H, W	645–4325	1,940	84–1,983	614	149–2426	663	<20–<90
HIV– (healthy control) Cohort	92	23–75	53	AA, AI, H, W	980–4420	2,102	171–2,119	696	115–786	338	NA

A, Asian; AA, African American; H, Hispanic; MA, Mexican American; NA, not applicable O, other; W, white.

Cytokine measurement

Our study measured nonstimulated circulating cytokine levels, including IL-1β, IL-6, IL-10, IL-17A, IFN-γ, and TNF-α in the plasma of 173 HIV⁺ individuals and 92 healthy controls. These circulating cytokine levels were measured using Invitrogen Ready Set GO enzyme-linked immunosorbent assay (ELISA) kits (Invitrogen by Thermo Fisher Scientific Inc., Carlsbad, CA, USA). All cytokines were analyzed following the manufacture's protocols. Briefly, Corning TM Costar TM 9018 ELISA plates were coated with 100 mL/well of capture antibody in coating buffer. The plates were sealed with parafilm and incubated overnight at 4°C. The wells were aspirated and washed three times with >250 mL/well of wash buffer. The wells were incubated with 200 mL of ELISA/ELISASPOT diluent buffer at room temperature for 1 h. Standards were prepared with addition of ELISA/ELISASPOT diluent and allowed to incubate for 30 min, to ensure homogeneity. The wells were washed once following addition of blocking reagent.

Twofold serial dilutions of the top standards were performed to make a standard curve for a total of eight points. IL-17A, TNF-α, and IFN-γ all had a high standard of 500 pg/mL, IL-10 a high standard of 300 pg/mL, IL-6 a high standard of 200 pg/mL, and IL-1β a high standard of 150 pg/mL, including a sensitivity of 2 pg/mL for all cytokines. HIV⁺ individuals' and healthy controls' plasma (100 mL/well) were added in duplicate in wells and ELISA/ELISASPOT diluent (100 mL) and sterile water was added in duplicate as two blank controls. The plates were sealed and incubated overnight at 4°C for maximum sensitivity. The wells were aspirated and washed 3–5 times with >250 mL/well of wash buffer. Detection antibody (100 mL/well) was added to all of the plates and incubated at room temperature for 1 h.

Plates were aspirated and washed 3–5 times with >250 mL/well of wash buffer followed by addition of 100 mL of avidin-HRP to each well and incubated at room temperature for 30 min. Wells were aspirated and washed 5–7 times with >250 mL/well of wash buffer. TMB (3,3′,5,5′ tetramethylbenzidine) solution (100 mL/well) was added to each well and incubated for 15 min on a plate shaker followed by addition of stop solution (50 mL/well) and incubated for 15 min. The plates were read at 450 and 570 nm, and the 570 nm value was subtracted from the 450 nm value before analysis.

The eight-point standard curve for each plate was plotted using a four-parameter variable slope asymmetric sigmoidal curve, where X is the log concentration (GraphPad Prism 7, GraphPad Software, Inc., LA Jolla, CA, USA). Each duplicate optical density values were plotted along the asymmetric sigmoidal curve and the log concentration was interpolated using GraphPad Prism 7. Values were then converted to picogram per milliliter and duplicate values were averaged to obtain the final sample concentrations.

Statistical analysis

Overall population totals were graphed using medians and/or means with 95% confidence intervals. An unpaired, nonparametric Mann–Whitney test was used to determine statistical significance between the healthy control population totals and HIV⁺ population totals, due to non-Gaussian distribution. Linear regression models were used for all linear plots comparing a population to CD4 T cell counts, CD8 T cell counts, and age of the individuals. Statistical significance was determined by comparing the linear regression slope with a slope of zero. In the event that both linear regression slopes were significantly nonzero, the slopes were compared with each other to see if they differed significantly. Software used was GraphPad Prism 8 (LA Jolla, CA, USA). p-Values of 0.05 or lower were considered significant, *p < 0.05, **p < 0.01, ***p < 0.0001, and ****p < 0.00001.

