Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Summary

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Abstract

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Introduction

The human body is covered with an extremely high number of microorganisms. The skin surface is the habitat of almost one million bacteria per square centimeter¹. This number, however, does not reflect the full variety of microorganisms that colonizes the skin. In addition to bacteria, the human body is colonized by many fungal species, including C. albicans, which is able to survive both at the mucosal and the skin level².

In recent years, the percentage of people diagnosed with fungal infections has enormously increased. This is mainly due to the higher number of immunocompromised pe....

Protocol

All the procedures were approved under the Institutional Animal Care and Use Committee (IACUC) and operated under the supervision of the department of Animal Resources at Children's Hospital (ARCH) at Boston Children's Hospital or were approved by the Italian Ministry of Health and performed under the Institutional Animal Care Committee at the University of Milano-Bicocca.

1. C. albicans Preparation

1. Culture C. albicans, strain CAF3-1¹³, in tubes containing rich medium (yeast extract peptone dextrose (YEPD), 1% (w/v) yeast extract, 2% (w/v) of Bacto Peptone, 2% (w/v) glucose) supp....

Results

Through the injection of the pathogen directly in the deep derma, the structural morphology of the tissue remains intact (Figure 1A).

The maintenance of skin structural integrity allows the detection of immune cells and their localization at the site of infection. The higher magnification shown in Figure 1B reveals that the abscess is mainly composed of polymorphonuclea.......

Discussion

Here we described a method of C. albicans infection to study the inflammatory process that is initiated upon fungal entrance in the deep derma.

Even though skin abscess formation is a relatively rare event upon C. albicans infection¹⁵, injection of the fungus directly in the deep derma allows not only the study of fungal-driven abscess formation, but also the analysis of specific immune cells that participate to contain fungal spread. With the method d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents
PBS	Euroclone	ECB4053L	warm in 37 °C bath before use
H-OCT compound	histo-line laboratories	R0030
Gill's Hematoxilyn	histo-line laboratories	09-178-2
Eosin Y solution, alcoholic	histo-line laboratories	09-209-05
Ethanol absolute	scharlau	ET00232500
Citro-HISTOCLEAR	histo-line laboratories	R0050
Eukitt, mounting medium	bio-optica
Acetone	sigma-aldrich	179124
PAS staining system	sigma-aldrich	395B-1KT
Bacto Peptone	BD	211677
Bacto Yeast Extract	BD	212750
D(+)-Glucose anhydrous for molecular biology	Applichem PanReac	50-99-7
Uridine	Merck Millipore	8451
HEPES	Applichem PanReac	A1070,0500
Safe-Lock tubes 2 mL	eppendorf	30121597
TRIzol Reagent	Life Technologies	15596018	Toxic, corrosive and mutagen. Use all precaution needed
Rneasy Mini Kit	QIAGEN	74104
liquid nitrogen			Wear eye protection
Instrument
Coulter Counter-Particle Count	Beckman Coulter
Centrifuge 5415 R	eppendorf
MC 3000 Microtome Cryostat	histo-line laboratories
TissueLiser	QIAGEN
Materials
0.3 ml Insulin Syringe with a 30G x 8mm needle	BD	324826
Surgical forceps
Surgical scissors
Base mould disposable	histo-line laboratories	2781
Positively charged bio microscope slides	bio-optica	09-2000
Cover slips 24 x 50 mm	thermo scientific	11911998