A new and clinically relevant murine model of solid-organ transplant aspergillosis

Abstract

Invasive fungal infections (IFIs) are a major cause of death in organ transplant patients. The murine hydrocortisone-mediated immunosuppression model of pulmonary aspergillosis is commonly used to characterise IFIs in these patients. However, this model does not take into account the effects of calcineurin inhibitors on transplant immunity to IFIs or the fungal calcineurin pathway, which is required for both virulence and antifungal drug resistance. To address these two issues, a new and clinically relevant transplant immunosuppression model of tacrolimus (FK506) and hydrocortisone-associated pulmonary aspergillosis was developed. We first characterised IFIs in 406 patients with a lung transplant. This showed that all of the patients with pulmonary aspergillosis were immunosuppressed with calcineurin inhibitors and steroids. Murine pharmacokinetic studies demonstrated that an ideal dose of 1 mg/kg/day of FK506 intraperitoneally produced blood trough levels in the human therapeutic range (5-12 ng/ml). There was increased mortality from pulmonary aspergillosis in a transplant-relevant immunosuppression model using both FK506 and hydrocortisone as compared with immunosuppression using hydrocortisone only. Lung histopathology showed neutrophil invasion and tracheobronchitis that was associated with reduced lung tumour necrosis factor-α (TNFα), JE (homologue of human MCP-1) and KC (homologue of human IL-8) at 24 hours, but increased lung TNFα, JE and KC at 48 hours when fungal burden was high. Furthermore, FK506 directly impaired fungal killing in alveolar macrophages in vitro, with FK506-mediated inhibition of the radial growth of Aspergillus fumigatus in vitro occurring at the low concentration of 5 ng/ml. Taken together, these findings show that the immunosuppressive activity of FK506 outweighs its antifungal activity in vivo. These observations demonstrate that FK506 impairs innate immune responses and leads to an incremental increase in susceptibility to IFIs when it is combined with steroids. This new and clinically relevant mouse model of invasive aspergillosis is a valuable addition to the further study of both fungal immunity and antifungal therapy in organ transplantation.

Fig. 1.

Determination of the effect of FK506 on A. fumigatus growth at a human-therapeutic trough concentration by radial growth assay. (A,B) Radial growth of A. fumigatus strains CEA10, AF293 and ATCC46645 was measured at defined time points after inoculation of 1×10³ conidia onto Sabaroud (A) or RPMI (B) media with or without 5 ng/ml FK506 and incubation at 37°C. Data are shown for triplicates measurements for each of the three strains tested. The detection limit indicates the width of the Petri dish.

Fig. 2.

Determination of ideal i.p. dosage of FK506 to produce human-therapeutic trough concentrations in blood. Whole blood levels of FK506 were measured by LC-MS at 12 hours after the third injection of FK506 i.p. daily. All FK506 dosages were given according to body weight. Mice received 1, 2.5, 5 or 7.5 mg FK506/kg/day. Data are shown as mean ± s.e.m. of triplicate measurements. Dotted lines indicate the upper and lower limits for the trough therapeutic ranges for the drugs in transplant recipients.

Fig. 3.

Survival from pulmonary aspergillosis and fungal burden in BALB/c mice immunosuppressed with FK506 and hydrocortisone. (A) Survival from pulmonary aspergillosis. Two groups of eight BALB/c mice were immunosuppressed with either hydrocortisone (125 mg/kg s.c. every 3 days) or with hydrocortisone (125 mg/kg s.c. every 3 days) in combination with FK506 (1 mg/kg i.p. daily) from day −3. They were then intranasally inoculated with 5×10⁶A. fumigatus CEA10 conidia. Data are shown as mean from the two independent experiments. Survival differences were determined by Kaplan-Meier analysis. (B) Time course of pulmonary fungal burden. Groups of three mice were either untreated or immunosuppressed with FK506, hydrocortisone or both and sacrificed at 6, 24 and 48 hours post-infection and lungs harvested. Total DNA was isolated and A. fumigatus fungal burden estimated by semi-quantitative RT-PCR in comparison with murine β-actin. Statistical significance was determined by linear regression analysis. Data are shown as mean ± s.e.m. (C) Killing of A. fumigatus by murine alveolar macrophages. The alveolar macrophage cell line MH-S was pre-treated either with 10 ng/ml FK506 or with carrier. The ability to kill A. fumigatus CEA10 was assayed at 6 hours post-infection at an MOI of 1 by RT-PCR for fungal RNA after normalisation to control. p.i., post-infection; HC, hydrocortisone; Mac, alveolar macrophage; AF, A. fumigatus.

Fig. 4.

Incremental mortality in the FK506-hydrocortisone immunosuppression model is independent of murine or fungal strain. Groups of eight (A,B) male BALB/c and (C) male C57BL/6 mice were immunosuppressed with hydrocortisone (125 mg/kg s.c. every 3 days) alone or in combination with FK506 (1 mg/kg i.p. daily) from day −3, and then intranasally inoculated with 5×10⁶A. fumigatus AF293 (A), ATCC46645 (B) or CEA10 (C) conidia. Survival differences were determined by Kaplan-Meier analysis. p.i., post-infection; HC, hydrocortisone; FK, FK506.

Fig. 5.

Immunosuppression with FK506 and hydrocortisone leads to increased inflammation in invasive pulmonary aspergillosis. Groups of three mice were either untreated or immunosuppressed with FK506, hydrocortisone or both and culled at 48 hours after infection. Lungs were fixed and three PAS stained sections from each mouse scored with ImageJ software by threshold analysis for the amount of inflammation. (A) Percentage of lung area inflamed (histopathology). (B) Representative Grocott-stained (right) and H&E-stained (left) lung sections for the combined FK506-hydrocortisone model, demonstrating airways occluded by inflammatory cells and fungal germlings, together with inflammation in and around the airway and damage to the bronchial epithelium. IC, immunocompetent; HC, hydrocortisone.

Fig. 6.

Immunosuppression with FK506 and hydrocortisone leads to increased levels of TNFα, JE and KC in the lung in invasive aspergillosis. (A) Groups of three mice were either untreated or immunosuppressed with hydrocortisone or FK506 plus hydrocortisone and culled at 24 and 48 hours after infection with 5×10⁶A. fumigatus CEA10 conidia. Saline controls were uninfected. Lungs were homogenised and supernatant levels of TNFα, JE and KC measured by Luminex MAP analysis. Statistical significance was determined by one-way ANOVA and Tukey’s test. (B,C) Groups of three mice were either untreated or immunosuppressed with FK506, hydrocortisone or both and culled at 24 hours after infection with 5×10⁶A. fumigatus CEA10 conidia. (B) FACS analysis for neutrophil influx into the bronchoalveolar space. There were no significant differences between infected groups. (C) FACS analysis for macrophages in the bronchoalveolar space. There were no significant differences between infected groups. Sham, uninfected group; HC, hydrocortisone; IC, immunocompetent; BAL, bronchoalveolar lavage. Data are shown as mean and s.e.m.