A cell-impermeable cyclosporine A derivative reduces pathology in a mouse model of allergic lung inflammation

Abstract

Although the main regulators of leukocyte trafficking are chemokines, another family of chemotactic agents is cyclophilins. Intracellular cyclophilins function as peptidyl-prolyl cis-trans isomerases and are targets of the immunosuppressive drug cyclosporine A (CsA). Cyclophilins can also be secreted in response to stress factors, with elevated levels of extracellular cyclophilins detected in several inflammatory diseases. Extracellular cyclophilins are known to have potent chemotactic properties, suggesting that they might contribute to inflammatory responses by recruiting leukocytes into tissues. The objective of the present study was to determine the impact of blocking cyclophilin activity using a cell-impermeable derivative of CsA to specifically target extracellular pools of cyclophilins. In this study, we show that treatment with this compound in a mouse model of allergic lung inflammation demonstrates up to 80% reduction in inflammation, directly inhibits the recruitment of Ag-specific CD4(+) T cells, and works equally well when delivered at 100-fold lower doses directly to the airways. Our findings suggest that cell-impermeable analogs of CsA can effectively reduce inflammatory responses by targeting leukocyte recruitment mediated by extracellular cyclophilins. Specifically blocking the extracellular functions of cyclophilins may provide an approach for inhibiting the recruitment of one of the principal immune regulators of allergic lung inflammation, Ag-specific CD4(+) T cells, into inflamed airways and lungs.

FIGURE 1. Extracellular cyclophilins are generated throughout leukocyte recruitment phase of allergic lung inflammation

Groups of OVA primed/challenged mice were sacrificed every other day starting at day 6 through day 16 of the inflammatory response. (A) Numbers of effector/memory CD4⁺ T cells and eosinophils were determined in BAL fluid of individual mice by flow cytometric analysis. Data show mean ± SE cell numbers, with n=10 mice per time point. (B) Western blot analysis was conducted on cleared fluid from individual BAL samples to determine levels of extracellular CypA and CypB. Data show mean ± SE gel band density (GBD) for each time point as determined by densitometric analysis. These data are representative of 3 independent experiments.

FIGURE 2. NIM811 intervention reduces leukocyte numbers in the lung tissue and airways of allergic mice

Mice were primed/challenged with OVA, with some groups treated i.p. with NIM811, diluent alone, or untreated, on days 7, 9 and 11 of the regimen. (A) NIM811 reduces the number of effector/memory CD4⁺ T cells in the lung tissue (38%) and airway BAL (50%). (B) NIM811 reduces the number of eosinophils in the lung tissue (29%) and airway BAL (48%). Data show mean ± SE cell numbers for each group (n=12 mice per group) with a Student’s t-test analysis used to determine statistical significance. *** = P<0.001, ** = P<0.01, and * = P<0.05 levels of statistical difference comparing OVA + NIM811 to OVA + diluent groups. These data are representative of 3 independent experiments.

FIGURE 3. MM218 intervention significantly reduces leukocyte numbers in the lung tissue and airways of allergic mice

Mice were primed/challenged with OVA, with some groups treated i.p. with MM218 or PBS on days 7, 9 and 11 of the regimen. Cells were also collected from naïve BALB/c mice to serve as a baseline. (A) MM218 significantly reduces the number of effector/memory CD4⁺ T cells in the lung tissue (75%) and airway BAL (88%). (B) MM218 reduces the number of eosinophils in the lung tissue (79%) and airway BAL (88%). Data show mean cell numbers for each group (n=6) ± SE with a Student’s t-test analysis used to determine statistical significance. *** = P<0.001 and ** = P<0.01 levels of statistical difference comparing OVA + MM218 to OVA + PBS groups. These data are representative of 6 independent experiments.

FIGURE 4. MM218 intervention reduces inflammation in the lung tissue of allergic mice

Mice were primed/challenged with OVA, with some groups treated i.p. with MM218 or PBS on days 7, 9 and 11 of the regimen. Whole lungs isolated from naïve, OVA + PBS and OVA + MM218 mice were paraffin-embedded and 6 μm sections cut and stained with H&E or PAS. Images (10X magnification) show tissue areas surrounding bronchioles. Arrows on H&E sections denote inflammatory foci. The magnification bar represents 10 μm. These data are representative of 3 independent experiments.