Results

Evaluation of circulating cytokine levels in the plasma of virologically controlled HIV⁺ aging adult individuals on long-term ART

We determined the secretory levels of both proinflammatory and anti-inflammatory cytokines in the plasma of HIV⁺ individuals and HIV^– or healthy controls. First, we analyzed the lymphocyte counts in HIV⁺ and healthy control cohorts and found that there was no significant difference in the lymphocyte counts (median) between our HIV⁺ cohort (2032.19 cells/mL) compared with HIV^– or healthy controls (2092.16 cells/mL), as shown in Figure 1A. In addition, the median circulating CD4 T cell numbers (cells/mL) were lower in the HIV⁺ cohort (645.34 cells/mL) compared with the healthy control cohort (747.45 cells/mL) but not significantly different; however, there was a significant increase (p ≤ 0.0001) in median circulating CD8+ T cell counts in HIV⁺ (781.18 cells/mL) compared with the healthy controls (361.06 cells/mL), as shown in Figure 1B.

FIG. 1.

(A) Lymphocyte count (cells/mL) of HIV⁺ cohort with controlled viremia on ART and HIV^– cohort. (B) Lymphocyte count (cells/mL) of HIV⁺ and HIV^– (healthy controls) cohorts separated by CD4 and CD8 counts (n = 173 HIV⁺, n = 92 HIV^–) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). ART, antiretroviral therapy.

Next, we determined the concentration of circulating cytokine levels (pg/mL) in our HIV⁺ cohort compared with our healthy control cohort. As shown in Figure 2, there was a significant increase in the levels of proinflammatory cytokines, including TNF-α (HIV⁺: 12.32 pg/mL, HIV^–:7.28 pg/mL, p < 0.0001, Fig. 2A), IFN-γ (HIV⁺:14.03 pg/mL, HIV^–: 8.68 pg/mL, p < 0.0001, Fig. 2B), IL-6 (HIV⁺: 5.23 pg/mL, HIV^–: 4.36 pg/mL, p = 0.0058, Fig. 2C), IL-1β (HIV⁺: 3.60 pg/mL, HIV^– (healthy controls): 1.18 pg/mL, p < 0.0001, Fig. 2D), and the anti-inflammatory cytokine IL-10 (HIV⁺: 4.57 pg/mL, HIV^–: 3.76 pg/mL, p < 0.00011, Fig. 2F), but no significant difference was seen in proinflammatory cytokine IL-17 (Fig. 2E).

FIG. 2.

Plasma levels of cytokines (pg/mL) in HIV⁺ cohort (n = 173) with controlled viremia on ART compared with HIV^– (healthy controls) cohort (n = 92), including (A) TNF-α, (B) IFN-γ, (C) IL-6, (D) IL-1β, (E) IL-17, (F) IL-10 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

We also calculated the fold increases in the levels (median values) of these secreted cytokines in our HIV⁺ and healthy control cohorts, and found that there was a 1.7-fold increase in TNF-α, 1.62-fold in IFN-γ, 1.2 in IL-6, 3.05-fold in IL-1β, 1.2-fold in IL-10, and a slight decrease in IL-17 in HIV⁺ cohort compared with the healthy control cohort, as shown in Figure 3.

FIG. 3.

Fold increase in plasma cytokine levels in HIV⁺ cohort (n = 173) with controlled viremia on ART compared with HIV^– (healthy controls) cohort (n = 92), including TNF-α, IFN-γ, IL-6, IL-1β, IL-17, and IL-10 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Evaluation of circulating cytokine levels in the plasma of virologically controlled HIV⁺ aging adult individuals on ART with increasing CD4⁺ T cell counts

Next, we evaluated the levels of circulating cytokines in our virologically controlled HIV⁺ aging individuals on ART with increasing CD4+ T cell counts (Fig. 4A–F). Figure 4A shows that there was no significant difference in the levels of TNF-α in HIV⁺ and healthy control (HIV^–) cohorts with increasing CD4 T cell counts, although the levels were higher in HIV⁺ compared with healthy controls as also shown in the overall median levels (Fig. 2A). We observed a slightly decreasing level of circulating IFN-γ with increasing CD4 T cell counts, although not statistically significant, in both HIV⁺ and healthy control cohorts (Fig. 4B), supporting the notion that controlled viremia in HIV⁺ has a reduced need of IFN-γ release with increasing CD4 T cell counts.

