Pneumocystis carinii Pneumonia in scid Mice Induced by Viable ...

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Jul 30, 1996 - JAMES M. BECK,1,2* ROBERT L. NEWBURY,3,4. AND BRENT E. PALMER3. Division of Pulmonary and Critical Care Medicine, Department of ...
INFECTION AND IMMUNITY, Nov. 1996, p. 4643–4647 0019-9567/96/$04.0010 Copyright q 1996, American Society for Microbiology

Vol. 64, No. 11

Pneumocystis carinii Pneumonia in scid Mice Induced by Viable Organisms Propagated In Vitro JAMES M. BECK,1,2* ROBERT L. NEWBURY,3,4

AND

BRENT E. PALMER3

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine, University of Michigan Medical Center,1 and Pulmonary and Critical Care Medicine Section, Department of Veterans Affairs Medical Center,2 Ann Arbor, Michigan 48105, and Pulmonary and Critical Care Medicine Section, Department of Veterans Affairs Medical Center,3 and Department of Medicine, University of California,4 San Francisco, California 94121 Received 19 June 1996/Returned for modification 30 July 1996/Accepted 9 August 1996

Pneumocystis carinii pneumonia remains a major cause of morbidity and mortality in human immunodeficiency virus-infected individuals, despite the widespread use of prophylaxis and the development of new chemotherapeutic agents. The study of P. carinii and of pulmonary host defenses directed against it has been limited by lack of reliable, reproducible methods to obtain pure populations of organisms in useful quantities. While recent success has been achieved with cultures of rat P. carinii organisms, cultures of mouse P. carinii organisms have not been successful. Experiments were performed to determine whether P. carinii organisms derived from mice could be propagated in vitro. Mouse P. carinii organisms, obtained from the lungs of chronically infected athymic mice, were inoculated into spinner flasks containing HEL299 feeder cells seated on microcarrier beads. The numbers of mouse P. carinii organisms increased significantly over 7 days in culture. To test the viability and pathogenicity of these cultured organisms, P. carinii organisms were harvested after 7 days of culture and were inoculated intratracheally into susceptible scid mice. Four weeks after inoculation, scid mice developed uniformly severe P. carinii pneumonia. These studies demonstrate for the first time that mouse P. carinii organisms can be propagated in vitro. Furthermore, cultured mouse P. carinii organisms maintain their pathogenicity in an in vivo model system. This culture system will have important applications in the study of P. carinii biology and in the study of host defenses directed against this important opportunistic pathogen. genicity of the cultured P. carinii organisms must also be established. Rat P. carinii organisms, propagated in vitro, remain pathogenic when inoculated into steroid-immunosuppressed rats (15) or athymic rats (1). However, P. carinii organisms are highly host species specific and organisms obtained from different host species demonstrate substantial antigenic variation (9). Therefore, rat P. carinii organisms are unlikely to be useful for studies involving mice or other hosts. Although the steroid-treated-rat model has provided useful information about the biology and immunology of P. carinii, recent studies using immunosuppressed-mouse models have provided important new information about the host defense against P. carinii (22). A culture system to produce mouse P. carinii organisms in large numbers and in high purity would accelerate progress with these models. These experiments were performed to determine whether P. carinii organisms derived from mice could be propagated in vitro. We found that the numbers of organisms increased significantly over 7 days in culture. Furthermore, we performed experiments to verify the pathogenicity of the cultured organisms in an in vivo model. Cultured P. carinii organisms, inoculated intratracheally into scid mice, induced severe P. carinii pneumonia.

Pneumocystis carinii pneumonia remains a major cause of morbidity and mortality in human immunodeficiency virusinfected individuals, despite the widespread use of prophylaxis and the development of new chemotherapeutic agents (14). Additionally, the widespread use of immunosuppressive therapy has resulted in an increasing number of reported P. carinii infections in immunosuppressed individuals not infected with human immunodeficiency virus (20). An improved understanding of the biology of this important opportunistic pathogen would result in improved chemotherapy directed against the organism. In addition, an improved understanding of pulmonary host defenses against P. carinii could result in novel immunotherapeutic approaches to treatment of this pneumonia (16). The study of P. carinii and of pulmonary host defenses directed against it has been limited by a lack of reliable, reproducible methods to obtain pure populations of organisms in useful quantities (23). Continuous culture systems could produce large numbers of pure organisms for study, but experimental success to date has been quite limited (2). While axenic cultures of rat P. carinii organisms have been reported (7), serial passage of organisms was unsuccessful. Recent success with a culture system, using microcarrier beads and fibroblast feeder cells in spinner flasks, resulted in large numbers of rat P. carinii organisms of high purity (12). To date, however, no reports of successful culture of mouse P. carinii organisms have appeared. In addition to increases in numbers of organisms, the patho-

