Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

The overall goal of this procedure is to develop a model of infection dependent oral inflammation that is mediated by pathologic TH 17 cells. This is accomplished by first injecting th 17 cells into immunodeficient mice. The next step is to grow the candida albicans fungus, collect the blasto spores, and prepare them in PBS.

The final step is to orally inject the mice with the blasto spores to drive the proliferation of injected th 17 cells and induce oral inflammation. The results show that TH 17 injected mice succumb to weight loss and tongue inflammation based on flow cytometry and immunohistochemistry analysis of the tongue. So the implications of this technique extend toward oral immunotherapy as this mouse model.

TH 17 inflammation model can also be used to study TH 17 inflammation in the context of other oral diseases, demonstrating the procedure will be me and nitrogen vascular, and a post-structural fellow in my laboratory. This procedure requires fact sorted, naive T cells and optionally treg cells to establish TH 17 cells culture CD four positive, CD 44, low CD 62 L, high CD 25 negative naive T cells in UBO 96, well plates at 30, 000 cells per well in TH 17 conditions for three to four days as a control naive T cells can be cultured along with 30, 000 CD four positive CD 25 positive FOX P three positive treg cells in the same culture conditions to generate TH 17 cells. After four days of culturing, collect the TH 17 cells using a pipette.

Set aside a small aliquot to examine their viability. Suspend these cells in half a milliliter of RPMI and add 100 nanograms of propidium iodide to them. For fax analysis, the main bulk of the cells is harvested and prepared for cell injection.

The main bulk of the cells should be washed in PBS and centrifuge then prepared for injection into the mice. At 10 million cells per milliliter in cold PBS inject the mice with 100 microliters of the cell suspension intraperitoneal using a 25 gauge needle. Other mice should be injected with PBS or with control TH 17 treg co cultured cells plan to infect the mice with C albicans in three to five days the day before.

Infecting the mice with C albicans, disperse five colonies of the CAF two dash one lab strain in 100 microliters of sterile PBS and add the suspension to 20 milliliters of sterile broth. Let the inoculated media grow overnight at 30 degrees Celsius with 130 RPM of agitation. The next day, collect 10 milliliters of the culture in a 15 milliliter tube, then centrifuge the Eloqua at 1900 G for five minutes.

At room temperature, the pellet contains the blasto spores and the sedant should be discarded. Pool all the collected blasto spores into 10 milliliters of sterile PBS and transfer a 20 microliter Eloqua to a micro fuge tube. Add 20 microliters of two XPFA to the Eloqua and after 15 to 20 minutes of fixation, count the blasto spores using a hemo cytometer during the fixation time.

Wash the bulk of the blasto spores with PBS at least two more times. Store the blasto spore pellets in an AB SL one hood at room temperature until the count is known. Then resus suspend the blasto spores and room temperature PBS at 200 million cells per milliliter.

Keep the cells at the ready to infect the mice. This method has been reviewed previously, but will be revisited briefly in this section. First, anesthetize the mice confirm their anesthetized state and apply ophthalmic ointment.

Application of should be repeated after 45 minutes. Next subcutaneously. Inject a milliliter of saline for hydration working one mouse at a time.

First infect the sham group and then the candida group. Prepare a three millimeter diameter cotton ball by saturating it with 50 microliters of PBS or blasto spore suspension. Then access the base of its tongue and put the cotton ball there.

Make sure the tongue is withdrawn inside and away from the teeth so it is not lacerated. Then transfer the mouse to its own recovery cage on a warm pad. Corn cob bedding should not be used in the cage or the heat pad will not transfer sufficient heat four feet above the recovery cages position a heat lamp.

The cotton ball must stay in place for 90 minutes. Each mouse should have its own 90 minute countdown timer. During this time, each mouse should be flipped side to side every 15 minutes to prevent lung congestion.

So the single most critical step in the whole procedure is to make sure that the cotton ball stays in place, the wholly inoculation time. If a mouse begins to recover from the anesthesia prematurely, re anesthetize it using only ketamine. After 90 minutes, remove the ball and observe the animal's recovery.

Once recovered, administer an additional milliliter of saline at two locations on the back for added hydration. After the procedure, up to five sham inoculated, mice may share a cage using naive T cells derived from thigh 1.1 or CD 45.1 mice. The described procedure was followed and the injected T cells were tracked using their respective markers.

Co transfer Tregs were derived from CD 45.2 mice and were also tracked by that marker only. The candida infected mice exhibited recruitment and expansion of the injected TH 17 cells. After infection, weight loss was tracked.

The mice injected with only TH 17 cells lost weight progressively and some had to be euthanized. Whereas weight loss in other groups eventually stopped. Mice that were injected with the Tregs co-culture showed far fewer TNF alpha producing cells four days after the infection.

Prolonged presence of TNF Alpha producing cells shows that the TH 17 only injected mice had unresolved infections and inflammation. The tongues of the tregs injected mice also showed far fewer inflammatory infiltrates and less fungal growth At the same time point, prolonged presence of inflammatory infiltrates shows that the TH 17 only injected mice had unresolved infections and inflammation Once mastered. This infection technique can be done in three hours for four mice, plus additional 15 minutes for every mouse to be infected.