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.
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 (Tregs) 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.
Oral infections and inflammation have been related to cancer and cardiovascular diseases, and have dramatic impact on overall human health2,3. 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 disease8,9, 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 weakened13-15. 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 models12,18.
Denne model er baseret på at inducere oral C. albicans infektion afhængig Th17 inflammation. På grund af fraværet af T regs den Th17 celle inflammation er uhæmmet og fører til dårligt løst immunpatologi. In vitro afledt naive CD4-celler polariseret som Th17 celler blev anvendt til adoptiv overførsel. 40-50% af den dyrkede CD4 + celler viser påviselig IL-17A ekspression omkring dag 3 (Th17 celler), og derfor blev anvendt til injektion i mus. Den største fordel ved modellen er, at T…
The authors have nothing to disclose.
We thank Dr. Helene Bernstein for providing us with access to her flow cytometer. We also thank CFAR flow cytometry facility for the flow cytometry services. This project was in part supported by CTSC core utilization funding and STERIS corporation/University Hospitals-Division of Infectious diseases grant to PP.
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 |