参见作者原研究论文

本实验方案简略版
Oct 2021

本文章节


 

Ex vivo Human Skin Infection with Herpes Simplex Virus 1
人皮肤单纯疱疹病毒1的体外感染   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Although herpes simplex virus 1 (HSV-1) is a well-studied virus, how the virus invades its human host via skin and mucosa to reach its receptors and initiate infection remains an open question. For studies of HSV-1 infection in skin, mice have been used as animal models. Murine skin infection can be induced after injection or scratching of the skin, which provides insights into disease pathogenesis but is clearly distinct from the natural entry route in human tissue. To explore the invasion route of HSV-1 on the tissue level, we established an ex vivo infection assay using skin explants. Here, we detail a protocol allowing the investigation of how the virus overcomes mechanical barriers in human skin to penetrate in keratinocytes and dermal fibroblasts. The protocol includes the preparation of total skin samples, skin shaves, and of separated epidermis and dermis, which is followed by incubation in virus suspension. The ex vivo infection assay allows the visualization, quantification, and characterization of single infected cells in the epidermis and dermis prior to viral replication and the virus-induced tissue damage. Hence, this experimental approach enables the identification of primary viral entry portals.


Graphical abstract:




Keywords: Human skin, Human epidermis, Human dermis, Herpes simplex virus 1, Ex vivo infection, Viral entry, ICP0

Background

Herpes simplex virus 1 (HSV-1) is one of the most prevalent human pathogens. During primary infection, the virus invades skin and mucocutaneous sites, which is followed by the latent infection of sensory neurons. Upon reactivation, HSV-1 travels back to near the initial infection site and can cause lesions. As the skin and mucosa form a protective barrier to infection, the general assumption is that the virus infects its host organism via epithelial breaks due to mechanical injuries or pathophysiological modifications. Viral replication largely takes place in the epithelium, and the extent of productive infection is restricted by the immune response. How HSV-1 enters single cells in culture is extensively studied; however, we know less about the viral invasion in human skin and mucosa to initiate infection in skin cells. Thus, the conditions under which the virus overcomes the epithelial barriers, either during primary or recurrent infection, and gains access to its receptors on the skin cells in vivo, are yet to be elucidated.


To address HSV-1 infection in tissue, initial studies were performed in 3-dimensional (3D) skin cultures giving first insights into how the virus may spread and how efficiently differentiating human keratinocytes can be infected (Syrjänen et al., 1997; Visalli et al., 1997; Hukkanen et al., 1999). More recently, human mucosa and skin explants have been used as ex vivo HSV-1 infection models, which allow studying various steps of viral infection much closer to the in vivo situation than 3D cultures or animal models. To analyze the determinants of viral invasion, we ex vivo infected human oral mucosa and detected no infected cells in complete mucosa samples but only invasion in the basal epithelial layer once the connective tissue and the basement membrane were removed (Thier et al., 2017). Intriguingly, mechanical wounding of the mucosa samples is insufficient for HSV-1 to overcome the epithelial barriers (Thier et al., 2017). However, successful HSV-1 infection was observed in human abdominal skin after ex vivo infection of microneedle-pretreated skin, thus providing a model to study the efficiency of antiviral drugs (Tajpara et al., 2018). Tajpara and colleagues (2018) infected thin dermatome-cut skin with lesions partly penetrating both epidermis and dermis and showed the pathogenesis of HSV-1 4 days after infection. The assumption is that viral invasion occurs via the sample edges and at those lesions that cross the skin sample, which, in turn, leads to extensive tissue damage over time. To analyze the initial sites of HSV1 invasion in human skin upon wounding and address the susceptibility of the epidermal layers and the dermis, we developed the protocol described here (De La Cruz et al., 2021). The procedure is based on our ex vivo infection studies in murine skin and human oral mucosa (Rahn et al., 2015a, 2015b; Thier et al., 2017; Wirtz et al., 2020; De La Cruz and Knebel-Mörsdorf, 2021). Our focus is on the early events of infection to understand how the virus overcomes the distinct epidermal barriers, identify which cells are initially infected, and determine how the virus spreads in tissue. As murine and human total skin samples are protected against ex vivo infection from the apical site confirming the protective barrier of healthy skin (Rahn et al., 2015b; De La Cruz et al., 2021), we dissected the skin samples to investigate how susceptible the epidermis and dermis are. After separation of the dermis from the epidermis by dispase II treatment, the basement membrane is disrupted, and remaining components are only occasionally found on the apical site of the dermis and the basal layer of the epidermis. Infection of the separated human epidermis with high virus dose leads to infection of basal and suprabasal keratinocytes, which results in cytopathic effects at 24 h postinfection (p.i.) (De La Cruz et al., 2021). The onset of viral infection in single basal keratinocytes and viral spreading in suprabasal layers can be visualized by immunostaining of very early expressed viral genes such as ICP0 and ICP4. Alternatively, virus particles could be stained or tagged HSV-1 strains could be used for infection. The challenge is to visualize single particles in tissue prior to viral gene expression, to differentiate between noninfectious and infectious particles, and to localize particles in- or outside of cells. As the human epidermis is highly susceptible to HSV 1, virus titers can be determined at various times p.i. In contrast to the epidermis, the infection protocol results only in single infected fibroblasts in the human dermis even at 24 h p.i. (De La Cruz et al., 2021).


