In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection
Summary
Here, we demonstrate the in vivo function of cutaneous dendritic cell subsets in Th17 immunity of deep dermal Candida albicans infection.
Abstract
The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.
Introduction
The skin is the outermost barrier organ, which protects the body from invading external pathogens and stimuli1. Skin is composed of two distinct layers, including the epidermis-a stratified epithelium of keratinocytes-and the underlying dermis-a dense network of collagen and other structural components. As a primary epithelial barrier tissue, the skin chiefly provides physical barriers and contributes to additional immunological barriers as it contains numerous resident immune cells2,3. Among the cutaneous immune cells, dendritic cells (DCs) are a type of professional antigen-presenting cells, which actively take up self- and non-self-antigens and migrate to the regional lymph nodes (LNs) to initiate antigen-specific T cell responses and tolerance according to the nature of antigens4.
The skin harbors epidermal antigen-presenting cells, namely the Langerhans cells (LCs) and at least two types of DCs, including dermal type 1 conventional DCs (cDC1) and dermal type 2 conventional DCs (cDC2)5. Epidermal LCs are of embryonic monocytic origin and maintain their cell number by self-perpetuation under homeostatic conditions6. In contrast, dermal cDC1 and cDC2 are of hematopoietic stem cell origin and are continuously replenished by DC-committed progenitors5. Cutaneous DCs are characterized by their surface markers, roughly divided into Langerin+ (including LCs and cDC1) and CD11b+Langerin– populations (mainly cDC2). In addition, this group has revealed that the CD11b+Langerin– DC population is further classified into two subsets according to CD301b expression7.
The important functional features of cutaneous DCs are centered on a division of labor, determined mainly by the intrinsic nature of each subset of DCs, in situ locations of the DCs, the tissue microenvironment, and local inflammatory cues8. These functional characteristics of cutaneous DCs necessitate the investigation of the role of specific subsets of DCs during certain types of immune response of the skin. Upon antigenic stimulation by cutaneous DCs in the draining LNs, naïve CD4+ T cells differentiate into specific subsets of helper T cells, which produce a set of defined cytokines for exerting their effector function9. Among the CD4+ helper T cell subsets, interleukin-17 (IL-17)-producing Th17 cells play a crucial role in autoimmune diseases and antifungal immunity10. In this regard, cutaneous fungal infection has been a robust model to study Th17 immunity in vivo11,12,13. When tape-stripped skins are epicutaneously exposed to the Candida albicans (C. albicans) yeast, epidermal LCs play a pivotal role in driving antigen-specific Th17 differentiation14.
Protective immunity against intradermal C. albicans infection requires innate immunity such as the fibrinolytic activity of fibroblasts and phagocytes15. However, little is known about the role of cutaneous DC subsets in establishing Th17 immunity in deep dermal C. albicans infection. This paper describes a method of intradermal skin infection of C. albicans, which produces local and regional Th17 immune responses. The application of diphtheria toxin (DT)-induced DC subset depletion mouse strains revealed that CD301b+ dermal DCs are crucial for Th17 immunity in this model. The approach described here allows for the study of the Th17 response to deep dermal invasive fungal infection.
Protocol
Representative Results
Discussion
This paper describes a method of intradermal C. albicans infection that allows the study of the role of cutaneous DCs in Th17 immune response in vivo. By applying multiparametric flow cytometric analysis with DT-induced mouse strains, we found that CD301b+ dermal DCs are a crucial cutaneous DC subset for initiating Th17 immunity against deep dermal C. albicans infection. Moreover, the results showed that the IL-17-producing T cell response was mainly produced by CD4+ but n…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This research was supported by Samjung-Dalim Faculty research grant of Yonsei University College of Medicine (6-2019-0125), by a Basic Science Research Program through the National Research Foundation of Republic of Korea funded by the Ministry of Education (2019R1A6A1A03032869) and Ministry of Science and Information and Communications Technology (2018R1A5A2025079, 2019M3A9E8022135, and 2020R1C1C1014513), and by Korea Centers for Disease Control and Prevention (KCDC, 2020-ER6714-00).
