Alternative Models for Studying Aspergilli

1. Alternative models for studying Aspergilli Dr Peter Warn School of Translational Medicine University of Manchester peter.warn@manchester.ac.uk

2. First The Good News • This should be the only slide you need to take notes from. • This presentation will be available at http://www.aspergillus.org.uk/ • Any SOPs referred to will be available through the same link • Additional SOPs will be available through the IAAM website http://www.sacmm.org/iaam.html

3. Why do we need models of aspergillosis? • To provide a bridge between in vitro studies and clinical research • Models have been the bedrock of research under pinning many research areas • Understanding Innate and adaptive immunity • Pathogenesis • Virulence • Drug discovery

4. Desirable attributes of animal models 1 • Mirror diseases seen in humans as closely as possible • Predictive of clinical outcomes • Models are standardized • Reproducible • Easy to set-up and require little specialist equipment • Reasonable cost Chamilos et al. Lancet Infect Dis 2007; 7: 42 -55. Clemons & Stevens. Med Mycol 2005; 43: S101-10.

5. Desirable attributes of animal models 2 • Amenable to studies including * Evaluation of therapeutics * Evaluation of host response * Evaluation of pathogen virulence factors * Assessment of in vivo gene expression Chamilos et al. Lancet Infect Dis 2007; 7: 42 -55. Clemons & Stevens. Med Mycol 2005; 43: S101-10.

6. Weaknesses of Animal Models • Will never fully replicate human disease • No single model answers all questions • May not mimic all structural features e.g. the structure of mouse lung • Additional effort with drug studies to ‘humanize’ PK and metabolic effects • Animal models can be acute and expensive Chamilos et al. Lancet Infect Dis 2007; 7: 42 -55. Clemons & Stevens. Med Mycol 2005; 43: S101-10.

7. Potential sites of infection in mammals Eyes Air Pocket  Subcutaneous chamber  Intranasal/sinus  Skin and hair Heart valve  Inhaled or tracheal  Peritoneal? Oral GI tract Vaginal Claw/nail Bladder Footpad Intravenous/ disseminated 

8. Modulators of fungal infection – host factors • Age of animal –in general younger animals more susceptible • Genetic background inbred v outbred – only mice • Immune status • Immunocompetent: • Immunocompromised: neutropenic vs. non-neutropenic • Tissue damage • Sex - Hormone status • Site of infection - route of infection/ method of infection • Pre exposure to whole fungi- hyphae or spores – immune status • Sensitization with fungal allergens

9. Modulators of fungal infection – fungal factors • Inoculum level • Stage of growth • Lag / log /stationary • Infection form • Spore v hyphae • Intrinsic virulence factors of the fungus • Virulence factors suitable for infection site • Time between infection and treatment

10. Housing and Husbandry Clean dedicated animal housing Day/Night light cycles Controlled temperature/humidity Room sterilization possible between models Waste disposal

11. Housing and Husbandry

12. Immunosuppression Normally required to establish an infection at the site of interest Make a model more ‘reproducible’ More closely replicate human disease Cytotoxic drugs (render animals neutropenic) Steroids (inhibit functions of immune cells) Hormones (change conditions at site of infection) Irradiation (render animals neutropenic) “Knock-out” / transgenic strains (potential to effect immune function/receptors/ cytokine response etc)

13. Model types • Lethal v non-lethal models • Lethal models: • Animals challenged with increasing inocula till death occurs. Outcome is time of death • Death can be due to multiple causes • Non-lethal models: • Animals infected with lower doses to develop a persistent high level infection with very mild symptoms • Samples can be collected at defined time-points allowing multiple surrogate markers of disease • Infected at a site which avoids systemic dissemination.