In addition, the skin samples could be mechanically wounded or stimulated with cytokines prior to ex vivo infection. Further modifications of the skin surface could be achieved by pre-incubation with bacterial or viral pathogens.


Our infection protocol is most suitable for investigating the early steps of HSV-1 infection in tissue. These include the interaction with epidermal barriers and with components of the extracellular matrix, the role of cellular receptors that allow viral penetration, and the cellular response to initial infection. The dissection of the human skin in skin shaves, epidermis, and dermis offers various experimental tools to address questions from different perspectives.

Materials and Reagents

  1. Feather® sterile disposable scalpels, stainless steel blade with plastic handles (blade No. 21)

  2. Suture/surgical scissors (from any qualified supplier)

  3. Filtered micropipette tips (Sapphire, various sizes, e.g., 10 μL, 300 μL, and 1,250 μL, Greiner, catalog numbers: 772363, 738265, 750265)

  4. 5 and 10 mL serological pipettes (Greiner, catalog number: 606 180)

  5. 50 mL conical centrifuge tubes (Greiner, catalog number: 227261)

  6. Syringe (various sizes, e.g., 5–20 mL from any qualified supplier)

  7. Millex® disposable syringe filter units, disposable 0.22 μm pore size (Merck Chemicals, catalog number: SLGP033RK)

  8. 40 μm cell strainers (Corning, catalog number: 352340)

  9. Dispase II (Roche, catalog number 4942078001), stored at 4°C

  10. Tissue culture plates, 24-well, flat bottom (Corning Incorporated, catalog number: 3524)

  11. Tissue culture plates, 6-well, flat bottom (Corning Incorporated, catalog number: 3516)

  12. DMEM-high glucose-GlutaMAX (GIBCO, catalog number: 10566016), stored at 4°C

  13. Fetal calf serum (same batch of FCS used for a complete series of experiments to avoid variable effects of FCS components)

  14. Penicillin/streptomycin solution (from any qualified supplier)

  15. formaldehyde solution (37%) (Sigma-Aldrich, catalog number: 8187081000), store at RT, prepare working solution freshly

  16. Peel-A-Way embedding Molds, truncated-T8 (Polysciences, catalog number: 18985-1)

  17. Tissue embedding and processing cassettes (from any qualified supplier)

  18. Sakura FinetekTM Tissue-TekTM O.C.T. Compound (ThermoFisher Scientific, catalog number: 12351753)

  19. Paraffin No. 3/6/9 (Richard-Allan-Scientific, ThermoFisher Scientific, catalog number: 8335)

  20. phosphate buffered saline (PBS, sterile) (from any qualified supplier)

