A pilot study to examine the effect of chronic treatment with immunosuppressive drugs on mucociliary clearance in a vagotomized murine model

Abstract

Background: Previously, we have demonstrated that mucociliary clearance (MCC) is diminished within the first months after surgery in lung transplant patients and the explanation for the reduction in MCC is unknown. We hypothesized that chronic treatment with a commonly prescribed regimen of immunosuppressive drugs significantly impairs MCC. We tested this hypothesis in a murine model of lung transplantation.

Methods: Fifteen C57BL/6 mice underwent vagotomy on the right side to simulate denervation associated with lung transplantation in humans. For 6 days, seven mice (controls) were intraperitoneally injected with three 100 µL doses of phosphate buffered saline and eight mice (immunosuppressed) were injected with three 100 µL injections of tacrolimus (1 mg/kg), mycophenolate mofetil (30 mg/kg), and prednisone (2 mg/kg) once daily. Then, mice inhaled the radioisotope (99m)technetium and underwent gamma camera imaging of their lungs for 6.5 hrs. Counts in the right lung at 1-1.5 hrs and at 6-6.5 hrs were first background-corrected and then decay-corrected to time 0 counts. Decay-corrected counts were then divided by time 0 counts. Retention at each time point was subtracted from 1.00 and multiplied by 100% to obtain percent removed by mucociliary clearance.

Results: Although there was a slowing of MCC at 1-1.5 hrs for the immunosuppressed mice, there was no statistical difference in MCC measured at 1-1.5 hrs for the two groups of mice. At 6-6.5 hrs, MCC was significantly slower in the immunosuppressed mice, compared to controls, with 7.78±5.9% cleared versus 23.01±11.7% cleared, respectively (p = 0.006).

Conclusions: These preliminary results suggest that chronic treatment with immunosuppressive medications significantly slows MCC in vagotomized C57BL/6 mice. These findings could shed light on why MCC is reduced in lung transplant patients whose lungs are denervated during surgery and who are chronically treated with immunosuppressive drugs post surgery.

Figure 1. Mean MCC (±SD) from the right lung between 1–1.5 hours and 6–6.5 hours in 7 C57BL/6 control mice (dark bar) and 8 drug-treated C57BL/6 mice (light bars).

There was a trend toward slower clearance in the drug-treated mice, compared to controls, at 1–1.5 hours, but differences in MCC were not statistically significant. Mucociliary clearance was statistically significantly slower in the drug-treated mice, compared to controls, at 6–6.5 hours (p = 0.006).

Figure 2. Representative examples of ciliated epithelial cells in the trachea of control and drug-treated mice.

Tissue was evaluated at 100 X and at 40 X magnification for the control (A and B) and drug-treated mice (C and D), respectively. Black arrows point to cilia. Qualitatively, the presence of ciliated epithelial cells in the airways in each of the representive animals is similar.

Figure 3. PAS staining of seromucous cells in tracheal glands of vagotomized mice.

A. Trachea of mouse treated with immunosuppressive agents. B. Trachea of mouse not treated with immunosuppressive agents. Black arrows point to PAS staining in seromucous cells. Qualitatively, both the drug-treated animals and control mice appeared to have similar PAS staining in the seromucous cells of the tracheal glands.

Figure 4. iNOS staining in airways of vagotomized mice.

A. Increased iNOS staining (brown) in bronchial epithelium of adult mouse treated with immunosuppressive agents (Black arrow). iNOS staining was qualitatively increased in the bronchial epithelium of five of the five drug-treated mice. B. Minimal iNOS staining in airway of adult mouse that was not treated with immunosuppressive agents. Minimal iNOS staining was observed in five of the five mice not treated with immunosuppressive agents. C. No first antibody control.