The tick salivary protein sialostatin L inhibits the Th9-derived production of the asthma-promoting cytokine IL-9 and is effective in the prevention of experimental asthma

Abstract

Ticks developed a multitude of different immune evasion strategies to obtain a blood meal. Sialostatin L is an immunosuppressive cysteine protease inhibitor present in the saliva of the hard tick Ixodes scapularis. In this study, we demonstrate that sialostatin L strongly inhibits the production of IL-9 by Th9 cells. Because we could show recently that Th9-derived IL-9 is essentially involved in the induction of asthma symptoms, sialostatin L was used for the treatment of experimental asthma. Application of sialostatin L in a model of experimental asthma almost completely abrogated airway hyperresponsiveness and eosinophilia. Our data suggest that sialostatin L can prevent experimental asthma, most likely by inhibiting the IL-9 production of Th9 cells. Thus, alternative to IL-9 neutralization sialostatin L provides the basis for the development of innovative therapeutic strategies to treat asthma.

Figure 1. SialoL impairs primary T cell-derived production of IL-9 but not T cell activation and proliferation

Naïve CD4⁺ T cells from BALB/c mice were stimulated (anti-CD3/CD28) in the presence and absence of different concentrations of sialoL (0.75 μM, 1.5 μM, 3 μM) under Th9-skewing conditions. Production of IL-9 was determined by (A) ELISA, (B) FACS, and (C) qRT-PCR. (D) T cell activation was determined by FACS analyses of CD25 and CD122 cell surface expression. Shown is each time one representative from three independent experiments ± SD. (E) T cell proliferation was determined by counting the cell numbers on day five after primary activation.

Figure 2. Exogenous human (h)IL-2 cannot compensate for the IL-9-suppressive effect of SialoL

(A) Naïve CD4⁺ T cells from BALB/c mice were stimulated (anti-CD3/CD28) in the presence or absence of different concentrations of sialoL (0.75 μM, 1.5 μM, 3 μM) under Th9-skewing conditions. Production of IL-2 was determined by ELISA. (B) Naïve CD4⁺ T cells from BALB/c mice were stimulated (anti-CD3/CD28) solely under Th9-skewing conditions or in the presence of neutralizing murine (m)IL-2 mAb (S4B6.1: 20μg/ml) or human(h)IL-2 (100ng/ml) and a combination of both. (C) Naïve CD4⁺ T cells from BALB/c mice were stimulated (anti-CD3/CD28) solely under Th9-skewing conditions or in the presence of sialoL (3 μM) or hIL-2 (100ng/ml) and a combination of both. IL-9 was determined after 72h by ELISA. Shown is one representative from two independent experiments.

Figure 3. IL-1-mediated enhancement of secretion and expression of IL-9 is inhibited by sialoL

(A) Naïve CD4⁺ T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. IL-9 was determined after 72h by ELISA. (B) Naïve CD4⁺ T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions. After five days T cells were restimulated in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. IL-9 was determined after 48h by ELISA. (C) Naïve CD4⁺ T cells from BALB/c mice were stimulated (anti-CD3/CD28) under Th9-skewing conditions. After five days T cells were restimulated in the absence or presence of sialoL (3 μM) or IL-1β (10ng/ml) and a combination of both. Expression of Il9 was quantified after 48h by qRT-PCR. Similar results were obtained in four independent experiments.

Figure 4. OVA-induced AHR and eosinophilia are strongly alleviated by treatment with sialoL

BALB/c mice were sensitized by two injections (i.p.) of OVA (20 μg) and subsequently challenged with nebulized OVA in the absence or presence of sialoL (10 μg, i.v.) as outlined in (A). Airway resistance (B) and numbers of eosinophils in BAL fluid (C) were measured in mice 24h after a third airway challenge on day 30. In (B) bars represent mean ±SEM AHR and in (C) every dot represents the number of eosinophils from an individual mouse. In (B) and (C) * p<0.05 compared to chal and sens/chal + SialoL.

Figure 5. OVA-induced cellular infiltration and mucus production are significantly reduced by treatment with sialoL

BALB/c mice were sensitized by two injections (i.p.) of OVA (20 μg) and subsequently challenged with nebulized OVA in the absence or presence of sialoL (10 μg, i.v.) as outlined in Fig. 4A. Tissue inflammation was evaluated 24h following the last challenge using hematoxylin and eosin staining (H&E). Periodic acid-Schiff (PAS) staining was applied to determine goblet cell metaplasia in mice which were only challenged with OVA (chal), sensitized and challenged (sens/chal) and treated in addition with sialoL (i.v.; sens/chal + SialoL).

Figure 6. OVA-induced development of IL-9-producing T cells is strongly inhibited by treatment with sialoL

BALB/c mice were sensitized by two injections (i.p.) of OVA (20 μg) and subsequently challenged with nebulized OVA in the absence or presence of sialoL (10 μg, i.v.) as outlined in Fig. 4A. Lung cells were prepared from mice 24h following the last challenge (day 31) and stimulated (4×10⁶/ml, 24 well plate) with 2.5 μg/ml OVA peptide 323–339 for 48h (mRNA expression) and for 72h (protein production), respectively. (A) IL-9 production was determined in the supernatants by ELISA; n= 8 mice per group. **p = 0.0084. (B) IL-9 mRNA expression levels were analyzed by quantitative real time PCR; n= 8 mice per group. *** chal vs. sense/chal p < 0.0001; ***sense/chal vs. sense/chal + SialoL p=0.0009; ns: not significant.

Figure 7. Th9-induced AHR is significantly reduced by treatment with sialoL

Rag1^−/− mice were adoptively transferred with Th9 cells developed from DO11.10 mice and challenged with nebulized OVA in the absence or presence of sialoL as outlined in Materials and Methods and (A). Airway resistance (B) was measured in mice 24h after a final airway challenge on day 7. Bars represent mean ±SEM AHR; * p<0.05 compared to Th9 and Th9/chall + SialoL.