  21. Korsolex® basic (BODE Chemie, catalog number: 0022014) for instrument disinfection

  22. Wt HSV-1 (Glasgow strain 17+); purified from the supernatant of infected BHK cells via density gradient centrifugation (5–15% ficoll gradients); virus titer determined by plaque assays in Vero-B4 cells (see Grosche et al., 2019)

  23. whole-skin dissociation kit (Miltenyi biotec, catalog number: 130-101-540)

  24. DPX mounting medium (Sigma-Aldrich, catalog number: 06522)

  25. TryplE Select (GIBCO, catalog number: 12563029)

  26. Cell Dissociation Solution Non-enzymatic 1× (Sigma-Aldrich, catalog number: C5914)

  27. Cell culture medium (see Recipes)

  28. Epidermal cryosection blocking buffer (see Recipes)

  29. Dermal and total (full-thickness) skin cryosection blocking buffer (see Recipes)

  30. Dispase II solution (see Recipes)

  31. Whole Mount Blocking Buffer (see Recipes)

  32. PBS-Tween (see Recipes)

Equipment

  1. Precision forceps (curved and straight) (from any qualified supplier)

  2. 5% CO2 incubator, set at 37°C

  3. Paraffin microtome

  4. Cryostat

  5. Class II biosafety cabinet

  6. Fume hood

  7. FACS machine (Canto II BD Biosciences)

Software

  1. ImageJ-win64 (open source; Schneider et al., 2012)

  2. FacsDIVA v6.1.3 (BD)

  3. FlowJo v7.6.3 (Tree Star)

Procedure

Preparation and ex vivo infection of human tissue

  1. Preparation and ex vivo infection of total (full-thickness) human skin (Figures 1, 2)

    1. Directly after surgery, store and transport intact skin biopsies in DMEM-high glucose-GlutaMAX containing 10% FCS and 1% penicillin/streptomycin at RT. Avoid long storage times. Otherwise, store biopsies at 4°C until ready to use.

    2. Place skin biopsies in a clean working surface Class II biosafety cabinet and carefully remove subcutaneous fat using forceps and surgical scissors (Figure 1a).

    3. Once cleaned of fat and underlying dermis is visible, use a scalpel (#21) to cut intact skin into 5 × 5 mm pieces (Figure 1b).



      Figure 1. Preparation of (full-thickness) human skin (a, b) and skin shaves (c).


    4. Wash total (full-thickness) skin pieces 3× with PBS and place (1 per well) in a 24-well tissue culture plate for infection.

    5. Prepare virus suspension using filter tips in 1 mL of DMEM-high glucose-GlutaMAX containing 10% FCS and 1% penicillin/streptomycin with MOI of interest (we suggest using 10–100 PFU/cell). The calculation of virus dose can be determined by the approximate cell-count of the surface area of a 5 × 5 mm full-thickness skin (approximately 2.5 × 105 cells).

    6. Submerge the total (full-thickness) skin in virus-containing medium (1 mL) for infection.

    7. Infect for the desired amount of time/time-points (max. 24 h) in a 5% CO2 incubator, set at 37°C.

    8. At the desired time-point(s), remove the virus suspension using filter tips and wash once with PBS.

    9. Immediately embed in Truncated T8 Peel-A-Way embedding Molds containing Tissue-Tek O.C.T. compound and freeze at -80°C for preparation as cryosections.

    10. Alternatively, fix in 3.4% formaldehyde directly in the tissue culture plates for 2 h at RT or overnight at 4°C for preparation as paraffin samples.

    11. For whole mount analysis, follow steps C3–C8. Exception: avoid incubation at 37°C (see Note 5).



    Figure 2. Preparation and ex vivo infection of total (full-thickness) human skin.

