Figure 1.
TSLP enhances development of Th2 immune responses during initiation of early phase of Th2 responses, but is dispensable on established Th2 responses.
Wild type and TSLP−/− mice were exposed with ovalbumin (10 µg) by intranasal delivery every other day until day 5. (A) TSLP mRNA expression in the lungs of mice sensitized for 6 days with ovalbumin. (B) On day 6, supernatants from allergen-restimulated lung draining lymph nodes were analyzed for cytokines by Bioplex. (C) Naïve DO11.10 CD4 T cells were adoptively transferred into wild type or TSLP−/− mice and these mice were challenged with ovalbumin (5 µg) every other day until day 5. On day 6, lung draining lymph node cells were allergen-restimulated and supernatants analyzed by Bioplex. In a third set of animals Th2 skewed DO11.10 CD4 T cells were adoptively transferred into wild type and TSLP−/− mice and exposed to ovalbumin for 5 days as above. Subsequently, lungs from mice that received Th2 cells and ovalbumin were (D) stained with PAS and (E) assayed for gob5 and muc5ac expression by qPCR. Fold changes were calculated upon comparison to control lungs from naïve animals. (F) Cytokines were analyzed by Bioplex from supernatants of restimulated lung draining lymph node cells from mice with Th2 cell transfer. Data represent mean±SEM from 4 to 5 mice/group. Lymph nodes from naïve mice challenge with allergen elicit no detectable response.
Figure 2.
TSLP alters naïve CD4 T cell to express Th2 cytokines both on its effect on DCs and T cells.
(A and B) DO11.10 CD4 T cells were cocultured with wild type BMDCs (A) were incubated plus ova peptides (10 ug/ml) and +/− TSLP (15 ng/ml). Supernatants were collected after 48 hrs and assayed for cytokines by Bioplex. (C) DO11.10 CD4 T cells were cultured in Th2 biased conditions for 8 days, and restimulated with ova peptides +/− TSLP for 48 hrs and supernatant were assayed for cytokines by Bioplex. (D) BMDCs or (E) lung CD11c cells were incubated with ova peptides (10 ug/ml) along with either presence or absence of TSLP (15 ng/ml) overnight. Next day DCs were washed extensively and CD4 T cells from DO11.10 spleens were added into DC cultures. After 48 hrs, supernatants were assayed for cytokines by Bioplex. Data represent mean ± SEM from 3 repeated experiments with triplicate wells.
Figure 3.
Absence of TSLP does not impair development of cockroach antigen (CRA)-induced allergic response.
Mice were injected with CRA by intranasal exposure on day 1, 3, and 5. (A) On day 6, lymph node cells were restimulated with CRA and supernatants were analyzed by Bioplex. Mice were either sensitized i.p./s.c. (B and D) or i.n. (C and E) and challenged (See material and method section). Lymph nodes were taken one day after final challenge and (B and C) analyzed cytokines from allergen-restimulated lung draining lymph nodes by Bioplex. (D and E) lungs were stained with PAS. Data represent mean±SEM from 5 mice/group.
Figure 4.
Absence of TSLP does not impair cockroach allergen induced chronic allergic response.
After repeated challenges of allergen (see Materials and Methods) over a 4 week period animals were examined for their allergic responses. (A) Airway responses were measured after one dose of methacholine. Data are represented as the peak airway resistance in H2O/ml/s±SEM. (B) Lungs were taken one day after final challenge and were stained with PAS and (C) assayed for gob5 and muc5ac expression by real-time PCR. Fold changes were calculated upon comparison to control lungs from naïve animals. (D) Analysis of cytokines from allergen-restimulated lung draining lymph nodes was assessed by Bioplex. (E) Supernatants from lung homogenates were assayed for cytokines by Bioplex. (F and G) BAL fluid was collected 24 hours after the last CRA challenge and was analyzed (F) for the total number of cells recovered by BAL and (G) for the percentage of each cell type. Data represent mean±SEM from 5 mice/group.
Figure 5.
In vivo blockade of TSLP signaling at the late phase of allergic diseases does not affect cockroach allergen-induced allergic reaction.
(A) Model outline and timing of Ab injections during chronic model of cockroach antigen-induced allergic lung disease was based upon applying anti-TSLP (M702) in a therapeutic format at the end of the challenge period. (B) Airway responses were measured after one dose of methacholine in ventilated and anesthetized animals. Data are represented as the peak airway resistance in H2O/ml/s±SEM. Lungs were taken one day after final challenge and were (C) assayed for Gob5 and Muc5ac expression and (D) stained with PAS. (E) Analysis of cytokines from allergen restimulated lung draining lymph nodes by Bioplex. Data represent mean±SEM from 5 mice/group. Fold changes were calculated upon comparison to control lungs from naïve animals.
Figure 6.
In vivo blockade of TSLP signaling at the induction phase of allergic diseases does not attenuate cockroach allergen-induced airway reaction.
(A) Model outline and timing of Ab injections during the chronic model of cockroach antigen-induced allergic lung disease. (B) Airway responses were measured after one dose of methacholine in ventilated and anesthetized animals. Data are represented as the peak airway resistance in H2O/ml/s±SEM. Lungs were taken one day after final challenge and were (C) assayed for Gob5 and Muc5ac mRNA expression by QPCR and (D) stained with PAS. (E) Analysis of cytokines from 48 hr supernatants of allergen re-stimulated lung draining lymph nodes by Bioplex. Data represent mean±SEM from 5 mice/group. Fold changes in QPCR analyses were calculated upon comparison to control lungs from naïve animals.