Thymic stromal lymphopoietin activity is increased in nasal polyps of patients with chronic rhinosinusitis

Abstract

Background: Chronic rhinosinusitis with nasal polyps (CRSwNP) is associated with TH2-dominant inflammation. Thymic stromal lymphopoietin (TSLP) is a cytokine that triggers dendritic cell-mediated TH2 inflammatory responses and that enhances IL-1-dependent TH2 cytokine production in mast cells. Although increased TSLP mRNA levels have been found in nasal polyps (NPs), expression of TSLP protein and its function in patients with chronic rhinosinusitis (CRS) have not been fully explored.

Objectives: The objective of this study was to investigate the role of TSLP in patients with CRS.

Methods: We investigated the presence and stability of TSLP protein in NPs using ELISA and Western blotting and investigated the function of TSLP in nasal tissue extracts with a bioassay based on activation of human mast cells.

Results: Although TSLP mRNA levels were significantly increased in NP tissue from patients with CRSwNP compared with uncinate tissue from patients with CRS or control subjects, TSLP protein was significantly decreased in NP tissue, as detected by using the commercial ELISA kit. We found that recombinant TSLP was time-dependently degraded by NP extracts, and this degradation was completely inhibited by a protease inhibitor cocktail, suggesting that TSLP is sensitive to tissue proteases. Interestingly, NP extract-treated TSLP had higher activity in mast cells, although the amount of full-length TSLP was reduced up to 85%. NP extracts significantly enhanced IL-1β-dependent IL-5 production in mast cells compared with uncinate tissue homogenates, and responses were significantly inhibited by anti-TSLP, suggesting that NPs contain biologically relevant levels of TSLP activity.

Conclusion: TSLP and its metabolic products might play an important role in the inflammation seen in patients with CRSwNP.

Keywords: 2-ME; 2-Mercaptoethanol; CRS; CRSsNP; CRSwNP; Chronic rhinosinusitis; Chronic rhinosinusitis with nasal polyps; Chronic rhinosinusitis without nasal polyps; DC; Dendritic cell; Double-stranded RNA; IL-5; ILC2; IMDM; Iscove modified Dulbecco medium; Myeloid dendritic cell; NHBE; NMFF; NP; Nasal polyp; Normal human bronchial epithelial cell; Northwestern Medical Faculty Foundation; PIC; PNEC; Primary nasal epithelial cell; Protease inhibitor cocktail; SCF; Stem cell factor; T(H)2 cells; TLR; TSLP; TSLPR; Thymic stromal lymphopoietin; Thymic stromal lymphopoietin receptor; Toll-like receptor; Type 2 innate lymphoid cell; UT; Uncinate tissue; dsRNA; epithelial cells; mDC; mast cells; nasal polyps; proteases; thymic stromal lymphopoietin.

Figure 1

Increased expression of TSLP in nasal polyps. Total RNA was extracted from epithelial scrapings (A) and whole tissue (B–D) from nasal polyps (NP) and uncinate tissue (UT) from control subjects and patients with CRS without NP (CRSsNP) and CRS with NP (CRSwNP). Expression of mRNAs for TSLP and TSLPR was analyzed using real-time PCR (A–D). The correlations were assessed by using the Spearman rank correlation (D). Expression of TSLP protein in tissue homogenates was measured using ELISA (E). TSLP protein concentration was normalized to the concentration of total protein (E). The results are shown as the median (25–75% interquartiles). * p < 0.05, 1-way ANOVA.

Figure 2

TSLP protein was cleaved by NP extracts. Recombinant TSLP was incubated with 1 mg/ml NP tissue extracts or 1 mg/ml BSA (control) for 24–48 hours, and the stability of TSLP was determined by ELISA (A) (n=5). TSLP was incubated with 1 mg/ml BSA or 1 mg/ml NP tissue extracts from donor 1–7 (NP1–7) for 24 hours, and cleavage of TSLP was determined by western blot (B). TSLP was incubated with 1 mg/ml NP extracts for 0–24 hours (C). TSLP was incubated with NP extracts in the presence of 1% protease inhibitor cocktail (PIC) or 1% DMSO (vehicle control) for 24 hours. The results are representative of three separate experiments with separate donors (B–D). * p < 0.05, paired Student t test.

Figure 3

NP extract-treated TSLP had higher activity in mast cells. TSLP was preincubated with NP extract for 24 hours. Mast cells were then stimulated with NP-treated TSLP and untreated TSLP in the presence of 20 ng/ml IL-1β for 48 hours (n=8) (A). Mast cells were stimulated with 4 μg/ml NP extracts, 20 ng/ml IL-1β 10 ng/ml TSLP or their combination, but without preincubation, for 48 hours (n=5) (B). Concentration of IL-5 was measured by CBA. *; p<0.05 compared to IL-1β alone (A, red bar) or medium control (B). NS, not significant.

Figure 4

TSLP activity was elevated in NP. Mast cells were stimulated with 300 μg/ml UT tissue extracts or 300 μg/ml NP extracts in the presence (B) or absence (A) of 20 ng/ml IL-1β for 48 hours. Mast cells were stimulated with 300 μg/ml NP extracts and 20 ng/ml IL-1β in the presence of control mouse IgG1 or anti-TSLP (n=10) (C). Concentration of IL-5 was measured by CBA. Concentration of IL-5 in tissue extracts was subtracted. The results are shown as the median (25–75% interquartiles). * p < 0.05, 1-way ANOVA (A, B) and paired Student t test (C).

Figure 5

Correlation of TSLP and markers of Th1/Th2/Th17 inflammation in sinus tissue. Messenger RNA for TSLP, IL-5, IFN-γ and IL-17A in sinus tissue that was used in Figure 1B was assessed by real-time PCR (n=78). The correlations were assessed by using the Spearman rank correlation.