Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1

Summary

The skin is one target tissue of the human pathogen herpes simplex virus type 1 (HSV-1). To explore the invasion route of HSV-1 into tissue, we established an ex vivo infection model of murine epidermal sheets which represent the outermost layer of skin.

Abstract

To enter its human host, herpes simplex virus type 1 (HSV-1) must overcome the barrier of mucosal surfaces, skin, or cornea. HSV-1 targets keratinocytes during initial entry and establishes a primary infection in the epithelium, which is followed by latent infection of neurons. After reactivation, viruses can become evident at mucocutaneous sites that appear as skin vesicles or mucosal ulcers. How HSV-1 invades skin or mucosa and reaches its receptors is poorly understood. To investigate the invasion route of HSV-1 into epidermal tissue at the cellular level, we established an ex vivo infection model of murine epidermis, which represents the site of primary and recurrent infection in skin. The assay includes the preparation of murine skin. The epidermis is separated from the dermis by dispase II treatment. After floating the epidermal sheets on virus-containing medium, the tissue is fixed and infection can be visualized at various times postinfection by staining infected cells with an antibody against the HSV-1 immediate early protein ICP0. ICP0-expressing cells can be observed in the basal keratinocyte layer already at 1.5 hr postinfection. With longer infection times, infected cells are detected in suprabasal layers, indicating that infection is not restricted to the basal keratinocytes, but the virus spreads to other layers in the tissue. Using epidermal sheets of various mouse models, the infection protocol allows determining the involvement of cellular components that contribute to HSV-1 invasion into tissue. In addition, the assay is suitable to test inhibitors in tissue that interfere with the initial entry steps, cell-to-cell spread and virus production. Here, we describe the ex vivo infection protocol in detail and present our results using nectin-1- or HVEM-deficient mice.

Introduction

Herpes simplex virus (HSV) can cause a range of diseases in humans from mild uncomplicated mucocutaneous lesions to life-threatening infections. HSV type 1 (HSV-1) is dominantly associated with orofacial infections and encephalitis, whereas HSV type 2 (HSV-2) more likely causes genital infections¹. While there is significant progress in understanding how HSV enters cells in culture, initiates infection and produces viral progeny, we know little about the viral invasion pathway(s) into tissue at the cellular level². For studies of HSV skin or mucosal infections, mice, rabbits and guinea pigs have been used as animal models. Skin infection was established by intradermal injection or by scratching the skin in the presence of virus, and disease development was correlated with virus production. These methods helped to understand various aspects of disease pathogenesis, and are used to evaluate antiviral drugs. To study HSV infection at the tissue level, organotypic human skin models have been applied. As the rate of infection is restricted in these raft cultures, only a limited number of studies investigating infection, viral spread and the effects of antiviral components have been published^3-6.

In order to characterize cellular determinants that play a role during HSV-1 infection in the intact epithelium, we established a protocol for ex vivo infection studies of murine epidermis⁷. Skin was prepared from newborns or from the tails of adult mice. Since HSV-1 could not infect complete skin samples, which were submerged in virus-containing medium, we separated the epidermis from the dermis by dispase II treatment. After floating of the epidermal sheets on virus-containing medium, infected cells can be visualized in the epidermal basal layer at various times postinfection (p.i.)⁷. To visualize the initiation of infection in individual cells prior to viral replication and virus production, we stained with an antibody against the infected-cell protein 0 (ICP0), which is one of the first proteins expressed during HSV-1 infection. The cellular localization of ICP0 passes through distinct phases during early infection. While ICP0 is present in nuclear foci during an early stage of viral gene expression, relocalization of ICP0 to the cytoplasm indicates a subsequent phase of infection⁸.

We used the ex vivo infection assay of epidermal sheets from different mouse models to test the potential role of various cellular factors during infection. To address the impact of Rac1 as a key regulator of actin dynamics, we infected the epidermis of mice with a keratinocyte-specific deletion of the rac1 gene⁹. This model allowed us to study the consequences of deficient Rac1 on the efficiency of HSV-1 infection in epidermal keratinocyte layers. The comparison to infected epidermis of control littermates revealed no significant difference, indicating that the absence of Rac1 had no effect on the initiation of infection in the basal layer of the epidermis⁷. The use of further mouse models allowed us to address which cellular receptors mediate entry into the epidermis. Infecting epidermal sheets from either nectin-1- or HVEM-deficient mice with HSV-1 revealed that the initial viral entry into tissue strongly depends on the presence of nectin-1¹⁰. Furthermore, our results demonstrate that HVEM can also serve as receptor in murine epidermis, although less efficiently than nectin-1¹⁰.