    (a) Schematic illustration of the procedure described in A. HE-stained sections prior to infection and 24 h p.i. Scale bars, 50 μm. (b) Immunostainings of cryosections visualize infected (viral ICP0-expressing) (green) cells only at the sample edge at 16 h after infection at 100 PFU/cell. Collagen VII (colVII) (red) depicts the basement membrane, and DAPI (blue) serves as a nuclear counterstain. Transmission light (TL) visualizes an edge and middle part of a skin sample. Scale bars, 50 μm.


  2. Preparation and ex vivo infection of skin shaves (Figures 1, 3)

    1. Follow the above steps A1–A3.

    2. Use skin samples with a size of at least 15 cm2.

    3. Wrap the skin around your index finger to stretch the skin and, using a scalpel, shave off the apical part including the epidermis and the underlying apical part of the dermis. Take shaves of approximately 1 mm thickness (Figure 1c).

    4. Trim shaves into 3 × 3 mm pieces.

    5. Proceed with steps described in A4–A9.



    Figure 3. Preparation and ex vivo infection of skin shaves.

    (a) Schematic illustration of the procedure described in B. HE-stained sections prior to infection. Scale bar, 50 μm. (b) Immunostainings of cryosections show the middle part of a skin shave. Transmission light (TL) visualizes the morphology, collagen VII (colVII) (red) depicts the basement membrane, and DAPI (blue) serves as a nuclear counterstain. Scale bar, 50 μm.


  3. Preparation and ex vivo infection of isolated human epidermis (Figure 4)

    1. Follow the above steps A1–A3.

    2. Place total (full-thickness) skin pieces in 6-well plates (approximately 10–15 pieces can fit in one well) and wash 3× with PBS.

    3. Using a precision scale, weigh 4 U/mL dispase II in a 50 mL conical centrifuge tube and dissolve in PBS by vortexing.

    4. Prepare a new 50 mL centrifuge tube and filter sterilize the dispase II solution using a 0.20 μm syringe filter.

    5. Place 2 mL of the dispase II solution per well in a 6-well plate containing total skin pieces so that the skin pieces are floating in solution (ensure that the epidermis is facing up).

    6. Incubate overnight at 4°C.

    7. Wash the dispase II-treated skin pieces 3× with PBS.

    8. Using two forceps, gently peel off the epidermis from dermis by starting at the corners of the skin. If met with resistance, incubate samples in dispase II solution for an additional 15 minutes at 37°C and repeat if necessary (see video Rahn et al., 2015a).

    9. Place isolated epidermal samples in 1 mL of PBS in 24-well tissue culture plates while virus suspension is prepared.

    10. Prepare virus suspension in DMEM-high glucose-GlutaMAX containing 10% FCS and 1% penicillin/streptomycin with MOI of interest. The calculation of virus dose can be determined by the approximate cell-count of the basal layer of a 5 × 5 mm epidermal sample (approx. 3 × 105 cells).

    11. Replace the PBS in the wells containing the epidermal samples with virus-containing medium (1 mL). The epidermal sheets should be floating with the basal side facing down.

    12. Infect for the desired amount of time/time-points in a 5% CO2 incubator, set at 37°C.

    13. At the desired time-point(s), remove the virus suspension and wash once with PBS.

    14. Immediately embed in Tissue-Tek O.C.T. compound and freeze at -80°C for preparation as cryosections.

    15. Alternatively, fix piece in 3.4% formaldehyde for 2 h at RT or overnight at 4°C for preparation as paraffin samples or as whole mounts.



    Figure 4. Preparation and ex vivo infection of isolated human epidermis.

    (a) Schematic illustration of the procedure described in C. HE-stained sections prior to infection and 24 h p.i. Scale bars, 25 μm. (b) After infection at 100 PFU/cell for 6 h, immunostaining of epidermal whole mount visualizes infected cells with nuclear ICP0 punctae (green) and some cells with cytoplasmic ICP0 (green) in the basal layer. Immunostainings of cryosections show the epidermis with ICP0-expressing (green) cells at 6 h p.i. and no infected cells after mock-infection. DAPI (blue) serves as a nuclear counterstain, and loricrin (lor) (red) mainly depicts the granular layer. Scale bars, 50 μm.


