Azithromycin Differentially Alters TCR-Activated Helper T Cell Subset Phenotype and Effector Function

Abstract

In addition to their antibiotic activities, azithromycin (AZM) exhibits anti-inflammatory effects in various respiratory diseases. One of the potent anti-inflammatory mechanisms is through inhibition of CD4+ helper T (Th) cell effector function. However, their impact on specific Th subset is obscure. Herein, we demonstrate the cellular basis of phenotypic and functional alterations associated with Th subsets following AZM treatment in vitro. Using well-characterized Th subset specific chemokine receptors, we report significant suppression of T cell receptor (TCR)-stimulated hyperactivated CCR4+CXCR3+ (Th0) expansion compared to CCR4-CXCR3+ (Th1-like) and CCR4+CXCR3- (Th2-like) cells. Interestingly, this effect was associated with diminished cell proliferation. Furthermore, AZM significantly inhibited the inflammatory cytokines IFN-γ and IL-4 production, CCR4 and CXCR3 receptor expression, and viability of Th0, Th1-like, and Th2-like subsets. Our findings suggest that AZM differentially affects TCR-activated Th subsets phenotype and function, and CCR4 and CXCR3 downregulation and suppressed Th0 subset expansion could potentially influence their trafficking and differentiation into cytokine-producing effector cells.

Keywords: CCR4; CD4+ helper T cells; CXCR3; IFN-γ; IL-4; anti-inflammatory; apoptosis; azithromycin.

FIGURE 1

AZM suppresses bulk CD4+ T cells function. (A) Cell proliferation. CFSE-labeled purified CD4+ T cells were stimulated with anti-CD3 anti-CD28 in presence of indicated concentration of AZM as described in materials and methods. Live cells were gated to detect level of proliferation. Representative FACS plots show the percent CFSE+ cells and each streak in gated population represents a cell division. (D) Aligned dot plots show mean percentage ± SEM cell proliferation. Data shown are from five healthy individuals. (B) Cell viability. Anti-CD3/CD28 stimulated cells were treated with indicated concentration of AZM. On day 3 cell death assay was performed using Annexin V and 7-AAD labeling. Representative FACS plots show percent apoptosis. (E) Aligned dot plots show mean ± SEM of total Annexin V+ (apoptosis) cells. Data shown are from four healthy individuals. (C) Cytokine production. Anti-CD3/CD28 stimulated and unstimulated (US) cells were double-stained with anti-IFN-γ and anti-IL-4 mAbs as described in the section “Materials and Methods.” FACS plots show the percent cytokine production by AZM treated and untreated cells. Cells without anti-CD3 and CD28 stimulation were taken as control (US). Aligned dot plots show mean ± SEM of total IFN-γ+ (F) and IL-4+ cells (G). Data shown are from three healthy individuals. *P < 0.05, **P < 0.01, ***P < 0.001, ns stands for non-significant.

FIGURE 2

AZM inhibition of helper T cell subset expansion. CD4+ T cells were stimulated with plate-bound anti-CD3 and soluble anti-CD28 in presence of indicated concentration of AZM for 3 days. (A) Representative contour FACS plots showing percent frequencies of various Th subsets. (B) Aligned dot plots show means ± SEM of Th subsets, CCR4+CXCR3- (Th2-like), CCR4+CXCR3+ (Th0), CCR4-CXCR3+ (Th1-like), and CCR4-CXCR3- (DN) cells. Data presented are from five independent experiments performed on healthy individuals (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ns stands for non-significant.

FIGURE 3

AZM downregulates CCR4 and CXCR3 expression on bulk CD4+ T cells. (A) Purified CD4+ T cells were stimulated with anti-CD3 and CD28 in presence of indicated concentration of AZM for 3 days. Cells were stained with anti-CCR4 and anti-CXCR3 monoclonal antibodies and looked for expression using flow cytometry. Representative FACS overlay histogram showing mean fluorescence intensities (MFI) of AZM treated and untreated cells. TCR unstimulated cells (US) serve as control. (B) Aligned dot plots show mean ± SEM of total CCR4+ and CXCR3+ cells on bulk CD4+ T cells. Data presented are from five independent experiments performed on healthy individuals (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001, ns stands for non-significant.

FIGURE 4

AZM suppresses helper T cell subset proliferation. CFSE-labeled CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in presence of indicated concentration of AZM for 3 days. Cells without anti-CD3 and CD28 activation (US) were taken as control. Individual Th subsets (Supplementary Figure S1B) were gated to determine cell proliferation by flow cytometer. (A) Numbers in representative FACS histograms show percent proliferation based on CFSE dilution. (B) Aligned dot plots show mean ± SEM of CFSE dilution by different Th subsets. Data presented are from five independent experiments performed on healthy individuals (n = 5). *P < 0.05, **P < 0.01, ns stands for non-significant.

FIGURE 5

AZM inhibits IFN-γ and IL-4 production of helper T cell subsets. Anti-CD3 and CD28 stimulated cells were cultured at indicated concentration of AZM for 3-days. BFA was added in last 4 hr of the culture. Cells without anti-CD3/CD28 stimulation were taken as control (US). Intracellular cytokine staining was performed with anti-IFN-γ and anti- IL-4 mAbs and detected by flow cytometer. Th subset cytokine production was determined by gating on specific Th subsets. Aligned dot plots show mean ± SEM of total intracellular IFN-γ+ (A) and IL-4+ (B) production by each subset. Data presented are from four independent experiments performed on healthy individuals (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001, ns stands for non-significant.

FIGURE 6

AZM induces helper T cell subset apoptosis. Anti-CD3 and CD28 stimulated cells were treated with indicated concentration of AZM. On day 3 cell death assay was performed using Annexin V and 7-ADD labeling. Unstimulated (US) cells were taken as control. (A) Representative FACS plots show percent apoptosis by each Th subset. (B) Aligned dot plots show mean ± SEM of total Annexin V+ (apoptosis) cells. Data presented are from four independent experiments performed on healthy individuals (n = 4). *P < 0.05, **P < 0.01 and, ns stands for non-significant.

FIGURE 7

AZM induces apoptosis of FACS sorted Th subsets. Anti-CD3 and CD28 stimulated cells were treated with indicated concentration of AZM. (A) Representative FACS plots show percent apoptosis by Th2-like, Th0, and Th1-like subset. (B) Aligned dot plots show mean ± SEM of total Annexin V+ (apoptosis) cells. Data presented are from three independent experiments performed on healthy individuals (n = 3). *P < 0.05, **P < 0.01 and, ns stands for non-significant.