Tobacco smoking increases immune activation and impairs T-cell function in HIV infected patients on antiretrovirals: a cross-sectional pilot study

Abstract

Background: The influence of tobacco smoking on the immune system of HIV infected individuals is largely unknown. We investigated the impact of tobacco smoking on immune activation, microbial translocation, immune exhaustion and T-cell function in HIV infected individuals.

Method: HIV infected smokers and non-smokers (n = 25 each) with documented viral suppression on combination antiretroviral therapy and HIV uninfected smokers and non-smokers (n = 15 each) were enrolled. Markers of immune activation (CD38 and HLA-DR) and immune exhaustion (PD1, Tim3 and CTLA4) were analyzed in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Plasma markers of microbial translocation (soluble-CD14 - sCD14 and lipopolysaccharide - LPS) were measured. Antigen specific functions of CD4+ and CD8+ T-cells were measured, by flow cytometry, in PBMCs after 6 hours stimulation with Cytomegalovirus, Epstein-Barr virus and Influenza Virus (CEF) peptide pool.

Results: Compared to non-smokers, smokers of HIV infected and uninfected groups showed significantly higher CD4+ and CD8+ T-cell activation with increased frequencies of CD38+HLA-DR+ cells with a higher magnitude in HIV infected smokers. Expressions of immune exhaustion markers (PD1, Tim3 and CTLA4) either alone or in combinations were significantly higher in smokers, especially on CD4+ T-cells. Compared to HIV uninfected non-smokers, microbial translocation (sCD14 and LPS) was higher in smokers of both groups and directly correlated with CD4+ and CD8+ T-cell activation. Antigen specific T-cell function showed significantly lower cytokine response of CD4+ and CD8+ T-cells to CEF peptide-pool stimulation in smokers of both HIV infected and uninfected groups.

Conclusions: Our results suggest that smoking and HIV infection independently influence T-cell immune activation and function and together they present the worst immune profile. Since smoking is widespread among HIV infected individuals, studies are warranted to further evaluate the cumulative effect of smoking on impairment of the immune system and accelerated disease progression.

Figure 1. Higher cellular markers of T-cell activation in HIV infected and HIV uninfected smokers.

Immune activation (CD38, HLA-DR) markers were evaluated by flow cytometry in CD4+ and CD8+ T-cells of HIV⁺S (n = 25), HIV⁺NS (n = 25), HIV^negS (n = 15) and HIV^negNS (n = 15). (A–D) Box plots show the frequency of activated T-cells: (A) CD4+HLA-DR+, (B) CD4+CD38+HLA-DR+, (C) CD8+HLA-DR+ and (D) CD8+CD38+HLA-DR+. Box plots represent median with 25th and 75th percentile borders, error bars represent 10th and 90th percentile. The mean ± standard error of mean (SEM) for each group are given below the bar for that particular group.

Figure 2. Elevated levels of microbial products in bloodstream of smokers are associated with immune activation.

Plasma levels of LPS (Limulus amebocyte lysate chromogenic endpoint assay) and sCD14 (ELISA) were measured in HIV⁺S (n = 25), HIV⁺NS (n = 25), HIV^negS (n = 15) and HIV^negNS (n = 15). (A, B, C) Graphs depict the levels of sCD14 (ng/ml) in the 4 groups, HIV⁺S, HIV⁺NS, HIV^negS and HIV^negNS (A) and correlation of sCD14 levels with CD4+CD38+HLA-DR+ (%) (B) and CD8+CD38+HLA-DR+ (%) (C); (D, E, F) Graphs shows the levels of LPS (pg/ml) in the 4 groups, HIV⁺S, HIV⁺NS, HIV^negS and HIV^negNS (D) and correlation of LPS levels with CD4+CD38+HLA-DR+ (%) (E) and CD8+CD38+HLA-DR+ (%) (F). Box plots represent median with 25th and 75th percentile borders, error bars represent 10th and 90th percentile. Correlation between the two variables is indicated by the black continuous line in the correlation figures. The mean ± SEM for each group are given below the bar for that particular group.

Figure 3. Higher levels of immune exhaustion markers in smokers compared to non-smokers.

T-cell exhaustion markers (PD1, Tim3, and CTLA4) were evaluated by flow cytometry in CD4+ and CD8+ T-cells of HIV⁺S (n = 25), HIV⁺NS (n = 25), HIV^negS (n = 15) and HIV^negNS (n = 15). (A, B, C) frequency of exhausted CD4+ T-cells: CD4+PD1+ (A), CD4+Tim3+ (B), CD4+CTLA4+ (C) and (D, E, F) frequency of exhausted CD8+ T-cells: CD8+PD1+ (D), CD8+Tim3+ (E) and CD8+CTLA4+ (F). (G–L) Pie chart shows the comparison of frequencies of dual positive markers in CD4+ and CD8+ T-cells of HIV⁺S, HIV⁺NS, HIV^negS and HIV^negNS: CD4+PD1+Tim3+ (G); CD4+PD1+CTLA4+ (H); CD4+Tim3+CTLA4+ (I); CD8+PD1+Tim3+ (J); CD8+PD1+CTLA4+ (K) and CD8+Tim3+CTLA4+ (L). Box plots represent median with 25th and 75th percentile borders, error bars represent 10th and 90th percentile. The mean ± SEM for each group are given below the bar for that particular group.

Figure 4. Decreased frequency of IL-2 and CD107a producing CD4+ T-cells in smokers compared to non-smokers.

CD4+ T-cell function was analyzed by intracellular staining for cytokines IL-2, IFN-γ and degranulation marker CD107a using flow cytometry in the PBMCs of all four groups of participants [HIV⁺S (n = 25), HIV⁺NS (n = 25), HIV^negS (n = 15) and HIV^negNS (n = 15)] after 6hrs of stimulation with CEF and SEB in the presence of Brefeldin A and Monensin. (A, B, C) Box plots show the frequencies of CD4+ T-cells which produced IL-2 (A), IFN-γ (B) and CD107a (C) after stimulation with CEF in the four groups of participants. (D, E, F) Box plots show the frequencies of CD4+ T-cells which produced IL-2 (D), IFN-γ (E) and CD107a (F) after stimulation with SEB in the four groups of participants. Box plots represent median with 25th and 75th percentile borders, error bars represent 10th and 90th percentile.

Figure 5. Lower frequency of IL-2 and CD107a producing CD8+ T-cells in smokers compared to non-smokers.

CD8+ T-cell function was analyzed by intracellular staining for cytokines IL-2, IFN-γ and degranulation marker CD107a using flow cytometry in the PBMCs of all four groups of participants [HIV⁺S (n = 25), HIV⁺NS (n = 25), HIV^negS (n = 15) and HIV^negNS (n = 15)] after 6hrs of stimulation with CEF and SEB in the presence of Brefeldin A and Monensin. (A, B, C) Box plots show the frequencies of CD8+ T-cells which produced IL-2 (A), IFN-γ (B) and CD107a (C) after stimulation with CEF in the four groups of participants. (D, E, F) Box plots show the frequencies of CD8+ T-cells which produced IL-2 (D), IFN-γ (E) and CD107a (F) after stimulation with SEB in the four groups of participants. Box plots represent median with 25th and 75th percentile borders, error bars represent 10th and 90th percentile.