MATERIALS AND METHODS Mice. Mouse P. carinii organisms were obtained from the lungs of athymic mice (nu/nu on a BALB/c background; Simonsen Laboratories, Gilroy, Calif.), in which P. carinii organisms are propagated by serial passage in our laboratory (6). To test the pathogenicity of cultured organisms, virus-free, male C.B-17 scid/scid mice, purchased at 8 weeks of age (Taconic Laboratories, Germantown, N.Y.), received intratracheal inoculations of cultured organisms. All mice were housed in an isolation room in the Animal Care Facility of the Department of Veterans Affairs Medical Center, San Francisco, Calif., in filter-topped cages under laminar flow hoods. Mice received sterile rodent chow and sterile drinking water. Normal sentinel mice were routinely examined for the presence of unintended pathogens by culture and serology. This research was approved by the Animal

* Corresponding author. Mailing address: Pulmonary and Critical Care Medicine (111G), Veterans Affairs Medical Center, 2215 Fuller Rd., Ann Arbor, MI 48105-2300. Phone: (313) 761-7980. Fax: (313) 761-7843. Electronic mail address: [email protected]. 4643

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Studies Committee of the Department of Veterans Affairs Medical Center, San Francisco, Calif. Isolation of P. carinii organisms from lungs of athymic mice. Lungs from heavily infected athymic mice were removed aseptically after exsanguination during pentobarbital anesthesia (6). To prepare the inoculum for each culture, 100 mg of infected mouse lung was placed into 5 ml of minimal essential medium containing 10% fetal calf serum, 1% nonessential amino acids, 2 mM L-glutamine, 100 U of penicillin per ml, and 0.1 mg of streptomycin per ml (all from Sigma, St. Louis, Mo.). The lung was homogenized gently with a tissue grinder, and 5-ml aliquots of the homogenate were carefully applied to glass slides etched with circles 1 cm in diameter. These slides were stained with modified Giemsa stain (Diff Quik; AHS del Caribe, Aguada, P.R.) to count P. carinii organisms microscopically (6). To exclude the presence of bacterial or fungal organisms, smears of the inoculum were stained with Gram stain (Difco Laboratories, Detroit, Mich.). Cultures of the inoculum did not demonstrate the presence of organisms other than P. carinii. Propagation of P. carinii organisms in spinner flask culture. Mouse P. carinii organisms were cultured by a modification of methods described for rat organisms (12). As a substrate for feeder cells, Cytodex 3 microcarrier beads (Pharmacia, Uppsala, Sweden) were hydrated with phosphate-buffered saline (PBS) and were sterilized according to the manufacturer’s instructions. Cytodex beads (3 mg/ml) were added to 33 ml of medium (described above) in sterile, siliconized 125-ml spinner flasks (Corning Glass Works, Corning, N.Y.). Flasks were then inoculated with 106 human embryonic lung fibroblasts (HEL299; American Type Culture Collection, Rockville, Md.). Every 60 min, flasks were spun for 1 min at 30 rpm on an adjustable magnetic stir plate, which was placed into a 358C incubator with 4% CO2. After 4 h, the volume of each flask was brought to 100 ml with additional medium and then flasks were continuously stirred at 30 rpm. After 24 h, beads were allowed to settle and half the medium was exchanged. To examine the confluence of feeder cells on beads, 50-ml aliquots were sampled and morphology was observed. The confluence of feeder cells on beads occurred after 3 to 5 days of culture. Once HEL299 cells were confluent on beads, P. carinii inoculation of the flasks was performed. After counting organisms as described above, spinner flasks were inoculated with 3.5 3 107 organisms (3.5 3 105 organisms per ml of medium) and stirring was continued at 30 rpm. Enumeration of P. carinii trophozoites. At 24-h intervals after inoculation, the spinner flasks were allowed to settle and 2-ml aliquots of the supernatants were removed. Supernatants were centrifuged at 500 3 g for 15 min, and the pellet was washed once with sterile PBS and was then resuspended in 1 ml of sterile PBS. To count organisms, 10 ml of this