  4. Preparation and ex vivo infection of isolated human dermis (Figure 5)

    1. Follow the above steps C1–C8.

    2. Place isolated dermal samples in 1 mL of PBS in 24-well tissue culture plates while virus suspension is prepared.

    3. Prepare 1 mL of virus suspension in DMEM-high glucose-GlutaMAX containing 10% FCS and 1% penicillin/streptomycin with MOI of interest. The calculation of virus dose can be determined by the approximate cell-count in the surface area of a 5 × 5 mm dermis (approx. 2 × 105 cells).

    4. Infect for the desired amount of time/time points in a 5% CO2 incubator, set at 37°C.

    5. At the desired time point(s), remove the virus suspension and wash once with PBS.

    6. Immediately embed in Tissue-Tek O.C.T. compound and freeze at -80°C for preparation as cryosections or whole mount.

    7. Alternatively, fix piece in 3.4% formaldehyde for 2h at RT or overnight at 4°C for preparation as paraffin samples.



    Figure 5. Preparation and ex vivo infection isolated human dermis.

    (a) Schematic illustration of the procedure described in D. HE-stained sections prior to infection and 24 h p.i. Scale bars, 50 μm. (b) After infection at 50 PFU/cell, immunostainings of cryosections visualize only some ICP0-expressing cells (green) at 24 h p.i. with DAPI (blue) as a nuclear counterstain. Transmission light (TL) shows the morphology. Scale bars, 50 μm.

Postinfection procedures

Histological Analyses

  1. Preparation of paraffin-embedded tissue samples for Hematoxylin and Eosin (HE) staining of paraffin sections

    1. After the tissue is fixed in 3.4% formaldehyde, wash samples with PBS and store in tissue embedding and processing cassettes in a 70% ethanol solution.

    2. Once all samples of interest are fixed and stored in 70% ethanol solution, proceed with the infiltration of samples in paraffin.

    3. Embed samples in embedding molds.

    4. Prepare 8 µm thick sections at a microtome with the water bath at 42–45°C.

    5. Prior to staining, de-paraffinize samples and stain HE accordingly.

    6. Embed samples with glass coverslips using DPX mounting medium and allow to dry overnight under a fume hood.

    7. Acquire transmitted light images of HE-stained samples in a light microscope.


Visualization of infected cells

  1. Immunohistochemistry – cryosections

    1. Caution: cryo samples are not fixed!

    2. Prepare 8 µm thick sections at a cryostat 3050 (Leica) at -21°C.

    3. Prepare at least three sections per microscope slide of at least 100 μm distance per sample.

      Visualization of early viral proteins (e.g., ICP0):

    4. Fix in 1–2% formaldehyde for 10 min at RT.

    5. Incubate cryosections with epidermal cryosection blocking buffer (for epidermal samples; see recipes) or with dermal/total skin blocking buffer (for dermis, total skin, and shave samples; see recipes) for up to 6 h at room temperature.

      Incubate with primary antibodies of interest (e.g., ICP0, monoclonal antibody 11060; 1:60; Everett et al., 1993) overnight at 4°C (De La Cruz et al., 2021).

    6. Wash 3 × 5 min with the respective blocking buffer.

    7. Incubate with secondary antibodies and 4’,6-diamidino-2-phenylindole (DAPI) for 45 min at RT.

    8. Embed with Dako Fluorescence medium by covering sections with cover slides.

    9. Acquire images at a confocal microscope (Leica DM IRBE with TCS-SP5 device).


  2. Immunohistochemistry – whole mounts

    1. Wash FA-fixed epidermal whole mount samples with PBS.

    2. Using a 24-well plate, incubate epidermal sheets (with the basal side facing down) with whole mount blocking buffer (see Recipe 5) for 1 h at room temperature.

