概要

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

Published: July 20, 2021
doi:

概要

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

NOTE: All animal experiments were approved by the Institution Animal Care and Use Committee (IACUC, Approval ID: 2019-0056, 2019-0055). Seven to 9-week-old wild-type (WT) C57BL/6 female mice weighing 18-24 g were used for this study. Some studies were performed using female Langerin-diphtheria toxin receptor (DTR) and CD301b-DTR mice of the same age and weight. Four to six mice were used in each group for an experiment, and the data are representative of three independent experiments. This work was conducted under B…

Representative Results

Here, we demonstrated an intradermal infection model of C. albicans to study the role of cutaneous DC-mediated Th17 immune response in vivo. Following an initial intradermal injection with C. albicans into the footpad, the skin-draining LNs were enlarged (Figure 2A). During the sensitization period, the ratio of CD4+ to CD8+ effector T cells was notably increased (Figure 2B,C). Additionally, the e…

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…

開示

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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記事を引用
Park, J., Kim, S. H., Lee, J., Che, L., Roh, W. S., Park, C. O., Lee, M., Kim, T. In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection. J. Vis. Exp. (173), e62731, doi:10.3791/62731 (2021).

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