preparation was carefully placed onto 1-cmdiameter circles etched on microscope slides. After staining with Diff Quik, the numbers of trophozoite nuclei in two diameters were counted and the number of organisms in the parent preparation was extrapolated. Counts were done by two independent observers who were unaware of the culture conditions, with interobserver agreement always $90%. To evaluate the presence of P. carinii cysts, duplicate smears were stained with Gomori methenamine silver stain. P. carinii inoculation of scid mice. After 7 days of culture, supernatants from spinner flasks were removed and were centrifuged. The P. carinii organisms in the resultant pellet were counted as described above and were brought to a concentration of 2 3 106 organisms per ml. This preparation was used to inoculate scid mice intratracheally as previously described (6). Briefly, intratracheal inoculation of scid mice was performed during pentobarbital anesthesia (75 mg/kg [intraperitoneally]). The trachea was exposed with a midline incision, and then a blunted needle was passed through the mouth into the mid-trachea with visual confirmation of position. A polyethylene catheter was passed through this needle until the catheter tip was just distal to the needle, and 0.1 ml of the inoculum (2 3 105 P. carinii organisms) was injected. The inoculum was immediately followed by an injection of 0.6 ml of air to ensure adequate dispersion of the inoculum and clearance of the central airways. Then the incision was sutured, and mice were placed prone for recovery. As negative controls, other groups of scid mice were inoculated with HEL299 cells (2 3 105 cells in 0.1 ml of medium) or with medium alone (0.1 ml). Scoring of infection in vivo. Four weeks after inoculation, the intensity of P. carinii infection in scid mice was scored. After a lethal dose of pentobarbital (400 mg/kg [intraperitoneally]), mice were exsanguinated by aortic transection. Lungs were inflated with air through the trachea and were fixed in formalin. Paraffin-embedded specimens were sectioned (thickness, 5 mm) and were stained with Gomori methenamine silver stain and hematoxylin-eosin stain for histologic examination. To exclude concurrent infection with other organisms, lung specimens from selected mice were removed aseptically before formalin fixation. Gram stains of touch preparations did not show other microorganisms, and bacterial and fungal cultures of lung tissue were always negative. The extent of infection with P. carinii cysts, evaluated on sections stained with Gomori methenamine silver stain, was scored according to criteria previously described and validated (6). These scores range from grade 0 (no P. carinii cysts or foamy extracellular alveolar exudate in any section) to grade 4 (P. carinii cysts throughout the alveoli with abundant foamy extracellular alveolar exudate) and correlate highly with organism counts performed on homogenized lungs (5). Then slides in each grade were grouped and compared to ensure consistency. Finally, slides were regraded to ensure reproducibility. All scoring was performed without knowledge of treatment groups.

INFECT. IMMUN.

FIG. 1. Propagation of mouse-derived P. carinii organisms in vitro. P. carinii organisms, isolated from the lungs of athymic mice, were inoculated into spinner flask cultures containing HEL299 cells adherent to Cytodex beads. Supernatant aliquots were sampled at the times indicated, the numbers of P. carinii organisms were counted, and data were expressed as fold increases over the baseline. Data are the means 6 standard errors of the means of eight separate experiments. p, P , 0.001 by analysis of variance compared with the results on day 0.

Statistics. The data reported are means 6 standard errors of the means for scalar measurements and medians for ordinal measurements. Scalar comparisons were made by analysis of variance using the Newman-Keuls multiple range test, and ordinal comparisons were made by the Kruskal-Wallis test (24). Testing was performed with StatView software (Abacus Concepts, Berkeley, Calif.). Significance was accepted when P was less than 0.05.

