Th17 Inflammation Model of Oropharyngeal Candidiasis in Immunodeficient Mice

Summary

Although Candida infection models are available to study host immune resistance, a model to study T cell mediated immunopathology in the context of Candida infection is absent. Here we describe a method to establish Th17 immunopathology associated with oral Candida infection in immunodeficient mice.

Abstract

Oropharyngeal Candidiasis (OPC) disease is caused not only due to the lack of host immune resistance, but also the absence of appropriate regulation of infection-induced immunopathology. Although Th17 cells are implicated in antifungal defense, their role in immunopathology is unclear. This study presents a method for establishing oral Th17 immunopathology associated with oral candidal infection in immunodeficient mice. The method is based on reconstituting lymphopenic mice with in vitro cultured Th17 cells, followed by oral infection with Candida albicans (C. albicans). Results show that unrestrained Th17 cells result in inflammation and pathology, and is associated with several measurable read-outs including weight loss, pro-inflammatory cytokine production, tongue histopathology and mortality, showing that this model may be valuable in studying OPC immunopathology. Adoptive transfer of regulatory cells (T_regs) controls and reduces the inflammatory response, showing that this model can be used to test new strategies to counteract oral inflammation. This model may also be applicable in studying oral Th17 immunopathology in general in the context of other oral diseases.

Introduction

Oral infections and inflammation have been related to cancer and cardiovascular diseases, and have dramatic impact on overall human health^2,3. Opportunistic infections and inflammation caused by C. albicans are associated with primary immunodeficiencies (PID)^4,5, inflammatory disorders such as periodontitis ^6,7, Sjogren’s syndrome, and salivary gland disease^8,9, as well as oral squamous cell carcinoma ^10-12. C. albicans is a dimorphic commensal fungus that colonizes the mouths of 60% of healthy humans asymptomatically, yet it is the most common fungal pathogen causing infections when the host defense is weakened^13-15. It causes recurring and chronic infections and inflammation in patients with AIDS and PID, and also in other immunocompromised individuals. As a commensal, its colonization load is associated with the change in the diversity of the overall oral microbiome¹⁶. As a pathogen it causes several forms of oropharyngeal candidiasis such as acute pseudomembranous, acute atrophic, chronic atrophic, chronic hypertrophic/hyperplastic, and angular cheilitis.

Protection against C. albicans is determined not only by host immune resistance, but also by the ability to appropriately control Candida-induced immunopathology. Although commensals such as C. albicans contribute to modulation and exacerbation of other oral inflammatory conditions, the mechanisms by which dysbiosis occur during opportunistic infections are unclear. Besides the known role of adaptive Th17 cells in memory response to C. albicans¹⁷, their role in initiation and perpetuation of inflammation pathology during chronic infections remain unclear. Furthermore, oral inflammatory diseases such as Sjogren’s syndrome and periodontitis are associated with Th17 mediated pathology. Interestingly, these diseases are also strongly associated with frequent OPC. However, the interactions among Th17 cells, oral immunopathology of OPC and other oral inflammatory diseases are unstudied.

Although mouse models of primary and secondary infection of oral candidiasis are available, a mouse model to study Candida infection associated Th17 inflammation, especially in the context of immunodeficiency is unavailable. This study presents a method for establishing oral Th17 inflammation associated with oral Candida infection in mice. Candida infection in mice is characterized by fungal lesions, inflammation in the tongue, decreased food intake, weight loss and eventually a moribund state. Oral pathology resembles chronic candidal infection lesions, as well as epithelial dysplasia in mouse oral cancer models^12,18.

Protocol

NOTE: The experiments using mice were performed in accordance to the institutional animal welfare committee (IACUC) guidelines.

