Modeling Dysplastic and Functional Lung Alveolar Repair after Influenza Infection

Our research focuses on improving repair of the lungs after damage from infection or chronic disease. We study endothelial cells lining blood vessels in the lung that perform gas exchange. Advances in single-cell sequencing technologies allow us to identify remarkable heterogeneity in lung cell states, including in disease, but we still don't fully understand the function of these cell states.

Intranasal infection is a simple and non-invasive technical viral inoculation that follows the natural root of infection and allows the study of both adaptive and innate immune response against the virus. Influenza remains globally prevalent and cause severe respiratory illness. Therefore, understanding the viral infectivity and cellular response to infection and tissue injury is critical for developing effective treatments.

To begin, check the breathing of the anesthetized mouse. Perform a toe pinch and observe the reaction. Proceed only if the mouse reacts with a small convulsion or jump without moving its head.

Using a P200 pipette set to 50 microliters aspirate the diluted influenza A virus from the vial on ice. Set the pipette down on a tip box or paper towel, ensuring that the tip does not contact gloves or the surface of the biosafety cabinet. Now pick up the anesthetized mouse by scruffing, and hold it upright at a 45-degree angle.

Maintain a firm grip to elongate the trachea for better inhalation while ensuring that breathing remains shallow and consistent without gasping. Drop the viral dilution into the nostrils in a dropwise manner, alternating between nostrils to ensure the virus enters both sides of the nose. Alternatively, create a bubble of viral dilution spanning both nostrils by slowly and consistently pipetting while the mouse inhales the liquid.

Hold the pipette tip close to the mouse's nose without making contact. If the mouse begins to wake up, administer only half the dose. After administering the influenza A virus, hold the mouse upright for one to two minutes to keep its airway open and ensure effective delivery to the lungs.

Gently place one finger on each side of the mouse's chest to feel for crackles, which indicates successful delivery to the lungs. Once the mouse begins to recover, return it to its cage to recover fully. Monitor the mouse until it becomes ambulatory.

After euthanizing the mouse, dissect the chest cavity to expose the rib cage and the trachea. Cut open the diaphragm and gently separate it from the rib cage without puncturing the lungs. Then cut along both sides of the rib cage and remove it from the top.

Cut the clavicles and dissect away the surrounding bone and soft tissue from the front of the trachea. Next, dissect the muscle and tissue surrounding the trachea until the cartilage rings are clearly visible without puncturing the trachea. Using curved forceps, push the esophagus out from behind the trachea and dissect it completely.

Then thread a piece of suture behind the trachea and tie a loose knot. Clip a large blood vessel and perfuse the circulatory system by slowly injecting DPBS through the right ventricle of the heart. To inflate the lungs for histology or immunofluorescence, inflate the lungs in 2%paraformaldehyde and dehydrate through an ethanol series for paraffin embedding.

For lung inflation by hands, use a syringe and 21 to 23 gauge needle with or without tubing or affix a tube of 2%paraformaldehyde to a rings stand positioned 30 centimeters above the bench for gravity inflation. Insert the needle into the top of the trachea with the bevel facing up, being careful not to pierce the bottom of the trachea. Tighten the suture not around the trachea and needle before introducing the fixative.

To prepare a single-cell suspension for flow cytometry or single-cell sequencing, remove the lung tissue from the DPBS and mince with scissors. Chop the tissue further with a razor blade for two to three minutes to obtain evenly sized pieces. Dissociate the tissue in digestion buffer containing collagenase one, dispase and DNase in DPBS at 37 degrees Celsius for 30 to 35 minutes.

Then filter the dissociated tissue through 100 micrometer and 40 micrometer cell strainers. Lyse red blood cells using ammonium chloride potassium lysis buffer. Re-suspend the final cell pellet in 1%Bovine serum albumin in PBS for downstream analysis.

Influenza infected lungs showed heterogeneous structural damage compared to mock infected lungs. Tissue heterogeneity in influenza infected lungs quantified using a K-means clustering algorithm revealed multiple spatial clusters. Infected mice began losing weight three to four days post-infection with peak weight loss of approximately 25%occurring at eight to 10 days post infection.

By 14 days post infection, body weight had recovered to within 10 to 15%of baseline. Due to variability in infection response, survival rates among infected mice dropped steadily with some mice euthanized after losing more than 30%body weight. An interferon stimulated endothelial cell state emerged at six days post-infection and resolved by day 19.

While an injury induced capillary endothelial cell state appeared at day 11 and persisted through one year post-infection. The persistent injury induced capillary endothelial cell state was characterized by sustained expression of track B and was observed one year after infection. Transitional alveolar epithelial cell states, including immature alveolar type one cells emerged after infection and resolved as the tissue repaired.

Following infection, alveolar macrophages were depleted and reconstituted from both inflammatory monocytes and remaining macrophages resulting in distinct transcriptional states that persisted for at least one year.