Characterisation of Interspecies Differences between Human and Rat Airway Macrophage Responses in vitro

E. Hoffman1, A. Kumar2, R. Mahendran1, A Patel2, V. Millar3, M. Clements3, B. Forbes2, L. Dailey2,4, & V. Hutter1

University of Hertfordshire, College Lane, Hatfield, Herts, AL10 9AB, UK2Kings College London, Waterloo Campus, Franklin-Wilkins Building, 150 Stamford Street, London, SE1 9NH, UK

3GE Healthcare Life Sciences, Maynard Centre, Forest Farm, Whitchurch, Cardiff CF14 7YT, UK.4Institute of Pharmacy, Martin-Luther University Halle-Wittenberg, Wolfgang-Langenbeck Str. 4, 06120 Halle/Saale, Germany

BACKGROUNDFoamy macrophage response are often observed in histologicallung slices of rats from pre-clinical studies in vivo, which aretypically characterised by a high vacuolated appearance andlarger cell size1,2,3. The mechanism of induction of the foamyalveolar macrophage phenotype is not well known and it hasnot been explained if these observations are truly an adverseresponse or not.

1. CELL CULTURE

Rat macrophage cells (NR8383) and human monocyte cells (U937) wereseeded into 96-well plates at a density of 3 x 104 cells/well. U937 cellswere differentiated to a macrophage phenotype using 4nM PMA incomplete culture media for 96h followed by a 24h rest period in completeculture media.

2. INDUCTION AND CHARACTERISATION OF MACROPHAGE RESPONSES

Cells were incubated with amiodarone (0.003 – 100 µM) or staurosporine(0.0003 – 10 µM) in complete cell culture medium.FOR CELL HEALTH AND MORPHOLOGY ASSESSMENT, cells were stained withHoechst 33342 (10 µg/ml), MitoTracker Red (300 nM), Image-It DeadGreen (25 nM) for 30 min followed by fixation with 3.7 %w/vparaformaldehyde for 20 min. Fixed cells were then stained overnight withCell Mask Deep Red (diluted 1:1000).FOR THE DETERMINATION OF LIPID CONTENT, cells were incubated with HCSLipidTox Phospholipid Red (diluted 1:1000) for 24 h and fixed with 3.7%w/v paraformaldehyde containing Hoechst 33342 (10 µg/ml) for 20 min.Cells were then incubated with HCS LipidTox Green (diluted 1:1000) for 30min for detection of neutral lipids.Cells from both assays were imaged using the In Cell Analyser 6000 (GEHealthcare, Little Chalfont, Bucks, UK) with a 40x objective.

METHODOLOGY

RESULTS

AIMS• Develop in vitro cell culture assay to better characterise

alveolar macrophage responses• Characterise interspecies differences between human and

rat macrophage models in vitro

Figure 1 - Cell health, morphology and lipid profiling of rat and human invitro macrophage cell models. Multi-parameter charts profiling cell health,morphology and lipid changes to NR8383 rat macrophage cells (black line)and U937 human monocyte-derived macrophage cells (purple line). Data ispresented as the mean of n=6 wells per plate from three experiments withdifferent cell passage numbers.

Figure 2 - Multi-parameter charts profiling cell health, morphology andlipid changes to NR8383 rat macrophage cells (A) and U937 humanmonocyte-derived macrophage cells (B) exposed to amiodarone (0.1-10µM) for 24 h. Data is presented as the mean of n=1 wells per plate fromthree experiments with different passage number.

Figure 3 - Multi-parameter charts profiling cell health, morphology andlipid changes to NR8383 rat macrophage cells (A) and U937 humanmonocyte-derived macrophage cells (B) exposed to staurosporine (0.01-1µM) for 24 h. Data is presented as the mean of n=1 wells per plate fromthree experiments with different passage number.

CONCLUSION

• Similar baseline profile of untreated lung macrophages invitro between rat and human models.

• Observed differenced in macrophage responses to drugchallenges:

A typical profile for phospholipidosis (increased phospholipidscontent) was observed for both cell lines after amiodaronetreatment.A profile for apoptosis was observed (reduced viability,elevated cell permeability, increased cell area) was observed forboth cell lines after staurosporine treatment.• Characterising macrophages in this way may provide a

better understanding od the pathophysiology of airwayimmune responses to inhaled medicines.

1. BASELINE PROFILE OF UNTREATED MACROPHAGES IN VITRO

2. CELLULAR RESPONSE OF IN VITRO MACROPHAGES TO AMIODARONE

3. CELLULAR RESPONSE OF IN VITRO MACROPHAGES TO STAUROSPORINE

REFERENCES1) Forbes B, et al. Advanced drug delivery reviews. 2014;71:15-332) Lewis DJ, et al. J Appl Toxicol. 2014;34:319-3313) Hoffman E, et al. Mol Pharmaceutics, 2015; 12(8):2675-2687