Azithromycin targets the CD27 pathway to modulate CD27hi T-lymphocyte expansion and type-1 effector phenotype

Abstract

Macrolide antibiotic azithromycin is widely used in clinical practice to treat respiratory tract infections and inflammatory diseases. However, its mechanism of action is not fully understood. Given the involvement of the CD27 pathway in the pathophysiology of various T-lymphocyte-mediated inflammatory, autoimmune, and lymphoproliferative diseases, we examined the impact of AZM on CD27 regulation and potential consequences on CD4+ and CD8+ T-cell phenotypes. Using cellular immunology approaches on healthy donors' peripheral blood mononuclear cells, we demonstrate AZM-mediated downregulation of surface CD27 expression as well as its extracellular release as soluble CD27. Notably, AZM-exposed CD27high (hi) cells were defective in their ability to expand compared to CD27intermediate (Int) and CD27low (lo) subsets. The defective CD27hi subset expansion was found to be associated with impaired cell proliferation and cell division. At the molecular level, the CD27hi subset exhibited lower mTOR activity than other subsets. Functionally, AZM treatment resulted in marked depletion of helper CD4+ (Th1) and cytotoxic CD8+ T-lymphocyte (Tc1)-associated CXCR3+CD27hi effector cells and inhibition of inflammatory cytokine IFN-γ production. These findings provide mechanistic insights on immunomodulatory features of AZM on T-lymphocyte by altering the CD27 pathway. From a clinical perspective, this study also sheds light on potential clinical benefits observed in patients on prophylactic AZM regimens against various respiratory diseases and opens avenues for future adjunct therapy against Th1- and Tc1-dominated inflammatory and autoimmune diseases.

Keywords: CD27 subset; CXCR3; T-lymphocytes; azithromycin; inflammation; mTOR; type-1 immunity.

Figure 1

AZM suppresses CD27hi subset expansion. Anti-CD3/CD28-stimulated PBMCs were cultured for 3 days in the presence of 0 µg/ml, 20 µg/ml, and 40 µg/ml AZM. (A) Representative contour FACS plots showing the gating strategy; PBMCs were subsequently gated for singlet, live lymphocytes, CD3+, and CD4+ and CD8+ T-lymphocytes. (B) Representative contour FACS plots showing frequencies of CD27hi, CD27Int, and CD27lo subsets of CD4+ (upper panel) and CD8+ (lower panel) T cells. Numbers in each FACS plot denote the cell frequency in the respective CD27 gated subset. (C) Statistical plots showing percentage (mean ± SEM) of the CD27hi, CD27Int, and CD27lo subsets against indicated doses of AZM. Unstim stands for cells without anti-CD3/CD28 stimulation. Data presented are from n = 8 healthy individuals. Statistical significance was calculated using two-way ANOVA followed by Šidàk multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns stands for non-significant.

Figure 2

AZM preferentially arrests CD27hi subset cell division. CFSE-labeled PBMCs were stimulated with anti-CD3/CD28 in the presence or absence of 40 µg/ml AZM for 3 days. (A) Representative FACS histograms showing the levels of cell proliferation as determined by the CFSE dilution assay. Numbers in each histogram denote the percent proliferation (CFSElo cells) of CD27hi (top), CD27Int (middle), and CD27lo (bottom) subsets on CD4+ and (C) CD8+ T cells. (B) Scattered dot plots show percent (mean ± SEM) proliferation of respective CD27 subsets of CD4+ and (D) CD8+ T cells. (E) FACS histogram and schematic view of the gating strategy to determine the number of cell divisions. Div-0 (no division), Div-1 (one division), Div-2 (two divisions), and Div-3 (three divisions). (F, G) Statistical plots showing the proportion of proliferated cells per division (mean ± SEM) in CD27hi, CD27Int, and CD27lo subsets. Data presented are from n = 6 healthy individuals. Statistical significance was calculated using the Mann–Whitney (U) test for comparing two groups and two-way ANOVA followed by Šidàk multiple comparisons test for multiple groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, and ns stands for non-significant.

