TSLP directly impairs pulmonary Treg function: association with aberrant tolerogenic immunity in asthmatic airway

Abstract

Background: Even though thymic stromal lymphopoietin (TSLP) has been implicated in the development of allergic inflammation, its influence on immune tolerance mediated by regulatory T cells (Treg) have not been explored. We aimed to dissect the influence of TSLP on immunosuppressive activities of Treg and its potential consequences in human allergic asthma.

Methods: In vitro culture system was utilized to study the effects of TSLP on human Treg. The functional competency of pulmonary Treg from a cohort of 15 allergic asthmatic, 15 healthy control, and 15 non-allergic asthmatic subjects was also evaluated by suppression assays and flow cytometric analysis.

Results: Activated pulmonary Treg expressed TSLP-R and responded to TSLP-mediated activation of STAT5. TSLP directly and selectively impaired IL-10 production of Treg and inhibited their suppressive activity. In human allergic asthma, pulmonary Treg exhibited a significant decrease in suppressive activity and IL-10 production compared to healthy control and non-allergic asthmatic counterparts. These functional alterations were associated with elevated TSLP expression in bronchoalveolar lavage fluid (BAL) of allergic asthmatic subjects. Furthermore, allergic asthmatic BAL could suppress IL-10 production by healthy control pulmonary Treg in a TSLP-dependent manner.

Conclusions: These results provide the first evidences for a direct role of TSLP in the regulation of suppressive activities of Treg. TSLP mediated inhibition of Treg function might present a novel pathologic mechanism to dampen tolerogenic immune responses in inflamed asthmatic airway.

Figure 1

Pulmonary Treg express functional TSLP-R. A. (Top) TSLP-R expression at protein (% of positive cells) and mRNA levels in HC pulmonary Treg and Teff (n = 14). (Bottom) Representative FACS plots of TSLP-R expression by HC pulmonary Treg and Teff. B. (Left) Phosphorylated STAT5 (pSTAT5) expression, measured by ELISA, in HC pulmonary Treg in response to IL-2, IL-4, IL-7, and TSLP (n = 7). (Right) Representative FACS plots of pSTAT5 expression in HC pulmonary Treg in response to different stimuli. C. pSTAT5 expression, measured by ELISA, in HC pulmonary Teff in response to IL-2, IL-4, IL-7, and TSLP (n = 7). Wilcoxon tests were used for statistical analysis. Horizontal bars represented median values as indicated throughout the figure.

Figure 2

TSLP inhibits suppressive activity and IL-10 production by Treg. A. Suppressive activity of un-stimulated vs. TSLP-primed HC pulmonary Treg against Teff proliferation (n = 8) represented as thymidine counts in suppression assays (left) and percentage suppression of Teff proliferation (right). Percentage suppression of Teff proliferation was calculated by percentage decrease in thymidine uptake in co-cultures of Teff and Treg compared to cultures of Teff alone. B. (Top) Representative FACS plots of IL-10 production by PMA/Ionomycin (PMA/I) activated HC pulmonary Treg after being primed with IL-2, IL-7, and TSLP. (Middle) Expression of IL-10 by PMA/I activated HC pulmonary Treg after being primed with different cytokines (n = 7). (Bottom left) Representative FACS plots of IL-10 production by PMA/Ionomycin (PMA/I) activated HC pulmonary Teff after being primed with TSLP. (Bottom right) Expression of IL-10 by PMA/I activated HC pulmonary Teff after being primed with TSLP (n = 7). Data represented intracellular flow cytometric and ELISA results. C. (Top) Effects of exogenous IL-10 on suppressive activity of TSLP-primed HC pulmonary Treg against Teff proliferation (n = 7). (Bottom) Effects of neutralizing antibodies against IL-10 on suppressive activity of HC pulmonary Treg against Teff proliferation (n = 7). Data were represented as thymidine uptake in suppression assay cultures (left) as well as percentage suppression of Teff proliferation (right). Wilcoxon tests were used for statistical analysis. Bar graphs and horizontal bars represented median values as indicated throughout the figure.

Figure 3

Decreased IL-10 production and suppressive function of allergic asthmatic pulmonary Treg. A. (Top) IL-10 expression by PMA/I activated pulmonary Treg from AA, HC, and NA subjects (n = 13). Data represented intracellular flow cytometric and ELISA results. (Bottom) Representative FACS plots of IL-10 expression by pulmonary Treg from different subject groups. B. Suppressive activity of pulmonary Treg against autologous Teff from AA, HC, and NA subjects represented as thymidine counts in suppression assays (n = 13). C. Allogeneic suppression assays with pulmonary T cells from AA and HC subjects (n = 7). Treg and Teff were cultured at 1:1 and 1:4 ratios. Kruskal Wallis tests (A, B) and Wilcoxon tests (C) were used for statistical analysis. Horizontal bars represented median values as indicated throughout the figure.

Figure 4

Impaired allergic asthmatic pulmonary Treg function is associated with elevated airway TSLP. A. TSLP expression in BAL of AA, HC, and NA subjects (n = 15). B. Correlation of TLSP levels in BAL of AA subjects with IL-10 production and suppression activity of AA pulmonary Treg (n = 13). Straight lines represented best-fitted lines. Dotted lines represented 95% confident intervals. C. (Top) Effects of blocking TLSP-R antibodies on AA BAL-mediated inhibition of IL-10 production by HC pulmonary Treg (n = 5). (Bottom) Representative FACS plots of IL-10 production by HC pulmonary Treg in different stimulatory conditions. D. Effects of NA BAL on IL-10 production by HC pulmonary Treg (n = 5). Data represented intracellular flow cytometric and ELISA results. Kruskal Wallis tests (A), Spearman tests (B) and Wilcoxon tests (C) were used for statistical analysis. Horizontal bars represented median values as indicated throughout the figure.