FIG. 4.

Plasma levels of cytokines (pg/mL) in HIV⁺ cohort (n = 173) with controlled viremia on ART compared with HIV^– (healthy controls) cohort (n = 92) with increasing CD4 T cell counts, including (A) TNF-α, (B) IFN-γ, (C) IL-6, (D) IL-1β, (E) IL-10, (F) IL-17 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

While there was a slight decline in the levels of IL-6 in our HIV⁺ cohort with increasing CD4 T cell counts, there was a significant decline (p ≤ 0.001) in IL-6 levels in our healthy controls, as shown in Figure 4C. However, there was an increase in circulating IL-1β levels in the HIV⁺ cohort with increasing CD4 T cell counts, although not statistically significant because of a few outlier individuals' higher IL-1β levels, compared with no change seen in the healthy controls (Fig. 4D). We next analyzed the circulating levels of the anti-inflammatory cytokine, IL-10, and saw that there was a slight increase in the HIV⁺ cohort compared with a slight decrease in the healthy controls with increasing CD4 T cell counts and both were statistically nonsignificant (Fig. 4E).

We also looked at the levels of circulating IL-17 (IL-17A) with increasing CD4 T cell counts. Our HIV⁺ cohort showed a nonsignificant change in IL-17 levels with increasing CD4 counts (Fig. 4F). In contrast, our healthy controls demonstrated a significant decrease (p < 0.0001) in the circulating levels of IL-17 as their CD4 counts increased (Fig. 4F). Further evaluation using the clinical cutoff of CD4 counts below and above 500 cells/mL (normal >500) reveals that the HIV⁺ cohort <500 CD4 counts had a significant elevation of IL-17 in comparison with the healthy controls (p < 0.0001) as shown in Figure 5A, B, aligning with other proinflammatory cytokine data shown above. When examining median circulating levels of IL-17 in these cohorts with CD4 counts above 500, there was no longer a significant difference in IL-17 levels (Fig. 5D).

FIG. 5.

Plasma levels of IL-17 cytokine (pg/ml) in HIV⁺ cohort (n = 173) with controlled viremia on ART compared with HIV^– (healthy controls) cohort (n = 92) with CD4 T cell counts below 500 and above 500. (A) Linear graph of IL-17 by CD4 count <500 of HIV⁺ and HIV^– cohorts. (B) Column graph of IL-17 by CD4 count <500 of HIV⁺ and HIV^– (healthy controls) cohorts. (C) Linear graph of IL-17 by CD4 count >500 of HIV⁺ and HIV^– cohorts. (D) Column graph of IL-17 by CD4 count >500 of HIV⁺ and HIV^– cohorts. (E) Linear graph of IL-17 by CD4 count of HIV^– cohort. (F) Column graph of IL-17 by CD4 count of HIV^– cohort. (G) Linear graph of IL-17 by CD4 count of HIV⁺ cohort. (H) Column graph of IL-17 by CD4 count of HIV⁺ cohort (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

However, evaluation of the IL-17 levels of each cohort as the CD4 counts increase illustrates that the IL-17 levels in the healthy controls continue to significantly decline (p < 0.0001) with increasing CD4 counts, whereas the HIV⁺ cohort does not (Fig. 5C). These data suggest that in this cross-sectional cohort, HIV⁺ individuals on long-term ART with controlled viremia and increasing CD4 T cell counts retain similar circulating levels of IL-17 irrespective of CD4 count (Fig. 5H). Conversely, the healthy controls modulate the IL-17 levels with respect to CD4 counts once above a count of 500 (Fig. 5A vs. 5C, F).