Figure 3

AZM promotes apoptosis of CD27 subsets. PBMCs were stimulated with anti-CD3/CD28 in presence or absence of 40 µg/ml AZM, and apoptosis was performed on day-3 using Annexin V and 7-AAD labelling. (A) Representative FACS plots showing percent apoptosis (Annexin V+ cells) on CD27hi, CD27Int, and CD27lo gated cells of CD4+ and (C) CD8+ T cells. (B, D) Scattered dot plots display total Annexin V+ cells (mean ± SEM) in respective CD27 subsets. (E, F) Scattered dot plots show % survival (Annex V-7AAD- cells) of CD27hi, CD27Int, and CD27lo subsets of CD4+ and CD8+ T cells. Percent cell survival was calculated by considering AZM untreated cells survival as hundred percent. Data presented are from n = 5 healthy individuals. Statistical significance was calculated using Mann–Whitney (U) test. *P < 0.05, **P < 0.01, and ns stands for non-significant.

Figure 4

AZM downregulates CD27 expression on activated T-lymphocytes. Cells were stimulated with anti-CD3/CD28 for the indicated period in the presence or absence of 40 µg/ml AZM. Representative FACS histogram showing mean fluorescence intensities (MFIs) of CD27 on CD4+ and CD8+ gated T cells on day 1 (A) and day 3 (C). Scattered dot plots show fold change in the MFI (mean ± SEM) normalized to unstimulated cells of CD4+ and CD8+ T cells on day 1 (B) and day 3 (D). Soluble CD27 concentration was measured in the culture supernatant using ELISA. (E) Scattered dot plot showing the concentration of sCD27 pg/ml (mean ± SEM). Data represent unstim (n = 4), 0 AZM and 40 AZM (n = 7 individuals each). (F) Representative contour FACS plot showing activation status, CD69 expression levels on CD4+ (upper panel), and CD8+ T cells (lower panel). Unstim cells serve as negative control. (G) Scattered dot plot showing percent CD69+ (mean ± SEM) cells on CD4+ and CD8+ T cells. Data presented are from n = 6 (B, D, G) and n = 7 (E) healthy individuals. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, **P < 0.01,***P < 0.001, ****P < 0.0001, and ns stands for non-significant.

Figure 5

AZM attenuates mTOR activity of CD27hi subset. Cells were stimulated with anti-CD3/CD28 in the presence or absence of 40 µg/ml AZM for 24 h. mTOR activity was determined by measuring the levels of phosphorylated S6 ribosomal protein (pS6RP). Resting cells without anti-CD3/CD28 stimulation (Unstim) are taken as negative control and to set CD27hi subset gating. (A) Representative contour FACS plots show levels (%) of pS6RP in CD27hi, CD27Int, and CD27lo subsets of CD4+ (upper panel) and CD8+ T cell (lower panel). (B) Scattered dot plots displaying % pS6RP+ cells (mean ± SEM) in total CD27hi, CD27Int, and CD27lo subset of CD4+ and (C) CD8+ T cells. Data presented are from n = 5 healthy individuals. Statistical significance was calculated using the Mann–Whitney (U) test. *P < 0.05, and ns stands for non-significant.

Figure 6

AZM triggers depletion of the CXCR3+ CD27hi subset. PBMCs were stimulated for 3 days in the presence or absence of 40 µg/ml AZM. (A) Representative contour FACS plots showing percentage of CXCR3+ cells on bulk CD4+ (upper panel) and CD8+ T cells (lower panel). (B) Scattered statistical plots showing percent CXCR3+ cells (mean ± SEM) on respective CD4+ and CD8+ gated T cells. (C) Representative contour FACS plots showing levels of CXCR3+ cells on CD27hi, CD27Int, and CD27lo subsets of CD4+ (upper panel) and CD8+ T cells (lower panel). Unstimulated (Unstim) cells served as control and to set CD27hi gating. (D) Scattered dot plots showing percent CXCR3+ cells (mean ± SEM) on CD27hi, CD27Int, and CD27lo subset of respective CD4+ and CD8+ gated T cells. Data presented are from n = 6 healthy individuals. Statistical significance was calculated using one-way ANOVA followed by Tukey’s multiple comparison test. *P < 0.05, ***P < 0.001, ****P < 0.0001, and ns stands for non-significant.