FIGURE 5. MM218 intervention reduces T_H2 cytokine levels in the airways of allergic mice but does not affect T_H2 T cell priming

Mice were primed/challenged with OVA and some groups were treated i.p. with MM218 or PBS on days 7, 9 and 11 of the regimen. (A) BAL fluid was collected and pooled for each group, followed by 8x concentration. Levels of IL-5 and IL-13 cytokines were measured in the resulting fluid by ELISA. Data show the mean ± SE for replicate wells (n=4). Student’s t-test established *** P<0.001 for both cytokines comparing OVA + MM218 to OVA + PBS groups. (B) Spleens were collected from the same groups of mice and cell suspensions were generated and stimulated in vitro with different doses of OVA protein to induce cytokine production. After four days of culture, supernatants were collected and tested for levels IL-5 and IL-13. Data show the mean ± SE for replicate wells (n=4). (C) Serum was collected from individual mice and levels of OVA-specific IgE measured by ELISA. Data shown mean ± SE titers, with n=6 mice per group. These data are representative of 2–3 independent experiments.

FIGURE 6. MM218 intervention reduces airway hyperreactivity in allergic mice

Mice were primed/challenged with OVA with some groups treated i.p. with MM218 or PBS on days 7, 9 and 11 of the regimen. On day 12, mice were individually anesthetized with an i.p. injection of ketamine/xylazine, a tracheostomy tube inserted, and attached to a respirator. The mice were then sequentially challenged for 2 minutes with increasing doses of methacholine (1.5625 – 25 mg/ml). Measurements of airway resistance and compliance were determined using the Buxco FinePointe RC and associated software. Data show mean ± SE R_L and C_dyn measurements at individual doses of methacholine for each group of mice (n=10 per group). Student’s t-test analysis established *** P<0.001, ** P<0.01 and * P<0.05 levels of statistical difference at various doses of methacholine between OVA + MM218 and OVA + PBS groups. These data are representative of 3 independent experiments.

FIGURE 7. MM218 inhibits in vitro CD4⁺ T cell migration in response to extracellular cyclophilins

(A) Activated CD4⁺ T cells were generated in vitro and set up in Boyden chambers using recombinant CypA and CypB ± MM218. RANTES was used as a positive control for T cell chemotaxis. Data show mean ± SE chemotactic indices for each group (n=6 wells per group). Student’s t-test established ** P<0.01 and *** P<0.001 levels of statistical differences between + MM218 and no MM218 for CypA and CypB stimulation, respectively. (B) The same assay was used with activated CD4⁺ T cells set up either with RANTES or CypA ± MM218. Data show mean ± SE chemotactic indices for each group (n=6 wells per group). Student’s t-test established a *** P<0.001 statistical difference between + MM218 and no MM218 for CypA stimulation only. These data are representative of 3 independent experiments.

FIGURE 8. MM218 intervention inhibits the in vivo migration of OVA-specific CD4⁺ T cells to lung tissues and airways of allergic mice

Activated OVA-specific T_H2 CD4⁺ T cells were transferred into BALB/c mice. A separate group of mice received naïve OVA-specific CD4⁺ T cells. All recipient mice were challenged over four days in the airways with OVA. Groups of mice (n=6) were treated i.p. with MM218 or PBS during OVA challenge. Data show significant reductions in the number of OVA-specific (KJ1-26⁺) effector/memory CD4⁺ T cells in the airways and lung tissues of mice given activated CD4⁺ T cells. No response was induced in mice given naïve CD4⁺ T cells (white bars). Student’s t-test test established * = P <0.05 and *** P<0.001 levels of statistical difference in the airways and lung tissue, respectively when comparing OVA + MM218 to OVA + PBS groups given activated T cells. These data are representative of 2 independent experiments.

FIGURE 9. MM218 is effective when administered directly to the airways

Mice were primed/challenged with OVA, with groups of mice (n=6) given difference doses of MM218 by i.n. delivery during challenge. Data show a significant reduction in effector/memory CD4⁺ T cells and eosinophils in MM218 treated mice. Student’s t-test established *** P<0.001 and ** P<0.01 levels of statistical difference comparing OVA + MM218 to OVA + PBS groups. These data are representative of 2 independent experiments.