    3. Incubate with primary antibodies of interest (e.g., ICP0 1:60) overnight at room temperature, shaking.

    4. Wash for at least 4× 45 min with PBS-Tween (see Recipe 6).

    5. Incubate with secondary antibodies and 4’,6-diamidino-2-phenylindole (DAPI) overnight at 4°C.

    6. Wash again for at least 4× 45 min with PBS-Tween (see Recipe 6)

    7. For embedding, put epidermal sheets with the basal side facing up on slides and embed with Dako Fluorescence medium by covering the epidermis with cover slides. Use a dissection microscope during mounting if it is difficult to distinguish the basal from the apical side.


  3. Cell dissociation for the determination of receptor surface expression (before infection) via flow cytometry

    1. Nectin-1 surface detection – epidermal cells:

      1. Put epidermal sheets (preparation see C) on TrypLE Select solution for 30 min at RT.

      2. Stop reaction with twice the volume of DMEM and isolate the cells mechanically from the tissue with forceps.

      3. Filter the cells through 40 μm cell strainers and count with counting chambers.

    2. HVEM surface detection – epidermal cells:

      Put epidermal sheets (preparation see C) on enzyme-free dissociation solution for 30 min at RT (see Petermann et al., 2015).

    3. Receptor surface expression – dermal cells:

      1. Digest dermal sheets with whole-skin dissociation kit (Miltenyi) for 2.5 h shaking (180 rpm) at 37°C.

      2. Filter the cells through 40 μm cell strainers and count with counting chambers.

    4. Antibodies:

      1. Anti-nectin-1 (monoclonal antibody CK41; 1:100; Krummenacher et al., 2000). Nectin-1 was visualized with anti-mouse IgG-Cy5 (1:100; Jackson).

      2. anti-HVEM antibody conjugated to phycoerythrin (PE) (CD270-PE, REA247, 1:11; Miltenyi).

Data analysis

  1. Due to the high variation observed between skin samples originating from different patients and/or different body areas, the number of individual samples must not be less than three. Depending on the sample size, it is preferred that the experiments are performed as duplicates (or triplicates if enough sample allows).

  2. Image acquisition:

    1. Images are acquired using a confocal microscope (Leica DM IRBE with TCS-SP5 device).

    2. Mock-infected samples serve as staining control.

    3. Three sections per sample are viewed, and representative areas are captured as z-stack (0.17-0.5 µm stacks) or section.

  3. Analysis of infection efficiency: determined either by cell counting or mean fluorescence intensity measurements (MFI) using ImageJ-win64. Images analyzed for MFI measurements should be acquired with identical microscope settings around the same time.

Notes

  1. In contrast to synchronized HSV-1 infection of cultured cells, the virus-containing medium is not exchanged for fresh medium 1 h p.i., but remains until the end time point is reached. Depending on the skin thickness, its extensions, or other contributing factors, it can take different lengths of time until the virus reaches its target cells. In addition, tissue damage seems to precede virus attachment. Thus, keeping the samples in the virus suspension allows the virus to have access to skin cells at a later time point.

  2. Our infection studies of isolated epidermis were restricted to 24 h, and the samples were directly infected after separation from the dermis. In our experience, longer incubation times could affect the tissue in multiple ways. This includes, but is not restricted to, rearrangement of tight junction complexes, tissue disintegration, and loss of cells (De La Cruz et al., 2021). Mock-infected samples are needed to determine the contribution of these changes to the analyses of HSV-1 infection. To analyze the effects that could be induced by toxins/cytokines, we preincubated complete skin samples for up to 3 days, followed by infection for 24 or 48 h p.i.

  3. To correlate the outcome of infection and the status of the tissue, we always performed HE staining prior to infection and at various times after infection. Moreover, HE staining prior to infection allows the exclusion of skin samples from patient material that exhibit unusual skin characteristics (e.g., too thin or too thick epidermis, etc.) and ensures that our analyses are performed with samples that are at least comparable.