To address the spatial distribution of infected cells in the epidermal layers, we visualize ICP0 expression in tissue sections and epidermal whole mounts (Figure 1). In cryosections of complete skin, no ICP0-expressing cells are detected (Figure 1). In contrast, cryosections of epidermal sheets demonstrate cytoplasmic ICP0 expression in the basal layer already at 3 hours p.i. (Figure 1). At later times, viral spreading to suprabasal layers can be visualized. The spatial distribution of infected cells in the basal layer can be easily followed in epidermal whole mounts (Figure 1). Upon infection with HSV-1 at 100 PFU/cell, approximately 50% of the basal keratinocytes in the interfollicular epidermis show ICP0 expression at 1.5 hr p.i. At this time point most infected cells express nuclear ICP0. The relocalization of ICP0 to the cytoplasm indicating a later stage of early gene expression is present in nearly all cells at 3 hr p.i. (Figure 1). These modes of visualizing infected cells upon ex vivo HSV-1 infection provide a powerful assay to study the effect of inhibitors or of deleted/mutated cellular components on viral entry and spread in tissue.

Protocol

Ethics statement.

The preparation of epidermal sheets from sacrificed animals is carried out in strict accordance with the recommendations of the Guide of Landesamt für Natur, Umwelt and Verbraucherschutz, Northrhine-Westphalia (Germany). The study was approved by LANUV NRW (Number 8.84-02.05.20.13.018).

1. Preparation of Instruments and Culture Media

1. Cultivate epidermal sheets in Dulbecco’s modified Eagle’s medium (DMEM)/high glucose containing glutamine dipeptide, 10% FCS, 100 IU/ml penicillin, and 100 µg/ml streptomycin (hereinafter referred to as “DMEM”).
2. For the preparation of epidermal sheets, carefully dissolve dispase II powder (5 mg/ml) in heated PBS (up to 37 °C). Filter the solution through a 0.22 µm filter and use it directly for treatment.
3. For the preparation of epidermal whole mounts¹³, prepare blocking buffer with 0.5% milk powder, 0.25% gelatin from cold water fish skin and 0.5% Triton X-100 in TBS. Allow substances to dissolve by incubating the mixture for at least 2 hr at RT on a rotating mixer. Also prepare 0.2% Tween 20 in PBS as washing buffer. For fixation, prepare 3.4% and 4% formaldehyde in PBS.
4. For the immunostaining of cryosections, prepare blocking buffer with 5% normal goat serum and 0.2% Tween 20 in PBS. For fixation, prepare 0.5% formaldehyde in PBS.

