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 ho....

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 (polariz....

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