Evaluation of circulating cytokine levels in the plasma of virologically controlled HIV⁺ aging adult individuals on ART with increasing CD8⁺ T cell counts

We next examined the levels of secreted cytokines with increasing CD8 T cell counts as activated CD8 T cells continue to persist despite undetectable viral loads. As expected, and shown in Figures 1B and 6A–F, HIV⁺ individuals had higher levels of CD8 T cells compared with healthy controls. With respect to the circulating cytokine levels with increasing CD8 T cell counts, there was a nonsignificant decrease in the levels of TNF-α in HIV⁺ cohort compared with HIV⁻ individuals or healthy controls (p = 0.8389), as shown in Figure 6A. In case of IFN-γ, there was a nonsignificant (p = 0.8446) decrease (Fig. 6B) similar to IL-6 that did not show any significant (p = 0.7603) difference with increasing CD8 T cell counts (Fig. 6C).

FIG. 6.

Plasma levels of cytokines (pg/mL) in HIV⁺ cohort (n = 173) with controlled viremia on ART compared with HIV^– (healthy controls) cohort (n = 92) with increasing CD8 T cell counts, including (A) TNF-α, (B) IFN-γ, (C) IL-6, (D) IL-1β, (E) IL-10, (F) IL-17.

In addition, the IL-1β levels were slightly increased (p = 0.8702; Fig. 6D), whereas levels of IL-10 (p = 0.3509; Fig. 6E) and IL-17 (p = 0.2541; Fig. 6F) were not changed with increasing CD8 T cell counts in HIV⁺ and healthy control cohorts. These data indicate that despite improved CD4 counts of most individuals, CD8 T cell counts have remained elevated due to continuous activation of CD8 T cells likely due to residual viremia in our HIV⁺ individuals on long-term ART.

Evaluation of circulating cytokine levels in the plasma of virologically controlled HIV⁺ adult individuals on ART with increasing age

We also evaluated the levels of secreted cytokines in the plasma of our HIV⁺ and healthy control cohorts with increasing age. Although we did not find any significant changes in the levels of secreted cytokines with increasing age, there were differences between HIV⁺ individuals and HIV^– (healthy controls). In the analysis of TNF-α levels, there was a positive slope for both HIV⁺ and healthy control populations, with both cohorts showing a gradual increase in TNF-α levels with increasing age, although TNF-α levels were higher in HIV⁺ compared with healthy controls (Fig. 7A).

FIG. 7.

Plasma levels of cytokines (pg/ml) in HIV⁺ cohort (n = 173) with controlled viremia on ART compared with HIV^– (healthy controls) cohort (n = 92) with increasing age, including (A) TNF-α, (B) IFN-γ, (C) IL-6, (D) IL-1β, (E) IL-17, (F) IL-10.

On the contrary, levels of IFN-γ (Fig. 7B) and IL-6 (Fig. 7C) had negative slopes for both HIV⁺ and healthy control cohorts, although the levels of IFN-γ for HIV⁺ cohort were slightly higher than those of healthy control cohort, and decreased steadily with increasing age (Fig. 7B), whereas the IL-6 levels in HIV⁺ were closer to neutral compared with healthy controls (Fig. 7C).

Furthermore, IL-1β levels for HIV⁺ cohort had a positive slope increasing with age compared with healthy controls that showed a negative slope, while the levels of IL-1β were higher in HIV⁺ individuals than healthy controls (Fig. 7D). Levels of IL-10 for both HIV⁺ and healthy control cohorts had positive slopes, although levels for HIV⁺ individuals were slightly greater than healthy controls with increasing age. In addition, IL-17 levels for HIV⁺ cohort had a positive slope, showing that IL-17 levels slightly increased with increasing age compared with the healthy control cohort that showed a negative slope (Fig. 7F). However, on average, the levels of IL-17 were consistently lower in HIV⁺ cohort compared with healthy control cohort with increasing age (Fig. 7F).

