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 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 vivo^11,12,13. 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 Biosafety Level 3 conditions, which could also be carried out under Biosafety Level 2 conditions according to institutional guidelines (room temperature 23 °C ± 3 °C, humidity 50% ± 10%).

1. Preparation of Candida albicans

NOTE: Experiments in this section were performed in a biological safety cabinet.

1. Streak C. albicans strain SC5314 onto a yeast-peptone-dextrose-adenine (YPDA) agar plate using an inoculation loop and needle.
2. Incubate the plate upside down for 2 days at 30 °C.
   NOTE: The YPDA agar plate with C. albicans can be stored at 4 °C for up to 1 month.
3. Isolate a single colony from the plate for inoculation into 10 mL of YPDA medium in a 50 mL tube using a sterile pipette tip.
4. Incubate at 30 °C with shaking at 230-250 rpm for ~17 h.
5. Place the yeast suspension in a cuvette, and measure the optical density (OD) at 600 nm every 30 min using a UV-VIS spectrophotometer until the OD₆₀₀ reaches 1.5-2.0.
   NOTE: This step may take 16-18 h.
6. Spin the yeast suspension at 1000 × g for 5 min.
7. Discard the supernatant and resuspend the yeast cells in an appropriate amount of sterile phosphate-buffered saline (PBS).
8. Count the C. albicans cells using a hemocytometer and spin the suspension at 1000 × g for 5 min.
9. Discard the supernatant, and resuspend the C. albicans cells in PBS to a concentration of 1 × 10⁷ cells in 40 µL of PBS per footpad.
10. For the preparation of heat-killed (HK) C. albicans, kill the yeast cells by heating at 65 °C for 60 min using a heating mixer after determining the cell number.

2. Mouse footpad infection with C. albicans

Figure 1: Schematic diagram of intradermal Candida albicans infection model. (A) The hind footpads of mice were injected intradermally with 1 × 10⁷ C. albicans. After 7 days, the footpads of mice were re-exposed to 1 × 10⁷ HK C. albicans by intradermal injection, and the delayed-type hypersensitivity response was measured 24 h after antigen challenge. Local Immune response during C. albicans sensitization was analyzed after 7 days in skin-draining LNs. (B, C) Images of footpads before intradermal footpad injection with C. albicans. (D, E) Injection of C. albicans into the deep dermis of the right footpad. (F, G) Clinical signs of redness and swelling following footpad injection of the right footpad. (H) A sketch showing lymphatic pathways from the footpad to popliteal LNs following C. albicans injection. (I) Exposed popliteal LNs located behind the knee 7 days after C. albicans injection and (J) without injection. Abbreviations: HK = heat-killed; LNs = lymph nodes. Please click here to view a larger version of this figure.

1. Anesthetize the mice with isoflurane in an induction chamber until the mice have a slow respiratory rate and show no withdrawal responses to toe or tail pinches.
   NOTE: During anesthesia, eye ointment is recommended to prevent dry eyes, especially for anesthesia lasting longer than 5 min.
2. Remove the cap from a 31 G needle, and load the 0.3 mL insulin syringe with the prepared C. albicans from step 1.9 after mixing the cells.
3. Remove the anesthetized mouse from the induction chamber, and gently inject 40 µL of the yeast cells (1 × 10⁷ cells) into the deep dermis of the right footpad for C. albicans sensitization.
   NOTE: The maximum volume that can be injected into a footpad is 50 µL.
4. Withdraw the syringe needle slowly from the injection site.
5. Place each mouse alone in a cage until it has fully recovered from anesthesia and then return it safely to the home cage.
6. To develop an antigen-specific response to C. albicans , challenge the right footpad of the mice with the prepared HK C. albicans via intradermal injection 7 days after the sensitization, as described previously (1 × 10⁷ cells; 40 µL per footpad; repeat steps 2.1-2.5).
7. Harvest skin-draining LNs 7 days after C. albicans sensitization or lesional footpad tissues 24 h after antigen challenge after euthanasia in a CO₂ chamber.

3. Diphtheria toxin-induced dendritic cell depletion in vivo

NOTE: In this study, both Langerin-DTR and CD301b-DTR mice were treated with DT 1 day before and after intradermal sensitization to C. albicans.

