Caspase-9 as a Target for Pneumocystis Pneumonia Therapy MARK E. LASBURY, SHAO-HUNG WANG, CHEN ZHANG, CHUNG-PING LIAO, PAMELA J. DURANT, DENNIS TSCHANG and CHAO-HUNG LEE Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202 P NEUMOCYSTIS pneumonia (PcP) is a major opportunistic disease in AIDS patients. Although the advent of the highly active anti-retroviral therapy has significantly reduced the inci- dences of PcP it remains the leading cause of death in patients with AIDS. Several drugs such as trimethoprim/sulfamethoxa- zole, pentamidine, and clindamycin/primaquine that target Pneumocystis organisms are available. Unfortunately, these drugs have high levels of side effects (Allegra et al. 1988; Jung and Paauw 1994; Raviglione, 1990) and unacceptable rates of treat- ment failure (Lundberg, Davidson, and Burman 2000; Moorman et al. 1998). Also, Pneumocystis isolates have emerged that exhibit resistance to the most effective drug combination trimeth- oprim/sulfamethoxazole (Iliades, Meshnick, and Macreadie 2005); therefore, additional drugs are needed to treat the disease. Pneumocystis infection leads to defects in the function (Lasbury et al. 2004) and level (Lasbury et al. 2003) of the innate immune response. One potential PcP therapy is the restoration of the innate immune response to the organism. In this study, we found that alveolar macrophages undergo apoptosis during Pneumo- cystis infection due to activation of caspase-9 and that inhibition of this caspase-9 activity reduced the severity of PcP, suggesting that caspase-9 can be a target for therapy of PcP. MATERIALS AND METHODS Female Sprague–Dawley rats (120–140 g) were obtained from Harlan (Indianapolis, IN) and were divided into four groups. The first group of rats, referred to as Normal rats, were uninfected immunocompetent rats. The second group of rats consisted of Normal rats challenged with 7.8 Â 10 6 P. carinii organisms by transtracheal inoculation as described previously (Lasbury, Dur- ant, and Lee 2003). The third group of rats included dexametha- sone-treated rats (Dex); they were immunosuppressed by providing drinking water containing 1.8 mg/ml dexamethasone. The fourth group of rats consisted of dexamethasone-treated rats transtracheally inoculated with 7.8 Â 10 6 P. carinii organisms (Bartlett et al. 1988) and these were called Dex-Pc rats. Alveolar macrophages were obtained by bronchoalveolar la- vage (BAL) and identified based on their morphology and reaction with the anti-RMA antibody that is specific for alveolar macro- phages (Lasbury et al. 2003). To test the effects of BAL fluids or caspase inhibitors on apoptosis of alveolar macrophages, the cells were cultured in RPMI medium (Sigma-Aldrich, St. Louis, MO) supplemented with 10% fetal bovine serum, 1 mM pyruvate, 1% non-essential amino acids, 14 mM glucose, 17.9 mM NaCO 3 , 10 mM HEPES buffer, 100 U/ml penicillin and 0.1 mg/ml strepto- mycin. The cells were incubated in tissue culture plates for 1 h at 37 1C in 5% CO 2 to allow attachment of the macrophages and then the appropriate BAL fluid with or without 10 mg/ml of each caspase inhibitor was added for 48 h as described previously (Lasbury et al. 2006). The TUNEL assay to detect DNA fragmentation, a character- istic feature of apoptosis, was performed as described previously (Lasbury et al. 2006). Immunoblotting was performed to detect activated caspases using antibodies against activated caspase-3, -8, -9, or -10 as described previously (Lasbury et al. 2006). RESULTS AND DISCUSSION After inoculation of P. carinii organisms, five rats from each group were sacrificed and evaluated for alveolar macrophage numbers every 7 days for a total of 42 days. Results showed that the alveolar macrophage number in each rat remained constant at a level of approximately 4.5 Â 10 6 cells in uninfect- ed Normal or Dex rats. Alveolar macrophage numbers in Normal- Pc rats increased to approximately 7 Â 10 6 cells per rat within 14 days. The number started to decrease to normal levels 35 days after inoculation of the organisms. In Dex-Pc rats, alveolar macrophage numbers decreased to approximately 2.0 Â 10 6 cells per rat 7 days after organism inoculation and remained at this level until the study was terminated at 42 days. These results in- dicated that P. carinii infection causes a reduction in alveolar macrophage numbers. To determine whether the decrease in alveolar macrophage numbers was due to apoptosis, the TUNEL assay was performed. Results showed that an average of 11% alveolar macrophages from Dex-Pc rats undergo apoptosis, whereas only 1.1% and 1.4% of those from Normal and Dex rats, respectively, were apoptotic. To confirm apoptosis, Western immunoblot analyses were per- formed to detect activated caspases, and alveolar macrophages from Dex-Pc rats were found to have a significantly higher level of activated caspase-3 than those from either Normal or Dex rats. Western immunoblot results also revealed that caspase-9, but not caspase-8 or -10, was activated suggesting that this apoptotic event goes through the intrinsic apoptosis pathway. This possi- bility was confirmed by a fivefold increase in the level of cyto- plasmic cytochrome c in alveolar macrophages from Dex-Pc rats over those from uninfected rats (Lasbury, M.E., unpub. observ.). The possibility that certain substances in the lungs of P. carinii- infected rats induced this apoptosis was examined by incubating normal alveolar macrophages with BAL fluids from Dex or Dex- Pc rats. The BAL fluids from Dex-Pc rats were found to cause apoptosis in approximately 15% of the cells, while only 2% of those incubated with BAL fluids from uninfected rats undergo apoptosis (Lasbury, M.E., unpubl. observ.). To determine whether this apoptosis was also mediated by the intrinsic apoptosis path- way, inhibitors for caspase-8 (Z-IETD-FMK), caspase-9 (Z- LEHD-FMK), or caspase-10 (Z-AVED-FMK) were added to the BAL fluids from infected rats, and the caspase-9 inhibitor was found to inhibit the apoptosis induced by BAL fluids from infect- ed rats by approximately 45%. Both caspase-8 and -10 inhibitors had no inhibitory effect on the apoptosis induced by BAL fluids from P. carinii-infected rats. To determine whether the caspase-9 inhibitor also inhibit ap- optosis in vivo, Dex-Pc rats were giving 80 mg of the caspase-9 inhibitor by transtracheal instillation at the time of organism in- oculation and every 14 days after inoculation. Some Dex-Pc rats Corresponding Author: C. H. Lee, Department of Pathology and Lab- oratory Medicine, Indiana School of Medicine, 1120 South Drive, Fesler Hall Rm 419, Indianapolis, Indiana 46202, USA—Telephone number: 11-317-274-2596; FAX number: 11-317-278-4603; e-mail: chlee@ iupui.edu S138 J. Eukaryot. Microbiol., 53(S1), 2006 pp. S138–S139 r 2006 The Author(s) Journal compilation r 2006 by the International Society of Protistologists DOI: 10.1111/j.1550-7408.2006.00205.x