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 cells^2,3. Among the cutaneous immune cells, dendritic cells (DCs) are a type of professional antigen-presenting....

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 Biosafety Level 3 conditions, which could also be carried out under Biosafety Level 2 conditions according to institutional guidelines (room temperature 23 ....

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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)