β2-integrin LFA1 mediates airway damage following neutrophil transepithelial migration during respiratory syncytial virus infection

Respiratory syncytial virus (RSV) bronchiolitis is the most common cause of infant hospital admissions, but there is limited understanding of the mechanisms of disease, and no specific antiviral treatment. Using a novel in vitro primary transepithelial neutrophil migration model and innovative imaging methods, we show that RSV infection of nasal airway epithelium increased neutrophil transepithelial migration and adhesion to infected epithelial cells, which is associated with epithelial cell damage and reduced ciliary beat frequency, but also with a reduction in infectious viral load. Following migration, RSV infection results in greater neutrophil activation, degranulation and release of neutrophil elastase into the airway surface media compared to neutrophils that migrated across mock-infected nasal epithelial cells. Blocking of the interaction between the ligand on neutrophils (the β2-integrin LFA-1) for intracellular adhesion molecule (ICAM)-1 on epithelial cells reduced neutrophil adherence to RSV-infected cells and epithelial cell damage to pre-infection levels, but did not reduce the numbers of neutrophils that migrated or prevent the reduction in infectious viral load. These findings have provided important insights into the contribution of neutrophils to airway damage and viral clearance, which are relevant to the pathophysiology of RSV bronchiolitis. This model is a convenient, quantitative preclinical model that will further elucidate mechanisms that drive disease severity and has utility in antiviral drug discovery.


Isolation of primary AECs from nasal brushings
. Collect airway epithelial cells by performing a nasal brushing on a healthy adult donor as described elsewhere 14 . Commercially available nasal epithelial cells (i.e. from Lonza or Epithelix Sarl) can also be used. The methods shown here can be used for cells obtained from both nasal brushings and commercially available human nasal epithelial cells. 1.2. Process nasal brushings cells immediately (i.e. go to step 1.3) or store in the fridge overnight in 2 ml M199 media supplemented with 2.5g/ml amphotericin B, 100 U/ml penicillin and 100 g/ml streptomycin. 1.3. Collect the nasal epithelial cells by centrifugation at 600xg. 1.4. Resuspend in F-media as is described elsewhere 15 (F-media composition: Dulbecco's modified Eagle's medium (DMEM) and F12 at a 3:1 ratio with 100 U/ml penicillin and 100g/ml streptomycin and 7.5% (v/v) fetal bovine serum supplementedwith 5 mM Y-27632, hydrocortisone (25 ng/ml), epidermal growth factor (0.125 ng/ml), insulin(5 mg/ml), 0.1 nM cholera toxin, amphotericin B (2.50 g/ml)). 1.5. Cells are ready to be seeded as described in section 2.2

Expansion of primary AECs using co-culture with 3T3-J2 feeder cell layers
All steps should be performed in a sterile environment using a Class II safety cabinet.

Collagen coating permeable membrane inserts.
All steps should be performed in a sterile environment using a Class II safety cabinet.

Seeding AECs onto permeable membrane inserts.
All steps should be performed in a sterile environment using a Class II safety cabinet. 4.1. Invert a collagen coated 24 well membrane insert in a sterile 12 well plate (one membrane insert/ well). 4.2. Seed AECs onto the bottom of the 3m pore membrane insert membrane at a density of 300,000/cm 2 in 70 l of F-media. Place the lid over the plate and incubate at 37 o C 5 % CO2 for 4-6 hours. The 3m membrane insert from Greiner have 0.6 x 10 6 /cm 2 pores, this is a lower density than the other commercially available from Greiner and the Corning (2 x 10 6 /cm 2 ). We found that the higher pore density membrane inserts impair the ability to visualize cells on the membrane and to measure ciliary beat frequency. Prepare a control membrane insert by seeding cells onto the top of a 0.4 m or 3 m pore insert membrane at a density of 150,000/ cm 2 . Feed these cultures basolaterally with 350 l of ALI media every 1-2 days. 4.12.
Wash off any AECs that detach from the membrane during culture by adding 400 l of ALI media apically. Aspirate this media and discard. 4.13.
Primary AECs should be used after 4 weeks of ALI culture, when motile cilia are observed under an inverted microscope equipped with a S Plan Fluor ELWD 20x ADM objective with working distance of (8.2 -6.9mm) and numerical aperture 0.45 (Nikon) and an ORCA Flash 4.0 digital camera (Hamamatsu). Our images were acquired at a resolution of 0.22um/pixel. 5.1. Viral stocks preparation and quantification of viral titre is performed as described previously 12 . Propagate rgRSV expressing GFP in HEp-2 cells as described previously 4 .

