Anti-Asthma Simplified Herbal Medicine Intervention-induced long-lasting tolerance to allergen exposure in an asthma model is interferon-γ, but not transforming growth factor-β dependent

Abstract

Background: Chronic allergic asthma is the result of a T-helper type 2 (Th2)-biased immune status. Current asthma therapies control symptoms in some patients, but a long-lasting therapy has not been established. Anti-Asthma Simplified Herbal Medicine Intervention (ASHMI™), a Chinese herbal formula, improved symptoms and lung function, and reduced Th2 responses in a controlled trial of patients with persistent moderate to severe asthma.

Objective: We evaluated the persistence of ASHMI™ beneficial effects following therapy in a murine model of chronic asthma and the immunological mechanisms underlying such effects. Methods BALB/c mice sensitized intraperitoneally with ovalbumin (OVA) received 3 weekly intratracheal OVA challenges to induce airway hyper-reactivity (AHR) and inflammation (OVA mice). Additionally, OVA mice were treated with ASHMI™ (OVA/ASHMI™) or water (OVA/sham) for 4 weeks, and then challenged immediately and 8 weeks post-therapy. In other experiments, OVA mice received ASHMI™ treatment with concomitant neutralization of IFN-γ or TGF-β. Effects on airway responses, cytokine- and OVA-specific IgE levels were determined 8 weeks post-therapy.

Results: Before treatment, OVA mice exhibited AHR and pulmonary eosinophilic inflammation following OVA challenge, which was almost completely resolved immediately after completing treatment with ASHMI™ and did not re-occur following OVA re-challenge up to 8 weeks post-therapy. Decreased allergen-specific IgE and Th2 cytokine levels, and increased IFN-γ levels also persisted at least 8 weeks post-therapy. ASHMI™ effects were eliminated by the neutralization of IFN-γ, but not TGF-β, during therapy.

Conclusion: ASHMI™ induced long-lasting post-therapy tolerance to antigen-induced inflammation and AHR. IFN-γ is a critical factor in ASHMI™ effects.

Figure 1

A-Protocol: Mice were sensitized and challenged with OVA at times indicated. Daily oral ASHMI™ treatment lasted for 4 weeks. Mice were subsequently challenged 1 day-, 4- and 8 weeks post therapy.. B-AHR (left panel) and % of BAL eosinophils (right panel) were measured 48 hrs after 5^th OVA challenge (day 58) and the 9th OVA challenge (day 113). Data shown as Mean ± SD in individual samples (N=4–5 mice/group). *, p<0.05;**, p<0.01; *** p<0.001 vs. sham; #, p<0.05; # # #, p<0.001 vs naïve. C, panels i-iii- H&E staining of lung sections from formalin fixed unlavaged left lung. Inset in panel ii shows eosinophils indicated by arrows. C, Panels iv-vi- and inset shows lung sections stained with PAS which stains mucin with a magenta color. Bar indicates 100µM.

Figure 2

A-Cytokines in BALF harvested 48 hours after the final set of OVA challenges were determined by ELISA. B- OVA-specific IgE in serum that was collected from mice one day prior to ASHMI™ therapy, 4-weeks and 8-weeks after stopping ASHMI™ therapy was measured by ELISA. Results are expressed as Mean ± SD of pooled samples in triplicate for cytokines and individual samples for IgE. (n=4–5/group). Results were replicated at least once in a separate experiment. **, p<0.01 and ***p<0.001 vs. sham.

Figure 3

A- Mice were sensitized and challenged at times indicated. 6 weeks of daily oral ASHMI™ was given with or without neutralizing IFN-γ, TGF-β or isotype control antibodies. Neutralizing antibodies were also given to groups of sham treated mice. Responses were assessed 8 weeks post-therapy. B- AHR measured 48 hours after last OVA challenge. Data shown as Mean ± SD of individual APTI values (n=4). C-Percentages of inflammatory cells in BALF counted after differential staining. Data shown as Mean ± SD of percentages in individual samples. D-H&E staining of sections of unlavaged lung (n=4). Bars indicate 100µM. #, p<0.05; ###, p<0.001 vs Naïve. *, p<0.05; ***p<0.001 vs. Sham. +, p<0.05;+++, P<0.001 vs Naïve.

Figure 4

A (Left panel)- PAS staining of sections of unlavaged lung. Mucin-positive cells stain magenta. 4B shows percent of PAS-positive cells/airway. 4A (Right Panel)- Trichrome staining of lung sections. Collagen stains turquoise blue. 4C shows area of peribronchial collagen staining. All data shown as Mean ± SD of 3–5 airways per animal (N=4/group) Bars indicate 100µM. *, P<0.05.

Figure 5

BALF supernatants were pooled for each group of mice and cytokine levels at eight weeks post-ASHMI™ therapy were determined by ELISA (A)-IL-4, (B)-IL-5, (C)-IL-13, (D)-IFN-γ, (E)-IL-10, (F)-TGF-β. Results are expressed as means ± SD of pooled samples measured in triplicate. (N=4 mice/group). *, P<0.05 ; **, P<0.01; ***p<0.001

Figure 6

Serum harvested from mice at the time of AHR evaluation was assayed for OVA-specific IgE by ELISA. Data are shown as Mean ± SD of individual samples measured in duplicate. N=4 mice/group. ***, P<0.001