14. Review of the available models Disseminated infection Intravenous: The “unnatural” model • Easy model for lethal infection in mice and other species • Targets kidneys and spleen, much less the lungs – some strains invade brain – 2o effects can occur • Easy model for antifungal therapy • Can be easily modified to examine pathogen specific virulence factors • Bypasses many stages in the infection process

15. Systemic infections • Lethal v non-lethal models

16. Systemic infections • Lethal v non-lethal models

17. Systemic infections • Lethal v non-lethal models

18. Systemic infections • Endpoints • Death • Surrogate marker of imminent death (hypothermia/ torticollis/renal failure) • Euthanize animals at specific time-points • Organ culture (quantitative) over a predefined time range • Measurement of fungal products e.g. Chitin, Galactomannan • Measurement of fungal burden by qPCR (either DNA or RNA)/ assessment of fungal gene expression • Lethal v non-lethal models

19. Reviewof the available models Mice versus other rodents Advantages: • Can study disease in mice with specific host immune defects…potentially identifying the most critical • Can study disease in large numbers of fairly uniform inbred animals … increasing reproducibility of results • Less space for housing • Cost • Ease of handling Disadvantages: • Serial sampling not usually possible • Lung remodelling/airway narrowing differs from larger animals • Drugs are cleared from mice far more rapidly than in humans • Course of disease generally very acute, leading to death or recovery

27. Models of localized infections a) Invasive Pulmonary aspergillosis • Most models of IPA use infection by direct intranasal/intratracheal inoculation • Mice are anaesthetized and conidia suspension inhaled • Rats, Guinea pigs & Rabbits infected via tracheostomy/ intubation • Advantages • Relatively cost effective • Little specialist equipment required • Possible to infect large numbers from a single organism stock • Possible to test multiple strains in a single model

28. Models of localized infections a) Invasive Pulmonary aspergillosis • Most models of IPA use infection by direct intranasal/intratracheal inoculation • Mice are anaesthetized and conidia suspension inhaled • Rats, Guinea pigs & Rabbits infected via tracheostomy/ intubation • Drawbacks • Enormous mouse-mouse variation - direct methods better • Inter-laboratory studies difficult • Distribution may not be equal between lobes • Inoculum delivered in liquid – assumption that all of the inoculum delivered to lungs • Some animals develop bacterial pneumonia • Animals develop disease in trachea or sinuses • Therapeutic studies difficult

29. Models of localized infections a) Invasive Pulmonary aspergillosis There have been several attempts to standardize delivery of spores but none have been widely accepted Piggott and Emmons Adapted Inhalation chamber Hinners Inhalation Chamber SIDRANSKY and FRIEDMAN chamber SIDRANSKYand FRIEDMAN. 1959 Am.J.Pathol. 35:169-183.

30. Development and standardization of aerosol challenge model of invasive pulmonary aspergillosis • Mouse, rat, guinea pig • Provide samples and resources to other investigators • Supported by NIH / NIAID • UTHSCSA / Harbor-UCLA / University of Manchester http://www.sacmm.org/iaam.html

31. IPA Inoculation Chambers • Acrylic chamber • Conidia delivered via small particle nebulizer • Consistent inoculum level • 1 hour exposure • Madison chamber • Sealed chamber • Simultaneous exposure of large number of different species • Adjust inocula sizes and exposure period

32. IPA Inoculation Chambers – Mice, rats and guinea pigs

33. IPA Inoculation Chambers – Mice, rats and guinea pigs Difficult to clean after and between runs We use vaporized formaldehyde OR VHP Multiple strains = chambers needed Suitable for: 40 mice 12 rats 8 guinea pigs

34. Models of localized infections a) Pulmonary – Neutropenic Mice/Guinea pigs Cyclophosphamide + Cortisone Cyclophosphamide + Cortisone Cyclophosphamide + Cortisone if required Animals are severely immunocompromised Antibiotic prophylaxis is essential – in water if possible Severe weight loss is common Immature animals do not tolerate immunosuppression WBC / mm3 Infect Days Time Course of Immunosuppression for acrylic chamber http://www.sacmm.org/pdf/Murine%20Inhalational%20Pulmonary%20Aspergillosis.pdf

35. Key features of the neutropenic mouse/guinea pig model Animals: CD1 mice (>22g) BalbC mice>18g/ Hartley guinea pigs>450g Housing: HEPA filtered cages with sterile food and water Antibacterial Prophylaxis: Several antibiotics are suitable. Best if given in water Infection: Exposure to fungal spores as an aerosol (1-2 x 109 spores) Immunosuppression: Cyclophosphamide 250mg/kg, i.p., 2 days pre- and 200mg/kg 3 days post-infection plus cortisone acetate* 250mg/kg, s.c., 2 days pre- and post-infection Post Infection Burden: 1-2 x 104 cfu/g lung 48 hours post infection Survival: Untreated Animal succumb 4-6 days post infection 60-80% (mice) 100% (guinea pigs) mortality) *Cortisone acetate is given as a suspension. Has batch variability. Remains as solid beneath skin throughout model

