Blocking cyclophilins in the chronic phase of asthma reduces the persistence of leukocytes and disease reactivation

Abstract

Allergic asthma is characterized by acute influxes of proinflammatory leukocytes in response to allergen stimulation, followed by quiescent (chronic) periods between allergen challenges, during which sustained, low-level inflammation is evident. These chronic phases of disease are thought to be mediated by populations of leukocytes persisting within airways and tissues. The lack of any in situ proliferation by these cells, along with their limited lifespan, suggests that a continual recruitment of leukocytes from the circulation is needed to maintain disease chronicity. The mechanisms regulating this persistent recruitment of leukocytes are unknown. Although classic leukocyte-attracting chemokines are highly elevated after acute allergen challenge, they return to baseline levels within 24 hours, and remain close to undetectable during the chronic phase. In the present study, we investigated whether an alternative family of chemoattractants, namely, extracellular cyclophilins, might instead play a role in regulating the recruitment and persistence of leukocytes during chronic asthma, because their production is known to be more sustained during inflammatory responses. Using a new murine model of chronic allergic asthma, elevated concentrations of extracellular cyclophilin A, but not classic chemokines, were indeed detected during the chronic phase of asthma. Furthermore, blocking the activity of cyclophilins during this phase reduced the number of persisting leukocytes by up to 80%. This reduction was also associated with a significant inhibition of acute disease reactivation upon subsequent allergen challenge. These findings suggest that blocking the function of cyclophilins during the chronic phase of asthma may provide a novel therapeutic strategy for regulating disease chronicity and severity.

Figure 1.

Kinetics of leukocytic infiltration in a murine model of chronic allergic asthma. (A) Female BALB/c mice were sensitized by intraperitoneal injection of 100 μg ovalbumin (OVA) in alum on Days 0 and 13. Mice were then challenged with an aerosolized solution of 5% OVA in PBS (or PBS alone) seven times between Days 19–28, and then three times per week for 4 weeks. Groups of mice were either killed 1 day after the final aerosol challenge (Acute), or housed for an additional 4 weeks without OVA challenge (Chronic Phase). An acute response was then reactivated by an additional aerosolized OVA challenge (Reactivation). (B) Leukocytic infiltration was assessed in the airways by flow cytometric analyses of bronchoalveolar lavage (BAL) fluid collected at three time points: 24 hours after the final repetitive challenge (Acute), 3 weeks into the chronic phase (Chronic), and 24 hours after the final reactivation challenge (Reactivation). Data show the mean ± SEM for numbers of leukocytes at the Acute, Chronic, and Reactivation time points. A Student t test was used to establish significant differences between the OVA and PBS groups (n = 6–12 mice per group). **P < 0.005. ***P < 0.0005.

Figure 2.

Elevated concentrations of cyclophilin A (CypA) are present during the chronic phase of asthma. Concentrations of CypA, eotaxin, regulated upon activation, normal T-cell expressed and presumably secreted (RANTES), monocyte chemotactic protein (MCP)-1, and MCP-3 were measured in the BAL fluid of mice killed at the time points specified in Figure 1. Measurements were determined via Western blot analysis (CypA), ELISA (eotaxin), or cytometric bead array (RANTES, MCP-1, and MCP-3). Data show the mean ± SEM for concentrations of each chemoattractant in OVA-challenged versus PBS-challenged groups. A Student t test was used to establish statistically significant differences between OVA and PBS data at each time point (n = 4–12 mice per group). **P < 0.005. ***P < 0.0005.

Figure 3.

N-methyl-4-isoleucine-cyclosporin (NIM811) blocks the CypA-induced migration of leukocytes in vitro. Purified populations of activated CD4⁺ T cells, neutrophils, and monocytes were set up in Boyden chemotaxis chambers in the presence of recombinant CypA ± 2 μM NIM811 (NIM) or diluent alone (DIL). Data show the mean chemotactic indices ± SEM for each group (n = 6 wells/group), as calculated by comparing the number of cells migrating in each test group relative to medium alone. A Student t test was used to establish statistically significant differences between NIM811-treated and DIL-treated groups. ***P < 0.0001.

Figure 4.

Inhibiting the function of cyclophilins during the chronic phase of asthma reduces the numbers of leukocytes recruited upon acute allergen challenge. Chronic allergic asthma was induced as already described, and 200 μg of NIM811, or diluent alone, were administered by intraperitoneal injection twice per week during the 4-week chronic phase. A separate group of mice received PBS aerosol challenges throughout the regimen, without intervention, to provide baseline data. Five days after the final intervention injection, all mice received two aerosol challenges of either PBS or OVA to induce an acute asthmatic response (Reactivation). Numbers of leukocytes were assessed in BAL fluid by flow cytometry 24 hours after the second acute reactivation challenge. Data show the mean ± SEM for numbers of leukocytes (n = 14–15 mice per group). The percent reduction between NIM811-treated and diluent-treated groups is also shown. A Student t test was used to establish statistically significant differences between the NIM and DIL groups. **P < 0.005. ***P < 0.0001.

Figure 5.

Inhibiting the function of cyclophilins during the chronic phase of asthma reduces tissue pathology after an acute reactivation of asthma. Lung pathology was analyzed in mice killed 24 hours after their second acute reactivation challenge, using the same timeline described in Figure 4. (A) Whole lungs from PBS-treated, diluent-treated, and NIM811-treated mice were fixed and embedded in paraffin, and 6-μm sections were cut and stained with periodic acid-Schiff (PAS) (left) or Masson's Trichrome (right). Black arrows denote areas of inflammatory foci. Images are shown at ×10 magnification. Scale bar = 10 μm. (B) Changes in the deposition of mucus and collagen were scored, using a semiquantitative scale ranging from 0 (normal) to 4 (pronounced deposition of mucus or collagen). A Student t test was used to identify significant differences between NIM-treated and DIL-treated groups (n = 6–8 mice per group). ***P < 0.0001.

Figure 6.

Inhibiting the function of cyclophilins during the chronic phase of asthma significantly reduces airway hyperresponsiveness in mice with asthma. (A) Airway resistance was measured in NIM811-treated versus diluent-treated mice, 24 hours after their second reactivation challenge, using the same timeline described in Figure 4. A separate group of naive mice was analyzed to provide baseline data for nonasthmatic lung function. Individual mice from each group were anesthetized, and a tracheostomy tube was inserted for ventilation via the Buxco FinePointe RC system. The animals were then challenged with increasing doses of methylcholine, and measurements of maximum airway resistance (RI) were determined using Buxco FinePointe software. Data show the mean ± SEM at individual doses of methylcholine for each group (n = 7–10 mice per group). Two-way ANOVA was used to establish statistically significant differences between the NIM811 and DIL groups. **P < 0.001. ***P < 0.0001. (B) Peripheral blood was collected by cardiac puncture from mice killed at the Reactivation time point. Concentrations of the OVA-specific IgE present in individual serum samples were measured according to ELISA (MD Biosciences Inc., St. Paul, MN). Data show the mean ± SEM for concentrations of OVA-specific IgE (n = 7 mice per group).