1. Reconstituting the Rag-1^-/- Mice with In Vitro Cultured Th17 cells (Three days prior to Infection)

1. For establishing Th17 cells, culture CD4+ CD44low CD62Lhigh CD25- naïve T cells (3 x 104) in U-bottom 96 well plates alone, or co-culture them along with 3 x 104 CD4+CD25+Foxp3+Tregs in the presence of soluble  α-CD3 (1 µg/ml), α-CD28 (2 µg/ml) antibodies and antigen presenting cells, under Th17 conditions (polarized using IL-6 (25 ng/ml), TGF- β (2 ng/ml),  α-IFN-γ (2 µg/ml) and α -IL-4 (2 µg/ml)) for three to four days. 
   NOTE: Naïve cells and T_regs were sorted using fluorescence activated cell sorting (FACS) and magnetic cell isolation procedures and cultured as described previously^19-22.
2. On day 4, after resuspending the Th17 cells using the pipette, collect them from cultures, and centrifuge them under sterile conditions.
   1. Take a small aliquot of these cells to examine their viability and for phenotyping. Resuspend them in 500 µl of RPMI medium, add propidium iodide (200 ng/ml) and assess their viability immediately by flow cytometry. Perform phenotyping by flow cytometry assessment of IL-17A production after restimulation (see section 6.3).
3. Centrifuge and wash the main bulk of the cells in sterile PBS at 480 x g for 6 min at 4° C in all the steps unless otherwise specified.
4. Use these cells for adoptive transfer into 6-8 week old CD45.2 Rag1-/- immunodeficient mice.
   1. Resuspend the cells at 1 x 10⁷ cells/ml cell density using cold sterile PBS.
   2. Inject 100 μl of the cells by intraperitoneal injection, using 25 G needles on 1 ml tuberculin syringes, such that one mouse receives 1 x 10⁶ cells. Some mice will receive PBS or Th17 cells only, and other mice will receive Th17 cells that were co-cultured with Treg cells.
      NOTE: Perform all the steps aseptically.

2. Growing C. albicans for infection (one day prior to Infection)

1. Before inoculation, disperse five colonies of the CAF2-1 C. albicans laboratory strain in 100 µl of sterile PBS suspension, and add the suspension to the sterile broth.
2. Inoculate 5 colonies of the C. albicans in 50 ml of the Yeast nitrogen base (YNB without amino acids)/Peptone/Dextrose broth medium and incubate in a shaker incubator at 30 °C for 15–18 hr at 130 rpm.
3. Monitor the broth for cloudiness, as that is an indication for the growth of the fungus.

3. Candida Harvesting and Counting Procedure (On the Day of Infection)

1. Collect 10 ml of Candida broth in 15 ml tubes. For larger volumes, collect Candida in 50 ml tubes.
2. Centrifuge the blastospores at 1900 x g for 5 min at RT in all the steps unless otherwise specified. Pellet the blastospores by centrifugation, followed by the removal of the supernatant.
3. If there is more than one tube, pool the Candida blastospores from multiple tubes, adding sterile PBS to the pellets and resuspend them in 10 ml of PBS for counting.
4. Take 20 μl of blastospores in 1.5 ml microcentrifuge tubes, add 20 μl of the 2X paraformaldehyde and incubate at RT for 15-20 min. Count the fixed blastospores using the hemocytometer under the microscope.
   NOTE: Paraformaldehyde is carcinogenic. Open the undiluted solution only in the fume hood.
5. In the meantime, repeat washing the blastospores at least twice, by centrifuging them in PBS, and pelleting the blastospores . Keep the pellets in 15 ml tubes at RT, in the ABSL1 hood, ready for infection.
6. Leave some sterile PBS at RT for resuspending the blastospores for infection.
7. After counting, add sterile PBS to the blastospore pellet to adjust the yeast blastospore cell numbers to 2 x10⁸ of yeast cells/ml. This is the blastospore suspension that will be used to infect the mice.
   NOTE: Perform all the steps aseptically.

4. Mice Infection (3-5 Days after Cell Transfer)

NOTE: The basic infection procedure is performed as described previously^19,23-25.