2. Preparation of Epidermal Sheets from Murine Skin

1. Preparation of epidermis from newborn back skin for infection studies
   1. Place a decapitated mouse (1 to 3 days after birth) into a dissection dish and cut off extremities and the tail close to the torso to allow efficient removal of the skin from the complete torso. During removal of the extremities, try to keep the cuts as small as possible in diameter.
   2. Fix the torso with curved fine forceps and gently move blunt tip scissors underneath the skin from cranial to caudal along the back to separate the skin from the torso. Slit the skin along the back.
   3. For FACS analysis, isolation of keratinocytes or preparation of RNA, peel off the complete skin from the torso using forceps to fix the tissue and the blunt tip scissors to push off the torso. For the preparation of cryosections or whole mounts take only pieces of the back skin.
   4. Chop the back skin with a scalpel into 1-2 pieces of approximately 10 x 10 mm. Put the pieces in one well of a 6-well plate with ~1 ml sterile PBS. Make sure that the dermal side faces the bottom.
   5. Replace PBS by 1.5 ml dispase II (5 mg/ml PBS) and incubate for either 30 min at 37 °C (for whole mounts) or O/N at 4 °C. Wash 3 times with PBS. Gently remove the epidermis as an intact sheet using one forceps to fix the underlying dermis and the other forceps to lift the epidermis. Use a binocular to make this step easy. Transfer the epidermal sheet into DMEM with its basal side towards the bottom. Use the sheets immediately for infection experiments.
      NOTE: Use the preparation of epidermis from newborn skin mainly for FACS analysis, isolation of keratinocytes or preparation of RNA. Because of its fragility, the preparation of infected sheets for cryosections or whole mounts is less suitable.
2. Preparation of epidermis from adult tail skin for infection studies
   1. Euthanize the mice at the age of 1 to 3 months by cervical dislocation. Take tail instead of back skin since the long embedded hair follicles on the back hamper separation of intact epidermal sheets. Cut off the tail from sacrificed mice and slit it lengthwise with a scalpel.
   2. Peel off the skin from the bone and chop it with a scalpel into pieces of about 5 x 5 mm. Put up to 4 pieces in one well and proceed with the incubation in dispase II as described under 2.1.5
      NOTE: Use adult tail skin to prepare cryosections or whole mounts. Alternatively to the immediate use, tails can be stored or transported for approximately 24 hr at 4 °C without losing significant infection efficiency. If tails are kept up to 48 hr, epidermal sheets could be hardly infected with HSV-1.
3. Dissociation of epidermis for FACS analysis
   NOTE: Detection of surface protein expression by FACS analysis requires appropriate cell dissociation techniques that do not harm the cell surface and the protein of interest.
   1. Incubate newborn epidermal sheets in a 6-well plate with the basal side on 1.5 ml/well recombinant trypsin-based cell dissociation solution for 15 min at RT and for 15 min at 37 °C.
   2. Stop the incubation by adding 3 ml DMEM. Dissociate keratinocytes by using a curved fine forceps to gently move the epidermis in the 6-well plate so that the keratinocytes get dissolved. Collect the cell suspension and repeat this step 3 times always using fresh DMEM.
      NOTE: This dissociation procedure is appropriate to detect nectin-1 on the surface of keratinocytes.
   3. Alternatively, incubate the epidermal sheets on enzyme-free cell dissociation solution (CDS) for 15 min at RT and 15 min at 37 °C. Incubate another 15 min at RT to gently flush out the keratinocytes by pipetting CDS up and down.
   4. Collect the cell suspension and repeat the flushing step 3 times using fresh DMEM.
      NOTE: This dissociation is suitable to detect surface expression of HVEM. The disadvantage of the CDS solution is that fewer cells are dissociated than with recombinant trypsin-based cell dissociation solution. In addition to surface expression, nuclear or cytoplasmic expression such as ICP0 expression can be analyzed by FACS after dissociation of the infected epidermal sheets with recombinant trypsin-based cell dissociation solution.
4. Dissociation of epidermis to isolate keratinocytes or extract RNA
   1. Put newborn epidermal sheets in a 6-well plate with the basal side towards the bottom of the dish which contains 1.5 ml/well recombinant trypsin-based cell dissociation solution. Incubate for 30 min at RT. Add 3 ml DMEM and dissociate keratinocytes by using a curved fine forceps to gently move the epidermis in the dish.
   2. Collect the cell suspension and repeat this step 3 times using fresh DMEM. Use this cell suspension to extract RNA or to culture primary murine keratinocytes.

3. Infection of Epidermal Sheets with HSV-1

1. Prepare a solution of HSV-1 wt strain 17¹¹ in not less than 500 µl DMEM and replace the medium by the virus solution on which the epidermal sheets are floating. This time point is defined as time 0¹². Perform infection of an epidermal sheet in one well of a 24-well plate at 37 °C. The calculation of the virus titer is based on the estimated number of cells in the basal layer per epidermal sheet (approximately 2 x 10⁵ cells per 5 x 5 mm sheet).
2. Infect with HSV-1 at approximately 100 plaque forming units (PFU)/cell and replace the virus-containing medium by DMEM at 1 hr p.i.  
   NOTE: In case of epidermal sheets from newborn skin, keep in mind that sheets start to dissociate during incubation.