Evaluation of circulating cytokine levels in the plasma of virologically controlled HIV⁺ aging adult individuals on ART with increasing viral load

Although DHHS/CDC considers <200 HIV copies/mL and several clinical laboratories <50 HIV RNA copies/mL as undetectable viral load, most of our HIV⁺ individuals had viral load of <20 HIV RNA copies/mL and some between <30 and <90 copies of HIV RNA copies/mL. Since the viral load of our HIV⁺ individuals was very low (undetectable), there was not a significant difference in the levels of cytokines with increasing viral load of <20 to <90 HIV RNA copies/mL, although slight differences were seen (Fig. 8). Several proinflammatory cytokines were found at higher levels in HIV⁺ individuals, including TNF-α (Fig. 8A), IFN-γ (Fig. 8B), and IL-1β (Fig. 8D), but were not modulated with viral load increasing from <20 to <90 HIV RNA copies/mL.

FIG. 8.

Plasma levels of cytokines (pg/mL) in HIV⁺ cohort (n = 173) with controlled viremia on ART compared with HIV^– (healthy controls) cohort (n = 92) with increasing viral load ranging from 20 to 90 HIV copies/mL, including (A) TNF-α, (B) IFN-γ, (C) IL-6, (D) IL-1β, (E) IL-10, (F) IL-17.

Moreover, levels of other cytokines such as IL-6 (Fig. 8C), IL-10 (Fig. 8E), and IL-17 (Fig. 8F) were lower than TNF-α, IFN-γ, and IL-1β, but did not change with increasing HIV RNA copy numbers (>20 to >90).

Discussion

Here we have investigated the levels of proinflammatory and anti-inflammatory cytokines in the plasma of 173 HIV⁺ individuals, ages ranging from 22 to 81 years (median 55 years) on long-term ART with viral load ranging from <20 to <90 RNA copies/mL (median <20 HIV RNA copies/mL) and compared with 92 healthy controls (HIV^–), ranging in age from 23 to 75 years (median 53 years). We have shown that the median plasma levels of TNF-α, IFN-γ, IL-1β, IL-6, and IL-10 were higher (p < 0.001 to <0.0001) and IL-17 levels slightly lower (p = ns) in our HIV⁺ individuals with undetectable viral load on ART than healthy controls, but lower than previously described for HIV⁺ individuals with uncontrolled viremia (French et al., 2015; Osuji et al., 2018; Tudela et al., 2014).

While our study involved HIV⁺ aging adult individuals, our data on plasma levels of cytokines in HIV⁺ individuals with controlled viremia are consistent with previously described HIV⁺ individuals with uncontrolled viremia and individuals on ART (French et al., 2015; Osuji et al., 2018; Tudela et al., 2014). We also found that the plasma levels of these cytokines were generally not significantly changed, although some cytokines were modulated differently, with increasing CD4 and CD8 T cell counts and the increasing age of our HIV⁺ individuals, most likely due to ART-mediated controlled viremia (Behrens et al., 2020; Behrens et al., 2018). Taken together, our data show that HIV⁺ aging adult individuals with undetectable viral load on long-term ART still secrete pro- and anti- inflammatory cytokines, suggesting that some level of inflammation process persists in these HIV⁺ aging individuals most likely due to residual viremia.

Several studies have shown that HIV infection initially induces the proinflammatory cytokine production such as IL-1β, IL-6, IFN-γ, and TNF-α that helps in containing the intracellular HIV and later anti-inflammatory cytokines such as IL-10 that counters proinflammatory cytokines leading to HIV disease progression (Hokello et al., 2024; Osuji et al., 2018; Reuter et al., 2012; Stacey et al., 2009). Furthermore, the levels of IL-1β, IL-6, and TNF-α were significantly reduced in HIV⁺ individuals treated with ART compared with ART-naive HIV⁺ individuals but still higher than healthy controls (Okay et al., 2020; Osuji et al., 2018; Stacey et al., 2009; Vimali et al., 2022). Our data on higher levels of cytokines such as IL-1β, IL-6, TNF-α, IFN-γ, and IL-10 in HIV⁺ adult individuals with undetectable viral load due to ART compared with age-matched healthy controls are consistent with these earlier studies (Okay et al., 2020; Osuji et al., 2018; Stacey et al., 2009). Further analysis of some of these cytokines showed that our virologically controlled HIV⁺ adult individuals produced TNF-α 1.7-fold, IFN-γ 1.6-fold, IL-6 1.2-fold, IL-1β 3.1-fold, and IL-10 1.2-fold higher compared with healthy controls, similar to those seen in younger HIV⁺ individuals on ART but significantly higher in ART-naive HIV⁺ individuals with uncontrolled viremia (Osuji et al., 2018; Reuter et al., 2012; Okay et al., 2020).