1. Weigh the Rag-1-/- mice that received PBS or the cells three days earlier.
2. Calculate the dose of anesthetic agents using their body weight. Anesthetize the mice by administering ketamine/xylazine mixture (16.1 mg/ml and 1.6 mg/ml), using 25 G needles on 1 ml tuberculin syringes, by intraperitoneal injection.
   1. Administer 50 µl per 10 grams of body weight. (i.e., 0.8 mg of ketamine and 0.08 mg of xylazine/10 g of body weight or 80 mg of ketamine and 8 mg of xylazine per kg of body weight respectively). Observe for toe pinch response every 15 min after anesthesia.
      NOTE: This dosage will induce 60-90 min of anesthesia, which is enough for the infection procedure.
   2. Apply the ophthalmic lubricant ointment in the eyes of the mice to prevent the corneas from drying out. As the eye lubricant may dry out, repeat ophthalmic lubrication every 45 min.
3. Inject 1 ml of saline (0.9% NaCl) subcutaneously on the back adjacent to the forelimb, to help rehydration of the mice during anesthesia.
4. Obtain a new, clean cage. Follow the infection procedure one mouse at a time, placing each mouse into the new cage once infected. Perform the PBS/sham infection first,and then proceed to the Candida infection groups.
5. Pick up the anesthetized mouse and open the mouth wide to reveal the base of the tongue.
   1. Place a 3 mm diameter cotton ball saturated with 50 µl of PBS or blastospore suspension, sublingually in the oral cavity for 90 min. Flip the mice every 15 min front and back to prevent lung congestion, ensuring that the cotton balls don't move.
6. Set up a timer for every mouse to ensure 90 min of Sham or Candida inoculation in the oral cavity.
7. Keep them in a cage under the heat lamp (4 feet away), and each mouse on heat gel pads.
8. Make sure that the tongue is withdrawn inside and away from the teeth, to avoid teeth lacerating the tongue.
9. At the end of 90 min, remove the cotton ball from the mouth of the mouse. Watch for any mouse that may recover from anesthesia sooner than 75 minutes. In such a case, anesthetize them again with ketamine (40 mg/kg) only.

5. Post Procedure Monitoring (during the 90 min Anesthesia)

1. Use heat gel pads during the 90 min anesthesia.
   1. To maintain the body temperature and to prevent suffocation, do not allow mice to recover on the regular corn-cob mouse bedding.
2. After the recovery from anesthesia, administer an additional 1 ml of sterile 0.9% NaCl subcutaneously in two locations on the back.
3. Keep up to five PBS (sham) inoculated mice per cage. House only 1-2 infected mice per cage.
4. Fill out the procedure card with initials every day after the procedure to note down the changes in the body weight, grooming habits and overall health.

6. Assessing the Inflammation

6.1) Weight loss

1. Weigh the mice every day during the infection, starting on the day prior to infection procedure.
   NOTE: Typically, the immunodeficient mice injected with Th17 cells and with no T_regs succumb to 20-30% weight loss due to increased fungal burden¹⁹ and exacerbated inflammation.

6.2) Histology scoring of the tongue

1. Sacrifice the mice by CO₂ asphyxiation followed by cervical dislocation.
2. Open the jaws and dissect out the tongue with scissors and forceps. Use blunt forceps to hold the tongue, and using the scissors reach out to the back of the mouth to incise the back end of the tongue.
3. For immunocytochemical hematoxylin and eosin (H&E) staining of the tongue tissues, rinse the tongue tissues with ice cold PBS. Add 5 ml of 10% formalin for 2-3 tongues and fix them O/N in 15 ml tubes.
   1. The next day, remove the formalin and immerse the tissues in 5 ml of 70% ethanol to prevent hyper-fixation. Send them out to commercial service to continue with paraffin embedding, sectioning and staining of paraffin sections.
      NOTE: Commercial histology service is used to perform these steps.
4. Once the slides are back from the commercial histology service, grade the inflammation by observing the tissue sections under a light microscope.
   1. Score from 0 to 5, with 0 being no inflammation, and 5 being the most severe inflammation. Score the inflammation using Table 1.

6.3) Cytokine production by Th17 cells

NOTE: Excessive TNF-α production is one of the readouts for excessive immunopathology. When mice are adoptively transferred with Th17 cells only, it causes immunopathology that is associated with excessive TNF-α production in Th17 cells.

1. Collect a single cell suspension from spleen, cervical lymph nodes, axillary lymphnodes, and tongue, using previously described methods^19,26.
2. Restimulate the cells with phorbol myristate acetate (PMA) (50 ng/ml) and Ionomycin (500 ng/ml) for 4 hr with Brefeldin A (10 µg/ml) added in the last 2 hr. Wash the cells with PBS and fix them using a commercial Fixation/permeabilization kit according to manufacturer’s instructions.
3. Perform intra cellular cytokine staining using the fluorochrome conjugated anti-TNF-α, anti-IL-17A and ROR-γt antibodies as described previously¹⁹.
4. Briefly, resuspend the cells in the 1X permeabilization buffer with the cocktail of anti-TNF-α, anti-IL-17A and ROR-γt antibodies, each antibody at 2 - 3 µg/ml final concentration.
5. Incubate the cells for 1 hr at RT.
6. After the incubation, wash the cells with 1X permeabilization buffer.
7. Resuspend the cell pellet in 500 µl of PBS with 0.5% bovine serum albumin for flow cytometry analysis.