RESULTS P. carinii propagation in vitro. The numbers of mouse P. carinii organisms increased reproducibly over 7 days of culture, using feeder cells attached to microcarrier beads, with fourfold increases over this time course (Fig. 1). The increase in numbers of organisms was reproducible, occurring in eight separate cultures. Examination of smears from the flasks, prepared as described above, demonstrated that virtually all of the organisms were present in the trophozoite form after 7 days of culture. Duplicate slides, stained with Gomori methenamine silver stain, demonstrated abundant cysts at the time of inoculation but an absence of cysts after 7 days of culture (data not shown). In addition to increasing numbers of organisms, the spinner flask cultures produced highly purified P. carinii populations. The crude lung homogenates which were used to inoculate the spinner flasks contained large numbers of host cell nuclei and debris in addition to P. carinii cysts and trophozoites (Fig. 2A). In contrast, supernatants obtained from the spinner flasks showed sheets of P. carinii trophozoites (Fig. 2B). Few mouse lung cells or fragments were seen in the spinner flask supernatants after the first day of culture. Furthermore, little feeder cell contamination was present in the supernatants from the spinner flasks. Sampling of the microcarrier beads after settling demonstrated dense coating of the beads with confluent, adherent feeder cells. Induction of P. carinii pneumonia in vivo. To test the pathogenicity of cultured P. carinii organisms, we used organisms from the supernatants of the culture system after 7 days of propagation. These organisms were used to inoculate scid mice

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FIG. 2. Morphology of P. carinii organisms. (A) Crude lung homogenates used to inoculate the spinner flasks contained host cellular debris as well as clusters of P. carinii trophozoites (arrow) and P. carinii cysts (arrowheads). (B) Supernatant aliquots from spinner flask cultures contained P. carinii trophozoites of typical morphology (modified Giemsa stain). Magnification, 3625.

intratracheally. Four weeks later, the intensity of P. carinii infection in the lungs of scid mice was scored. Lungs from scid mice inoculated with P. carinii organisms demonstrated uniformly intense P. carinii infection (Fig. 3). As negative controls, lungs from mice inoculated with HEL299 cells or with medium did not demonstrate P. carinii infection. Histologically, lungs of scid mice infected with P. carinii organisms showed the foamy, eosinophilic extracellular exudate indicative of P. carinii (Fig. 4A). In contrast, lungs of mice inoculated with HEL299 cells were histologically normal, despite the inoculation of mice with feeder cells at concentrations that were at least 5 logs higher than those found in the P. carinii preparations (Fig. 4B). Therefore, feeder cells alone did not produce inflammation or alveolar damage in the lungs of recipients. Alveolar exudate demonstrated by hematoxylin-eosin staining always indicated the presence of P. carinii organ-

FIG. 3. Intensity of P. carinii infection after intratracheal inoculation. scid mice were inoculated with medium, HEL299 cells, or cultured P. carinii organisms. The intensity of P. carinii infection in lung tissue 4 weeks after inoculation was graded. Data are medians for 11 mice per group in two separate experiments. p, P , 0.005 by Kruskal-Wallis test compared with results for medium alone.

isms, which were present in abundance in sections stained with Gomori methenamine silver stain (Fig. 4C). DISCUSSION The results of these studies demonstrate for the first time that mouse P. carinii organisms propagate reproducibly in vitro. Using a spinner flask system, we obtained large numbers of mouse P. carinii organisms, and these organisms were free of significant contamination by lung host cells or by feeder cells. Furthermore, the propagated organisms were viable and pathogenic, as indicated by their ability to induce severe pneumonia in susceptible scid mice. Successful propagation of mouse P. carinii organisms has not been reported previously. The summary of a consensus conference, detailing previous attempts at P. carinii propagation, lists a single unsuccessful attempt at culture of mouse P. carinii organisms (23). However, limited success has been achieved with culture of rat P. carinii organisms. First reported for primary cultures of embryonic chick lung cells (18), rat P. carinii organisms have been propagated in short-term culture by using a variety of feeder cells (2). Both the isolation of large numbers of organisms and success of serial passage have been elusive goals (17). A recent and significant improvement has been the use of a culture system to propagate rat P. carinii organisms (12). Using this system, Lee et al. obtained fivefold increases in the numbers of P. carinii organisms by using spinner flasks and HEL299 cells for 5 days in culture. We chose HEL299 cells for mouse P. carinii culture on the basis of other investigators’ successes with rat P. carinii organisms, acknowledging that they differ significantly from the attachment of the organisms to type I alveolar epithelial cells in vivo. These feeder cells clearly serve as an attachment substrate for trophozoites (13), and cell lines to which the organisms tightly adhere are associated with improved growth (3). Additionally, feeder cells may provide essential molecules or nutrients to the organisms that are not present in cell-free culture (10). During 7 days of culture, the numbers of trophozoites increased significantly but the numbers of cysts declined. Similar observations have been made with rat culture systems (12). While the life cycle of P. carinii remains somewhat speculative, it is likely that trophozoites can replicate independently, without encystment (8). Although we did not carry our cultures