Our data suggested that production of IL-1β, IL-6, TNF-α, IFN-γ, and IL-10 is still higher in our HIV⁺ individuals despite an undetectable viral load due to residual viremia and activation of cytokine producing immune cells. A slightly higher level of anti-inflammatory cytokine, IL-10, is counteracting the proinflammatory cytokines.

Interestingly, another proinflammatory cytokine IL-17, which works in synergy with TNF-α (Friedrich et al., 2015; Guo et al., 2014; Lee et al., 2008), was found to be significantly lower in our HIV⁺ cohort on ART compared with healthy controls, strictly when comparing the cohorts with CD4 counts below 500. This subset of data is consistent with other studies that showed IL-17 is not completely restored in HIV⁺ individuals on ART (Caruso et al., 2019; Wiche Salinas et al., 2021), However, when CD4 counts rose above 500, we found that IL-17 levels remained unchanged in our HIV⁺ cohort on ART, whereas IL-17 levels in the healthy controls returning to the typical circulating levels (Caruso et al., 2019; Wiche Salinas et al., 2021).

In addition, when we evaluated the relative relationship of IL-17 production with increasing CD4 counts ranging from 500 to 2,000, there was a significant decrease in IL-17 production in the healthy controls (Fig. 5C p < 0.01), whereas IL-17 levels remained consistent in our HIV⁺ cohort on ART and demonstrated a slight increase in production with increasing CD4 counts (Fig. 5C, G). IL-17 producing Th17 cells are found in genital and gut mucosa where HIV replication occurs following transmission causing immune activation and release of IL-17, which works in synergy with Th1 cytokines to generate a robust immune response, but following depletion of Th17 cells by HIV, IL-17 levels are reduced in HIV⁺ individuals without ART.

However, prior studies have shown that HIV⁺ individuals who are successfully treated by ART show an elevation of IL-17, but not complete restoration and remains lower than healthy controls (Caruso, et al., 2019; Wiche Salinas et al., 2021). When stratifying the data by CD4 count, however, our study reveals that the diminished IL-17 production seen in the HIV⁺ cohort on ART compared with healthy controls only occurred for the individuals with CD4 counts below 500. Further studies, ideally longitudinal studies during ART therapy, are needed to elucidate the roles of Th17 cytokines in HIV infection, especially in HIV⁺ individuals who have aged due to long-term ART and maintained undetectable viral loads and increased CD4 T cell counts.

Our study also examined the plasma levels of these cytokines in our virologically controlled HIV⁺ cohort with increasing CD4 and CD8 T cell counts and found not to be significantly altered, although modulated differently. TNF-α, that is made by macrophages, NK cells, and CD4 and CD8 T cells, slightly increased with increasing CD4 counts but decreased with increasing CD8 T cell counts, suggesting that the contribution of T cells in producing TNF-α reduced due to diminished activation of T cells in our HIV⁺ individuals with controlled viremia, as also shown elsewhere (Amoani et al., 2021; Pino et al., 2021).

IFN-γ, which is produced by Th1 CD4 T cells, CD8 T cells, and NK cells, slightly decreased with both increasing CD4 and CD8 counts, suggesting reduced level of activation of these cells because of lack of HIV replication as a result of suppression by ART (Amoani et al., 2021; Pino et al., 2021). Similar results were also seen for IL-6, which is produced by macrophages, endothelial cells, in addition to T cells. IL-1β mainly produced by macrophages and endothelial cells increased with both increasing CD4 and CD8 T cell counts in our HIV⁺ cohort with undetectable viral load likely due to activation of macrophages by IFN-γ made by CD4 and CD8 T cells (Amoani et al., 2021; Pino et al., 2021).