36. Models of localized infections a) Pulmonary - Mice 104 103 Cfu per mouse 102 101 100 1 3 2 Experiment Reproducibility of infection excellent both between experiments and inter-lab

37. Models of localized infections a) Pulmonary – Neutropenic Mice Note- There is occasionally loss of controls (steroids) Note- This model does not lead to 100% mortality Murine Inhalational Model - Outcomes

38. Models of localized infections a) Pulmonary – Neutropenic Rats Prednisolone in a depo formulation is used IM Daily tail vein bleeds are possible (~1ml) Antibiotic prophylaxis is essential – in water if possible Severe weight loss is common Rats need a long acclimatization period

39. Key features of the neutropenic rat Model Animals: Sprague Dawley rats, Male 225-250g Housing: HEPA filtered cages with sterile food and water Antibacterial Prophylaxis: Baytril (enrofloxacin), 4 days pre-infection to prevent secondary bacterial pneumonia & urinary tract infection. Infection: Exposure to fungal spores as an aerosol (1 x 109 spores) Immunosuppression: Cyclophosphamide 75mg/kg, i.p., 2 days pre- and post-infection plus Depo-medrone (prednisolone) 15mg/kg, i.m., 2 days pre-infection Post Infection Burden: 3 x 104 cfu/g lung 48 hours post infection Survival: Untreated Animal succumb 4-6 days post infection (100% mortality)

40. Models of localized infections a) Pulmonary – Neutropenic Rat

41. Aspergillus Burden Changes During Infection • CFU of infected untreated rats showed little difference in the burden over time • Serial lung histopathology shows progressive Afu hyphae invading lung parenchyma

42. Characteristic disease progression in rats Experimental endpoints: Major causes of death Weight loss >25% Laboured breathing Bloody nasal discharge Unable to reach food and water

43. Models of localized infections a) Pulmonary – Non-neutropenic Rats The non-neutropenic model is similar in rats and mice The dose of cortisone is limited by toxicity Antibiotic prophylaxis is essential – in water if possible Severe weight loss is common Uninfected animals have large numbers of white cells in lungs at the end of the study *Danger of Pneumocystis in rats*

44. Disease Progression – Non-neutropenic rats The lung pathology following infection in non-neutropenic hosts is dominated by white cell recruitment resulting in loss of lung function

45. Neutropenic vs. Non-neutropenic Wiederhold N TIMM 2009 Berenguer et al. Am J RespCrit Care Med 1995; 152: 1079. Balloy et al. Infect Immun 2005; 73: 494.

46. Models of localized infections a) Pulmonary – Chronic infection mice C57BL/6 mice infected intratracheally with 1 x 105 spores of A. fumigatus embedded in agarose. Disease is restricted to the lungs with no tissue invasion. Infections possible for >20 days Don Sheppard IAAM Workshop 2008

47. Chronic Aspergillus Models - Tissue Chambers • Chambers (1cm x 0.3cm) inserted subcutaneously • Aspergillus is separated from cellular responses and unable to invade beyond the chamber. • Sampling possible though silicon membrane • Complex ‘biofilms’ develop in chamber. • Suitable for antifungal efficacy/ development of resistance/ host adaption studies Silicon rubber membrane 1cm Osmotic membrane Animal need ~1 week recovery post surgery Antibiotic prophylaxis post-op Chambers can remain in situ for up to 6 weeks Volume recovered during sampling is small

48. No time to discuss other models • Rabbits – great for drug and imaging studies • Transgenic/knockout mouse models – fantastic for understanding disease mechanisms • Non-mammalian hosts • Sinus models • Allergy/Asthma

49. Acknowledgements • Andrew Sharp • Raghdaa Shrief • Jayesh Majithiya • Joanne Slater • David Denning • University of Manchester • IAAM Contract team • Fungal Research Trust