Results

In this model, both the Candida-infected mice and uninfected mice in Rag-1^-/- immunodeficient background were adoptively transferred with Th17 cells 3-5 days prior to the infection. A total of 10-12 mice were used for the experiments, with 2 mice in each Sham infected groups, and 4-5 in each of the Candida infected groups. Naive cells were derived from congenic Thy1.1 or CD45.1 mice, so that injected Th17 cells were tracked in vivo using Thy1.1 or CD45.1 staining respectively (

Discussion

This model is based on inducing oral C. albicans infection dependent Th17 inflammation. Because of the absence of T_regs, the Th17 cell induced inflammation is unrestrained and leads to poorly resolved immunopathology. In vitro derived naïve CD4 cells polarized as Th17 cells were used for the adoptive transfer. 40 - 50% of the cultured CD4+ cells show detectable IL-17A expression around day 3 (Th17 cells), and therefore were used for the injection in mice. The major advantage of the model...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CAF-2	University of Pittsburgh (Sarah Gaffen)	-	Candida culture
U-bottom 96 well plates	Fisher	055588	Used for cell culture
a-CD3	eBiosciences	16-0031-85	Polarization of cells to Th17 conditions
a-CD28	eBiosciences	16-0281-85	Polarization of cells to Th17 conditions
m-IL-6	Bio Basic	RC232	Polarization of cells to Th17 conditions
h-TGF-b	R&D	240-B	Polarization of cells to Th17 conditions
a-IFN-g	eBiosciences	16-7311-85	Polarization of cells to Th17 conditions
a-IL-4	eBiosciences	16-7041-85	Polarization of cells to Th17 conditions
α-IL17A ef660	eBiosciences	50-7177-82	Used for cell culture - 1:50
α-TNFa-PE-Cy7	eBiosciences	25-7423-41	Used for cell culture - 1:100
α-RORgt PE	eBiosciences	12-6981-82	Used for cell culture - 1:50
YNB w/o amino acids)/Peptone/Dextrose broth medium	Bio Basic	S507.SIZE	Mediium for candida growth
Shaker incubator	New Brunswick Scientific	Innova 4300	Incubation growth for candida
15 ml tubes	Bio Basic	BT888-SY	Used for cell culture and candida growth
50 ml tubes	Bio Basic	CT 788-YS	Used for cell culture and candida growth
Table top Centrifuge	VWR International LLC	82017-654	For pelleting candida -900 g
Allegra Centrifuge	Beckman Coulter	392302	Cell culture - 480 g
1.5 ml eppendorf tubes	Bio Basic	BT620-NS	Preparing final concentration of candida
2x paraformaldehyde	Electron Microscopy Sciences	15710	Fixing candida for count
Hemocytometer	VWR International LLC	15170-172	Counting cells
PBS - Phosphate Buffered Saline	Bio Basic	PD8117	Preparation of buffers
Ketamine/xylazine	Case Western Reserve University - Animal Resource Center	-	Obtained from ARC approved protocol for anesthesia
Ophthalmic lubricant ointment	Allergan	-	Eye ointment for animals to prevent dryness
Saline (0.9% NaCl)	G Biosciences	786-561	Administerd to animals to prevent dehydration
3-mm-diameter cotton wool ball	VWR International LLC	BP7603	3 mm balls for candida infection
Heat gel pads	Case Western Reserve University - Animal Resource Center	-	for maintaining the body temperaturre and fast recovery
Hematoxylin and Eosin staining	Histoserv - MD	-	Tissue histology
10% formalin	Electron Microscopy Sciences	JC1111/MC	Tissue histology
70% ethanol	VWR International LLC	97064-490	Sterilization purpose
PMA - Phorbol 12-myristate 13-acetate	Sigma-Aldrich	P1585-1MG	Restimulation of cells
Ionomycin	Life Technologies	124222 1mg	Restimulation of cells
Fixation permeabilization kit	eBiosciences	E16913-106	Fixing cells for Flow cytometry
Tuberculin syringes	BD Biosciences	309659	Mice injection
25 G x 3/8 needles	BD Biosciences	309626	Mice injection
Rag1 -/- mice	Jackson laboratories	Stock no: 002216	Recipient mice
CD45.1 congenic mice	Jackson laboratories	Stock no:002014	Donor Th17 cells
CB17-SCID mice	Jackson laboratories	Stock no: 001303	Recipient mice
Balb/c mice	Jackson laboratories	Stock no: 000651	Donor Th17 cells