The anti-inflammatory cytokine, IL10, produced by CD4 T cells, B cells, macrophages, and dendritic cells, was not substantially altered with increasing CD4 and CD8 counts as proinflammatory cytokines have reduced due to undetectable viral load (Amoani et al., 2021; Behrens et al., 2020; Behrens et al., 2018; Pino et al., 2021). IL-17 made by Th17 cells remained unchanged with increasing CD4 and CD8 T cell count (Caruso et al., 2019; Wiche Salinas et al., 2021). Although there were some differences in several of these cytokines with increasing CD4 and CD8 T cell counts, mainly because our HIV⁺ cohort had an undetectable viral load and these cytokines were measured in the plasma without any extrinsic modification.

Similar to HIV infection, aging also increases inflammation, including secretion of proinflammatory cytokines (Behrens et al., 2020; Behrens et al., 2018; French et al., 2015; Reuter et al., 2012; Stacey et al., 2009; Tudela et al., 2014). Two of the proinflammatory cytokines, TNF-α and IL-1β, were found to be slightly increased with increasing age in our HIV⁺ cohort and were higher than healthy controls (Stowe et al., 2010), suggesting that residual viremia in our HIV⁺ individuals with undetectable viral load is contributing to the activation of cells that produce these cytokines. On the contrary, IL-6, IFN-γ, IL-10, and IL-17 remained unchanged with increasing age of HIV⁺ individuals most likely due to undetectable viral load and lack of threshold antigen levels for activation (Appay and Sauce, 2017; Kalayjian et al., 2003).

The copy numbers of undetectable viral load vary based on the laboratories and range from >20 copies to >200 copies of HIV RNA/mL. Our HIV⁺ cohort comprising 173 individuals with a median age of 55 years had a viral load ranging from mostly >20 to minimally up to >90 copies of HIV RNA/mL. Our HIV⁺ individuals showed no significant difference in the levels of these cytokines, although TNF-α and IL-10 slightly increased and IFN-γ, IL-1β, and IL-6 slightly decreased that were statically nonsignificant, suggesting that the threshold to activate cytokine producing cells was the same between >20 and >90 copies of HIV RNA/mL, as shown in previous studies (Amoani et al., 2021; Behrens et al., 2020; Behrens et al., 2018; Pino et al., 2021).

Having achieved mostly an undetectable viral load and restored CD4 T cell counts, HIV⁺ individuals on long-term ART are aging. However, their immune profile remains eschewed with a significantly inflated CD8 T cell pool and a moderately inflamed cytokine profile. Although clinically below our detection limits, their residual viremia either alone or combined with the disproportional CD8 T cell pool lay the foundation for a chronic, although subtle, inflammatory state. As a result of contributing to this inflammation, other cytokine producing cells such as macrophages, dendritic cells, NK cells, and endothelial cells continue to experience activation. Therefore, these HIV⁺ adult individuals experience a chronic level of inflammation with their increasing age (Behrens et al., 2020; Behrens et al., 2018).

Overall, our data showed that the levels of TNF-α, IFN-γ, IL-1β, IL-6, and IL-10 were higher and IL-17 production was dysregulated in our HIV⁺ cohort on ART compared with our healthy control cohort. One of the limitations of our study is that we were unable to enroll age-matched, ART-naive HIV⁺ individuals with uncontrolled viremia to compare the levels of these cytokines with our HIV⁺ individuals on ART with controlled viremia. To circumvent that, we compared our data on the HIV⁺ cohort on ART data with previously described data on ART-naive HIV⁺ cohort with uncontrolled viremia and our data from healthy controls. Importantly, our data show that some level of inflammation continues in HIV⁺ individuals on ART despite an undetectable viral load and improved CD4 counts increasing the risks of other comorbidities and necessitating the need for improving HIV treatment options.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

This work was supported by a grant to N.A. from the Arizona Biomedical Research Commission, Arizona Department of Health Services (Grant No. ADHS14-082984).