This protocol describes the establishment of a novel mouse model to study the skin fungus, Malassezia, and its interaction with the mammalian skin in vivo. In contrast to in vitro approaches, this infection model allows the study of innate and adaptive immune mechanisms against Malassezia in the full complexity and in a tissue specific manner. Malassezia is associated with various skin disorders, such as atopic dermatitis.
Understanding the immune response to fungus may provide novel strategies to treat such frequent and chronic inflammatory skin diseases. Experimental skin infection with Malassezia provides a long awaited model for studying how skin commensal fungi modulate the immune system and thereby impact the course and severity of various skin disorders. To prepare a Malassezia inoculum add 10 milliliters of liquid mDixon medium and three to five individual Malassezia colonies from an mDixon agar plate to a sterile 100 milliliter Erlenmeyer flask.
Then place the flask at 30 degrees Celsius and 180 rotations per minute for 48 to 96 hours. When the culture is cream colored and turbid, transfer two milliliters of the culture into a sterile two milliliter microcentrifuge tube and sediment the yeast by centrifugation. Resuspend the pellet in one milliliter of PBS for a second centrifugation followed by resuspension in one milliliter of fresh PBS with vigorous pipetting.
Dilute the Malassezia suspension 20 to 50 times with PBS to ensure that the reading is between 0.1 and one and measure the OD 600 on a spectrometer. Aliquot a volume of the Malassezia suspension in PBS that corresponds to an OD 600 of four into one sterile two milliliter tube per animal to be infected and sediment the yeast by centrifugation. Then resuspend the pellets in 200 microliters of native olive oil.
To initiate a Malassezia infection, confirm a lack of response to pedal reflex in an anesthetized six to eight week old female C57 Black6 mouse and apply ointment to the animal's eyes. Use a caliper to measure the thickness of two different areas of both ears to allow calculation of the average thickness per ear. Then apply a small piece of tape to the skin of one ear before quickly removing the tape from the ear skin five times using a new piece of tape each time.
After stripping, use a sterile pipette to apply 100 microliters of the Malassezia olive oil suspension to the dorsal side of each ear and inject 200 microliters of sterile, prewarmed 2%glucose solution subcutaneously into the nuchal fold to support metabolism and rehydration. Then allow the mouse to recover on a heating pad for 30 minutes with monitoring before returning the animal to its cage. At the appropriate experimental time points, use a caliper to measure two different areas of each mouse ear to allow calculation of the average thickness per ear.
To measure the fungal burden in the infected skin, add 500 microliters of sterile 0.05%NP40 in distilled water and an autoclaved five milliliter diameter steel ball to one two milliliter microcentrifuge tube per ear and weigh each tube on a balance. Next, harvest the ears from each animal at the base and place the tissues into individual tubes of NP40. Weigh each tube on the balance to determine the weight of each ear sample before homogenizing the ear tissues for six minutes at 25 Hertz.
Then dispense 100 microliters of each sample onto mDixon agar plates and distribute the suspensions homogeneously across the agar before their upside down incubation at 30 degrees Celsius. Here representative images for the growth of Malassezia sympodialis in liquid mDixon medium and on mDixon agar as demonstrated are shown. An increase in ear thickness can be observed after Malassezia furfur application to the skin that has been barrier disrupted compared to the unperturbed skin of wild type C57 Black6 mice.
Indeed, quantification of the increase in ear thickness over time reveals that the change in thickness is significant compared to vehicle treated skin. Further, the fungal burden in the ear skin on day two after infection with Malassezia pachydermatis is 100 to 1000 times above the detection limit. Malassezia tends to form aggregates and is not easily suspendable.
Therefore, ensure homogenization of the oil fungus suspension by extensive vortexing. Additional readouts may include, histology to determine skin pathology and the isolation of immune cells from the skin and draining lymph nodes to investigate the antifungal immune response. This model opens the opportunities to study the immune response against Malassezia and to investigate the role of the fungus as a commensal in the context of various skin disorders.
Malassezia species are classified as BSL-2 organisms in some countries. Be sure to follow the regulation of your local authorities and to use the appropriate safety measures.