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FIG. 4. Histology of lungs 4 weeks after inoculation with cultured P. carinii trophozoites or control preparations. (A) scid mouse inoculated with P. carinii trophozoites. Abundant eosinophilic exudate was present in alveolar spaces with large, foamy alveolar macrophages and neutrophils (hematoxylin-eosin stain; magnification, 3150). (B) scid mouse inoculated with HEL299 cells. No inflammation or alveolar damage was present. Identical histology was observed in mice inoculated with medium alone (hematoxylin-eosin stain; magnification, 3150). (C) Morphology of P. carinii cysts (Gomori methenamine silver stain; magnification, 3625).

beyond 7 days, it is possible that extended culture would result in encystment of some organisms. Additionally, it is possible that alterations in culture conditions (such as depletion of nutrients) could shift replication of organisms from trophozoite to cyst forms. Increases in the numbers of P. carinii organisms over time with any culture system are of limited benefit if the organisms obtained are not viable or are not pathogenic. However, few investigators have submitted cultured organisms to the ultimate test of viability and pathogenicity: the ability to induce infection in susceptible hosts. We chose scid mice as the recipients of the cultured organisms on the basis of previous work demonstrating that they develop severe pneumonia within 4 weeks of inoculation with organisms obtained directly from the lungs of athymic mice (4). We demonstrated that cultured mouse P. carinii organisms induced consistent, severe pneumonia when inoculated into scid mice. Inoculation of scid mice with medium alone or with feeder cells did not result in P. carinii pneumonia. Again, our data extend those available from recent studies of rat P. carinii organisms. Rat P. carinii organisms obtained in serial passage were demonstrated to induce pneumonia after the inoculation of rats immunosuppressed with corticosteroids (15). As those investigators noted, however, their system was not adequate for the production of large numbers of organisms and required serial passage onto fresh feeder cells every 3 days. The pathogenicity of cultured rat

P. carinii organisms has been confirmed recently by a report showing the infection of nude rats with cultured organisms (1). Given the increasing importance of immunosuppressedmouse models in understanding the biology of P. carinii and host defenses directed against it (22), the development of this culture system represents an important advance in P. carinii research. For example, recent studies using mice specifically depleted of CD41 lymphocytes and scid mice have provided important information about the roles of lymphocyte populations in host defense (5, 6, 11, 19, 21). Additionally, the availability of immunologic reagents for mice makes this host an attractive one in which to study host defense. The availability of transgenic mice will undoubtedly increase the importance of mouse models for the study of host defense against P. carinii. Our results are important for P. carinii research for three reasons. First, we have confirmed that modifications of techniques used to propagate rat organisms can be used to propagate and purify mouse organisms. Second, we have provided a useful method for the purification of mouse P. carinii organisms for metabolic and biochemical studies and for host defense investigations. Third, we have demonstrated that organisms propagated in vitro retain their viability and pathogenicity. Therefore, this culture system can provide a useful source of mouse P. carinii organisms to exploit both in vitro approaches to P. carinii biology and well-described immunodeficient-mouse models of infection. A reliable culture method for mouse P. carinii organisms, therefore, has important implications for further in vivo and in vitro studies of this important opportunistic pathogen. ACKNOWLEDGMENTS This work was supported by the Career Development Program and the Medical Research Service, Department of Veterans Affairs (J.M.B.) and by the AIDS Clinical Research Center, University of California, San Francisco. We thank Marilyn Bartlett for helpful advice, Angela M. Preston for expert technical assistance, and Steven R. Meshnick for discussion and review of the manuscript.

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