Inhaled iloprost suppresses the cardinal features of asthma via inhibition of airway dendritic cell function

Abstract

Inhalation of iloprost, a stable prostacyclin (PGI(2)) analog, is a well-accepted and safe treatment for pulmonary arterial hypertension. Although iloprost mainly acts as a vasodilator by binding to the I prostanoid (IP) receptor, recent evidence suggests that signaling via this receptor also has antiinflammatory effects through unclear mechanisms. Here we show in a murine model of asthma that iloprost inhalation suppressed the cardinal features of asthma when given during the priming or challenge phase. As a mechanism of action, iloprost interfered with the function of lung myeloid DCs, critical antigen-presenting cells of the airways. Iloprost treatment inhibited the maturation and migration of lung DCs to the mediastinal LNs, thereby abolishing the induction of an allergen-specific Th2 response in these nodes. The effect of iloprost was DC autonomous, as iloprost-treated DCs no longer induced Th2 differentiation from naive T cells or boosted effector cytokine production in primed Th2 cells. These data should pave the way for a clinical effectiveness study using inhaled iloprost for the treatment of asthma.

Figure 1. Local administration of iloprost suppresses asthma features.

Mice were sensitized by i.p. injection of OVA/alum (see Methods) on days 0 and 7 and were exposed on days 19–21 to OVA aerosols. Prior to each aerosol, mice received an i.t. injection of vehicle, CAY10449 plus vehicle (OVA/CAY+vehicle/OVA), 0.2 μg iloprost, or CAY10449 plus iloprost. Labels indicate sensitization/treatment/challenge. (A and D) BAL fluid was analyzed by flow cytometry. (B) May-Grunwald-Giemsa staining of lung sections. (C and E) Cytokine production in MLN cells restimulated in vitro for 4 days with OVA. (F) BHR to various doses of i.v. metacholine was assessed for changes in dynamic resistance and lung compliance and BHR to inhaled metacholine for PenH responses was assessed 24 hours after the last antigen exposure were measured. Data are mean ± SEM; n = 8 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2. Effect of iloprost treatment on distribution of DCs.

Single-cell suspensions from MLNs were stained for DCs (A) or B and T cells (B) and analyzed by flow cytometry. Experiments were set up as in Figure 1. Labels indicate sensitization/treatment/challenge. Data (mean ± SEM) were calculated as absolute number of cells. **P < 0.01; ***P < 0.001. (C) Iloprost inhibited the maturation of lung DCs in vivo. A single-cell suspension was prepared from the lungs, and CD11c⁺MHCII^hi lung DCs were analyzed for their expression of CD40, CD80, CD83, and CD86. Data from 1 representative experiment of 3 is shown.

Figure 3. Effect of iloprost on lung DC migration to the thoracic draining LNs.

(A) On day 0, naive mice were instilled i.t. with FITC-OVA with or without 0.2 μg iloprost. On day 1, the presence of FITC⁺ migrating DCs in thoracic draining LNs was analyzed by flow cytometry. (B) In the same mice, lung DCs were also enumerated. Lungs were enzymatically digested and stained for the presence of FITC⁺MHCII⁺CD11c⁺ DCs. Results are representative of 4 mice per group. Experiments were repeated 3 times with similar results. Data are mean ± SEM. **P < 0.01; ***P < 0.001.

Figure 4. Administration of iloprost prevents sensitization induced by DCs.

On day 0, mice received an i.t. injection of OVA in the presence or absence of iloprost. From days –1 to 2, mice were injected i.p. with anti–Gr-1 Abs to deplete pDCs or isotype control Abs. Ten days later, mice were exposed to 3 OVA aerosols. (A) BAL fluid was analyzed by flow cytometry. (B) Hematoxylin and eosin staining of lung sections. (C) MLN cells restimulated in vitro for 4 days with OVA, and cytokines were measured in the supernatant. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. Iloprost treatment of DCs inhibits their potential to prime for Th2 responses.

(A and B) On day 0, mice received an i.t. injection of vehicle-treated OVA-pulsed DCs (vehicle-OVA-DC), iloprost-treated OVA-pulsed DCs, or unpulsed DCs. From days 10–13, all mice were exposed to OVA aerosols. (A) BAL fluid was analyzed by flow cytometry. (B) Hematoxylin and eosin staining of lung sections. (C) On day –2, mice were injected i.v. with OVA-specific naive T cells from DO11.10 mice. On day 0, mice were instilled i.t. with vehicle-treated OVA-pulsed DCs, iloprost-treated OVA-pulsed DCs, or unpulsed DCs. On day 4, LN cells were collected and cultured in 96-well plates for 4 days. (D) Supernatants of bone marrow–derived DCs treated overnight with vehicle or different concentrations of iloprost were collected. The presence of IL-4, IL-5, IL-10, IL-12, TNF-α, IL-13, and IFN-γ in the supernatants was analyzed by ELISA. Data are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Iloprost-treated DCs fail to boost Th2 cytokine production in differentiated Th2 cells.

Bone marrow–derived OVA-pulsed DCs were treated with Iloprost or vehicle. DCs were collected and cocultured with in vitro differentiated DO11.10 OVA-specific Th2 cells, that were previously generated in the presence of IL-4, anti–IL-12, and anti–IFN-γ. Cytokines were measured in the supernatant 4 days after setting up the culture. Data are mean ± SEM. *P < 0.05.