RSV infection of differentiated AECs
All steps should be performed in a sterile environment using a Class II safety cabinet. 6.1. Prior to infection, rinse the apical side of the cells with 400l ALI media to remove any detached epithelial cells. 6.2. Measure trans-epithelial electrical resistance (TEER) using an epithelial voltohmeter to confirm cell integrity. Only cultures that give reading of >200Ω/cm 2 should be used. 6.3. Aspirate basolateral and apical media is from both sides of the membrane insert. 6.4. Remove membrane insert from the 24 well plate and place upside down in a fresh 12 well plate. 6.5. Dilute your virus stock in ALI media to give a multiplicity of infection (MOI) of 5 in 25 l.
To calculate the amount of virus required for infection based the calculation on their being roughly 1x 10 5 AECs present on the membrane insert membrane (a large number of cells dissociate during incubation for 4 weeks). 6.6. Pipette 25 l of virus preparation on to the cell-side of each membrane insert, being careful not to touch the AECs. Note: place 25 l of ALI media only onto the mock infected membrane inserts. 6.7. Replace the plate lid (ensuring the liquid does not touch the lid) and incubate membrane inserts at 37 o C 5 % CO2 for 1 hour. After incubation, wash cells in PBS to remove any excess liquid on the top of membrane inserts. 6.8. Place the membrane inserts in a fresh 24 well plate and add 100 l of ALI media to the basolateral side of the cells. The apical side of membrane insert remains at ALI. Incubate at 37 o C, 5 % CO2 for 24-72 hours to allow progression of viral infection.

Preparation of differentiated AECs for neutrophil migration assay
All steps should be performed in a sterile environment using a Class II safety cabinet. 8

Neutrophil transmigration assay
All steps should be performed in a sterile environment using a Class II safety cabine 10.1. Add 5x10 5 neutrophils in HBSS+ containing 1 % (v/v) autologous serum (collected from the whole blood see 8.3) to the basolateral side of each membrane insert. 10.2. Incubate plates at 37 o C, 5 % CO2 for 1-4 hours. Different time points can be used depending on the experiment. 10.3. After migration, collect neutrophils from the apical side of the epithelial cells (these are now referred to as the migrated neutrophils) and basolateral side (the non-migrated neutrophils) in a microfuge tube. 10.4. If the neutrophils were pre-stained, quantify the number migrated using a plate reader and compare to a standard curve of known numbers of stained neutrophils, as described previously 12 . 10.5. Collect supernatants from the basolateral and apical side of the membrane insert; aliquot and store at -20 o C for future analysis. 10.6. Fix membrane insert in 1 % (v/v) PFA and stain for neutrophil or epithelial cell markers of interest.
10 Analysis of ciliary function during neutrophil transmigration 10.1 To measure ciliary beat frequency, place membrane inserts a 24 well plate in an incubation chamber at 37°C and 5% CO2 attached to an inverted microscope system. 10.2 Equilibrate cells for at least 30 minutes before image capture.

10.3
Record beating cilia using a digital video camera at a rate of 200 frames per second and capture at least 512 frames. The video area (512 x 512 pixels) should contain at least 10 ciliated cells. For each condition, at least 5 areas of the membrane insert should be videoed for CBF analysis. 10.4 Save files as an .AVI and calculate CBF by fast Fourier transformation using opensourced ciliaFA software as previously described 16 .