4. Visualization of Infected Cells

1. Epidermal Whole Mounts¹³
   1. Fix epidermal sheets with 3.4% formaldehyde either for 2 hr at RT or O/N at 4 °C. Wash two times with PBS and block with 0.5% milk powder, 0.25% gelatin from cold water fish skin and 0.5% Triton X-100 in TBS for 1 hr at RT¹⁴.
   2. Incubate the sheets O/N with mouse anti-ICP0 (monoclonal antibody 11060)¹⁵ diluted 1:60 in blocking buffer at RT. To visualize the intermediate filament network incubate simultaneously with rabbit polyclonal anti-mouse keratin 14 (100 ng/ml).
   3. Wash 4 to 5 times with PBS-0.2% Tween 20 during 4 hr at RT. Incubate O/N with AF488-conjugated anti-mouse IgG (1 µg/ml), AF555-conjugated anti-rabbit IgG (1 µg/ml), and DAPI (4’, 6-diamidino-2-phenylindole) (100 ng/ml) in blocking buffer at RT.
   4. Wash with PBS-0.2% Tween 20 for 4 hr at RT. Mount epidermal sheets with their basal side on top of a specimen slide, embed in fluorescent mounting medium and cover with coverslips.
2. Epidermal Cryosections
   1. Adult epidermal sheets were fixed in 4% formaldehyde in PBS for 20 min at RT and washed 2 times with PBS. Embed fixed sheets in tissue freezing medium and freeze in liquid nitrogen. Cut 8-10 µm sections with a cryomicrotome.
   2. Fix the sections again with 0.5% formaldehyde in PBS for 10 min at RT, wash 2 times with PBS, block with 5% normal goat serum and 0.2% Tween 20 in PBS for 30 min at RT, and then stain for 60 min with mouse anti-ICP0 (monoclonal antibody 11060)¹⁵ diluted 1:60 in blocking buffer, followed by 3 times washing with the blocking buffer.
   3. Then incubate with AF488-conjugated anti-mouse IgG (1 µg/ml) and DAPI (100 ng/ml) in blocking buffer for 45 min at RT and wash 3 times. Embed sections in fluorescent mounting medium and cover with coverslips. 

Results

The challenge of the method is to prepare epidermal sheets into which HSV-1 can penetrate from the basal layer. The critical step is the separation of the epidermis from the dermis by dispase II treatment, which, depending on the mouse strain, needs to be adapted. The concentration of dispase II can range from 2.5 to 5 mg/ml, and the time of incubation from 20 to 45 min. The staining of the intermediate filament protein keratin 14 readily allows predicting whether the basal epidermal layer can be infected or whether it w...

Discussion

When epidermal sheets of adult skin from C57BL/6 are infected with HSV-1 at approximately 100 PFU/cell, we observe infection in nearly all cells of the basal layer in the interfollicular epidermis while lower virus doses correlate with less infected cells and a slower progress of infection. In general, hair follicles show a variable number of infected cells; while most of the rather small keratinocytes lining the developing hair follicles are infected, only the hair germ of the adult hair follicle is completely infected....

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
DMEM/high glucose/GlutaMAX	Life Technologies	31966047	needed for cultivation of epidermal sheets
dispase II powder	Roche	4942078001	has to be solved in heated PBS
enzyme-free cell dissociation solution	Sigma	C5914	needed for very gentle dissociation of epidermal sheets
TrypLE select cell dissociation solution	Life Technologies	12563-029	needed for dissociation of epidermal sheets
chelex 100 resin	Bio-Rad	142-2832	needed for chelation of polyvalent metal ions from the fetal calf serum
gelatin from cold water fish skin	Sigma	G7765	needed for minimization of non-specific antibody binding
Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)	Covance	PRB-155P	used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis
Mouse IgG (H+L) Secondary Antibody, Alexa Fluor 488 conjugate (conc.: 2 mg/ml)	Life Technologies	A-11029	used as secondary antibodies
Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 555 conjugate (conc.: 2 mg/ml)	Life Technologies	A-21429	used as secondary antibodies
4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)                             (conc.: 0.1 mg/ml)	Sigma	36670	used to counterstain the nucleus