Materials
| 0.3 mL (31 G) insulin syringe | BD | 328822 | |
| 1x Perm/Wash buffer | BD | 554723 | |
| 1 mL (30 G) syringe insulin syringe | BD | 328818 | |
| 24 well-plate | Falcon | 353047 | |
| 50 mL conical tube | Falcon | 50050 | |
| 70 μm strainer | Falcon | 352350 | |
| 70% ethanol | |||
| ABI StepOnePlus real-time PCR system | Applied Biosystems | ||
| Anesthesia chamber | Harvard Apparatus | ||
| Brefeldin A | BD | BD 555029 | |
| β-Mercaptoethanol | Gibco | 21985023 | |
| Candida albicans strain SC5314 | provided by Daniel Kaplan at Pittsburgh University | ||
| CD3 | BioLegend | 100216 | Clone 17A2 |
| CD301b-DTR mice | provided by Akiko Iwasaki at Yale University | ||
| CD4 | BioLegend | 100408 | Clone GK1.5 |
| CD44 | eBioscience | 47-0441-80 | Clone IM7 |
| CD8a | BD Biosciences | 553031 | Clone 53.6.7 |
| Centrifuge | |||
| Clicker counter | |||
| Cuvette | Kartell | KA.1938 | |
| Cytofix/Cytoperm solution | BD | 554722 | |
| Diphtheria toxin (DT) | Sigma | ||
| Dulbecco's phosphate-buffered saline (DPBS) | Welgene | LB001-02 | |
| FACS (Fluorescence-activated cell sorting) buffer | In-house | ||
| Fc receptor blocker | BD | 553142 | |
| Fetal bovine serum (FBS) | Welgene | S101-07 | |
| Forceps | Roboz | for harvesting sample | |
| Hemocytometer | Fisher Scientific | 267110 | |
| Hybrid-R total RNA kit | GeneAll Biotechnology | 305-101 | |
| hydroxyethyl piperazine ethane sulfonic acid (HEPES) | Gibco | 15630-080 | |
| IL-17A (intracellular cytokine) | BioLegend | 506912 | Clone TC11-18H10.1 |
| Ionomycin | Sigma | I0634 | |
| Isoflurane | |||
| Langerin-DTR | provided by Heung Kyu Lee at Korea Advanced Institute of Science and Technology | ||
| LIVE/DEAD Fixable Aqua Dead Cell Stain Kit | Invitrogen | L34957 | |
| Loop and Needle | SPL | 90010 | |
| Monensin | BD | BD554724 | |
| NanoDrop 2000 | Thermo Scientific | ||
| Penicillin | Gibco | 15140-122 | |
| Petri dish | SPL | 10090 | |
| Phorbol 12-myristate 13-acetate (PMA) | Sigma | P8139 | |
| PrimeScript RT Master Mix | Takara Bio | RR360A | |
| RPMI 1640 | Gibco | 11875-093 | |
| Scissors | Roboz | for harvesting sample | |
| Stainless Steel Beads, 5 mm | QIAGEN | 69989 | |
| Sterile pipette tip | |||
| SYBR Green Premix Ex Taq II | Takara Bio | RR820A | |
| TCRβ | BioLegend | 109228 | Clone H57-597 |
| ThermoMixer C | Eppendorf | ||
| TissueLyser | QIAGEN | ||
| UV-VIS spectrophotometer | PerkinElmer | ||
| Wild-type C57BL/6 mice | Orient Bio | 7- to 9-week-old mice were used | |
| Yeast-peptone-dextrose-adenine (YPDA) medium, liquid, sterile (1% yeast extract, 2% Bacto peptone, 2% dextrose) | |||
| YPDA agar plate, sterile (1% yeast extract, 2% Bacto peptone, 2% dextrose, 2% Bacto agar) |
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