1. Prepare a 10 µg/mL solution of DT in PBS.
2. Remove the cap from the needle of a 1 mL insulin syringe, mix the DT, and fill the syringe with the DT.
3. Properly restrain the mice in a head-down position.
4. Disinfect the ventral side of the mice with 70% ethanol.
5. Slowly inject each mouse with 100 µL of 1 µg DT intraperitoneally into the lower left quadrant of the abdomen to deplete specific dendritic cell subsets.
   NOTE: Be careful not to damage organs during the injection.
6. Wait for 5 s; then, slowly remove the needle.

4. Quantitative real-time polymerase chain reaction

1. Place the mouse in a CO₂ chamber until no breathing movement is observed.
2. Disinfect the mouse with 70% ethanol, and cut the lesion from the hind footpad skin into small pieces using forceps and scissors.
3. Completely immerse the sliced tissues in RNA isolation reagent.
4. Homogenize the samples using a tissue homogenizer according to the manufacturer's instructions (2 cycles of 3 min at 30 Hz).
   NOTE: Stainless steel beads were used for tissue lysis in this study.
5. Spin at 10,000 × g for 5 min, 4 °C.
6. Carefully transfer the supernatant to a fresh tube.
7. Isolate total RNA from the lesional skin using a total RNA isolation kit.
8. Determine the RNA concentration using a UV-Vis spectrophotometer.
9. Synthesize cDNA using a reverse transcription kit for quantitative real-time polymerase chain reaction (qPCR).
10. Perform real-time qPCR with the real-time PCR system by monitoring the synthesis of double-stranded DNA during PCR cycles using green fluorescent dye.
    ​NOTE: In this study, the results were normalized to the level of hypoxanthine-guanine phosphoribosyltransferase (Hprt). The primer sequences are listed in Table 1, and the PCR protocol is as follows: initial denaturation at 95 °C for 30 s, amplification for 42 cycles (95 °C for 5 s, 60 °C for 30 s).

5. Cell isolation and flow cytometric analysis

1. After CO₂ euthanasia, dissect the mice using forceps and scissors, carefully exposing and harvesting the footpad-draining LNs called popliteal LNs, located behind the knee.
2. Prepare single-cell suspensions from the footpad-draining LNs of each mouse by filtering the tissues through a 70 µm strainer after homogenization using the plunger of a 3 mL syringe.
3. Wash the cells with PBS and spin at 500 × g for 5 min at 4 °C.
4. Discard the supernatant, and wash the cells with PBS again.
5. Spin at 500 × g for 5 min at 4 °C.
6. Discard the supernatant and resuspend the cells in complete RPMI-10 medium containing 55 µM β-mercaptoethanol, 50 ng/mL phorbol 12-myristate 13-acetate (PMA), and 500 ng/mL ionomycin in a 24 well-plate for T cell stimulation.
7. After 1 h, add 10 µg/mL of brefeldin A and 1000x monensin to the cell suspension and culture for an additional 5 h.
8. Harvest the cells and wash them with fluorescence-activated cell sorting (FACS) buffer.
9. Spin at 500 × g for 5 min at 4 °C and discard the supernatant.
10. Stain the dead cells with a fixable dead cell-staining dye and incubate for 30 min at 4 °C.
11. Wash the samples with FACS buffer and spin them at 500 × g for 5 min at 4 °C.
12. Discard the supernatant, and stain the cells with fluorochrome-conjugated surface marker antibodies and Fc receptor blocker for 30 min at 4 °C.
13. Wash the samples with FACS buffer and spin them at 500 × g for 5 min at 4 °C.
14. Discard the supernatant and resuspend the pelleted cells in fixation and permeabilization solution for 15-20 min at 4 °C for intracellular staining.
15. Wash the samples with 1x washing buffer and spin them at 500 × g for 5 min at 4 °C.
16. Discard the supernatant, and perform intracellular cytokine staining for 30 min at 4 °C.
17. Wash the samples with 1x washing buffer and spin at 500 x g for 5 min at 4 °C.
18. Resuspend the cells in the appropriate volume (200-300 µL) of FACS buffer.
19. Analyze protein expression using flow cytometry.

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

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)