Trichostatin A abrogates airway constriction, but not inflammation, in murine and human asthma models

Abstract

Histone deacetylase (HDAC) inhibitors may offer novel approaches in the treatment of asthma. We postulate that trichostatin A (TSA), a Class 1 and 2 inhibitor of HDAC, inhibits airway hyperresponsiveness in antigen-challenged mice. Mice were sensitized and challenged with Aspergillus fumigatus antigen (AF) and treated with TSA, dexamethasone, or vehicle. Lung resistance (R(L)) and dynamic compliance were measured, and bronchial alveolar lavage fluid (BALF) was analyzed for numbers of leukocytes and concentrations of cytokines. Human precision-cut lung slices (PCLS) were treated with TSA and their agonist-induced bronchoconstriction was measured, and TSA-treated human airway smooth muscle (ASM) cells were evaluated for the agonist-induced activation of Rho and intracellular release of Ca(2+). The activity of HDAC in murine lungs was enhanced by antigen and abrogated by TSA. TSA also inhibited methacholine (Mch)-induced increases in R(L) and decreases in dynamic compliance in naive control mice and in AF-sensitized and -challenged mice. Total cell counts, concentrations of IL-4, and numbers of eosinophils in BALF were unchanged in mice treated with TSA or vehicle, whereas dexamethasone inhibited the numbers of eosinophils in BALF and concentrations of IL-4. TSA inhibited the carbachol-induced contraction of PCLS. Treatment with TSA inhibited the intracellular release of Ca(2+) in ASM cells in response to histamine, without affecting the activation of Rho. The inhibition of HDAC abrogates airway hyperresponsiveness to Mch in both naive and antigen-challenged mice. TSA inhibits the agonist-induced contraction of PCLS and mobilization of Ca(2+) in ASM cells. Thus, HDAC inhibitors demonstrate a mechanism of action distinct from that of anti-inflammatory agents such as steroids, and represent a promising therapeutic agent for airway disease.

Figure 1.

Experimental design. Animals were sensitized with two intraperitoneal (IP) injections on Days 0 and 14 with 20 μg of Aspergillus fumigatus antigen (AF). Three intranasal (IN) challenges of 25 μg AF were performed, once a day for the 3 days before the animal was killed. Animals were treated with an HDAC inhibitor, trichostatin A (TSA), or DMSO (diluent) alone by IP injection once a day for the 3 days before being killed on Day 28.

Figure 2.

Expression and activity of histone deacetylase (HDAC) in human and murine tissue. (A) Immunoblot analyses of lysates from human airway smooth muscle (HASM) cells, lung epithelial cells, and wild-type murine lung tissue were performed using anti-HDAC antibodies. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used to confirm equal protein loading. (B) Relative HDAC activity from lung tissue lysate was quantified. Data are reported as the ratio of measured HDAC activity to basal HDAC activity (Relative HDAC activity). Mice were sensitized and challenged with antigen or solvent (naive), and received vehicle treatment (DMSO) or TSA. Data represent means ± SD from five samples for each condition. *P < 0.05 (treatment versus basal concentrations). (C) Relative HDAC activity from HASM lysate was quantified. Data are reported as the ratio of measured HDAC activity to basal HDAC activity (Rel. HDAC activity). Cells were treated with TSA at concentrations of 1, 10, or 100 μM for 48 hours before lysis and assay. Data represent means ± SD from five samples for each condition. *P < 0.05 (treatment versus basal concentrations).

Figure 3.

TSA inhibits lung resistance (R_L) in naive and antigen-exposed mice. (A) R_L of naive (non–antigen-exposed), vehicle, TSA, or dexamethasone (Dex)–treated mice (basal condition). Methacholine (Mch) increased R_L to a maximum of 2.46 ± 0.1 cm H₂O/ml. Treatment with Dex exerted little effect on basal Mch responsiveness. TSA markedly inhibited basal responsiveness to Mch. Data represent the mean ± SD for each condition derived from of 12 measurements at each dose in three mice. *P < 0.05 (naive versus TSA-treated mice). (B) R_L of mice that were antigen-challenged and received vehicle (DMSO), TSA, or Dex. Maximum R_L in antigen-challenged mice was measured at 13.9 ± 0.7 cm H₂O/mL, that is, much higher than in naive mice. TSA was as effective as Dex in abrogating Mch-induced airway hyperresponsiveness (AHR). Data represent the means ± SD derived from 12 measurements at each dose in six mice for each condition. *P < 0.05 (AF + TSA versus AF). ⁺P < 0.05 (AF + Dex versus AF).

Figure 4.

TSA reversed antigen-induced effects on lung compliance. (A) Dynamic compliance was measured in naive mice and mice treated with vehicle (DMSO), TSA, or Dex in response to increasing doses of Mch measured by the Scireq FlexiVent system. TSA markedly inhibited Mch-induced decreases in dynamic compliance. Data represent the means ± SD derived from 12 measurements at each dose in three mice for each condition. *P < 0.05 (naive versus TSA-treated mice). (B) Dynamic compliance of mice challenged with antigen or solvent (naive), and of mice that received treatment (DMSO), TSA, or Dex. Data represent the means ± SD derived from 12 measurements at each dose in six mice for each condition. *P < 0.05 (AF + TSA versus AF).

Figure 5.

Dex, but not TSA, inhibits antigen-induced increases in BALF cell counts. (A) The total number of cells was obtained from bronchoalveolar lavage fluid (BALF) of mice that were sensitized and challenged with antigen or diluent (naive) and mice that received vehicle (DMSO), TSA, or Dex. TSA exerted little effect on numbers of inflammatory cells in the BAL. Data represent the mean ± SD for each condition, derived from the BALF of three mice. *P < 0.05 (naive versus treatment cohort). (B) The cellular profile of BALF from mice that were sensitized and challenged with antigen or solvent (naive) and mice that received vehicle (DMSO), TSA, or Dex. Data represent the mean ± SD for each condition, derived from the BALF of three mice. *P < 0.05 (naive versus AF). ⁺P < 0.05 (AF + Dex versus AF). (C) Percentages of cell subtypes. Data represent the mean ± SD for each condition, derived from the BALF of three mice. *P < 0.05 (baseline versus treatment cohort).

Figure 6.

Dex, but not TSA, inhibits antigen-induced increases of IL-4 concentrations in BALF. Concentrations of (A) IL-4 and (B) IL-6 in BALF were measured from mice that were challenged with antigen or diluent (naive) and mice that received vehicle alone (DMSO), TSA, or Dex. TSA exerted little effect on the secretion of cytokines in mice sensitized and challenged with antigen. Data represent the mean ± SD for each condition, derived from the BALF of three mice. *P < 0.05 (naive versus AF). ⁺P < 0.05 (AF + Dex versus AF). ^#P < 0.05 (AF + Dex versus AF + TSA).

Figure 7.

TSA inhibits the agonist-induced contraction of precision-cut lung slices (PCLS) by decreasing the agonist-induced mobilization of calcium in airway smooth muscle (ASM). (A) Carbachol induced contractions of PCLS after treatment with vehicle, TSA at 4 μm, or TSA at 20 μM. (B) Human ASM was treated with TSA (10 μM) for 12, 24, 48, and 72 hours, and then treated with 1 μM bradykinin for 1 minute, lysed, and assayed for active and total Rho by immunoprecipitation and immunoblot, respectively. (C) Fura-2 signal ratios from human ASM cells treated with TSA at 0 (untreated), 1, 5, and 10 μM for 48 hours in response to histamine (100 μM). In each group, approximately 20 cells were measured. In each graph, thin gray curves represent individual responses, and the thick dark curve represents the mean response.