  4. It may sometimes occur that the epidermis and dermis do not separate after dispase II treatment overnight. In these situations, we recommend incubating the samples at 37°C for additional time. Check every 15 min until the epidermis and dermis easily separate and no resistance between them is felt.

  5. To analyze infection patterns in the epidermis after ex vivo infection of total skin, the epidermis can be separated from the dermis by dispase II treatment after infection, and whole mounts can be prepared for staining and visualization of the basal cells (A11). For this approach, incubation with dispase II at 37°C should be avoided to prevent the infection from progressing.

  6. Additionally, virus titers can be determined after infection of epidermal sheets with 1 PFU/cell for 1 hour on ice followed by incubation at 37°C (5% CO2). Medium is refreshed at 1 h p.i. and supernatants are collected at 3 h p.i. representing the input virus. Samples are overlaid once again with fresh, warm medium, and supernatants are collected at 48 h p.i. to determine produced virus. Additional longer or shorter time points can be obtained; however, individual epidermis samples must be prepared for each desired time point (do not use one epidermal sheet to obtain supernatants for multiple time points). To determine the resulting virus titers, traditional plaque assays with the collected supernatants can then be performed.

  7. Use 3% Korsolex for 60 min to disinfect any reusable lab equipment after the infection procedure.

Recipes

  1. Cell culture medium (also used as virus suspension medium)

    DMEM (1×) + GlutaMAX supplemented with

    10% fetal calf serum

    100 μg/mL streptomycin

    100 U/mL penicillin


  2. Epidermal cryosection blocking buffer

    Reagent Final concentration Amount
    Normal goat serum 5% 500 μL
    Tween-20 0.2% 20 μL
    PBS (1×) a.d. 10 mL


  3. Dermal and total (full-thickness) skin cryosection blocking buffer

    Reagent Final concentration Amount
    Milk powder 0.5% 0.25 g
    Cold water fish skin gelatin (prewarmed to 40°C) 0.25% 125 μL
    Triton X-100 0.5% 250 μL
    Normal goat serum 5% 2500 µL
    Bovine serum albumin (10% w/v) 0.1% 500 µL
    PBS a.d. 50 mL


  4. Dispase II solution

    Prepare 4 U/mL freshly in 1× PBS.

    Filter sterilize solution before use.


  5. Whole Mount Blocking Buffer

    Reagent Final concentration Amount
    Milk powder 0.5% 0.25 g
    Cold water fish skin gelatin (prewarmed to 40°C) 0.25% 125 μL
    Triton X-100 0.5% 250 μL
    TBS (1×) a.d. 50 mL


  6. PBS-Tween

    Reagent Final concentration Amount
    Tween-20 0.2% 50 μL
    PBS (1×) a.d. 25 mL

Acknowledgments

This research was funded by the German Research Foundation (KN536/16-3), the Köln Fortune Program/Faculty of Medicine, University of Cologne, and the Maria-Pesch foundation. We thank Wolfram Malter (Dept. of Gynecology, University Hospital Cologne) and Max Zinser (Dept. of Plastic, Reconstructive and Aesthetic Surgery, University Hospital Cologne) for supplying human skin samples. This protocol was used in our previously published work (De La Cruz et al., 2021).

Competing interests

The authors declare no competing interests.

Ethics

Human skin specimens were obtained after informed consent from all patients. The study was approved by the Ethics Commission of the Medical Faculty, University of Cologne (approval no. 17-481).

References

  1. De La Cruz, N. C., Möckel, M., Wirtz, L., Sunaoglu, K., Malter, W., Zinser, M. and Knebel-Mörsdorf, D. (2021). Ex Vivo Infection of Human Skin with Herpes Simplex Virus 1 Reveals Mechanical Wounds as Insufficient Entry Portals via the Skin Surface. J Virol 95(21): e0133821.
  2. Everett, R. D., Cross, A. and Orr, A. (1993). A truncated form of herpes simplex virus type 1 immediate-early protein Vmw110 is expressed in a cell type dependent manner. Virology 197(2): 751-756.
  3. Grosche, L., Döhner, K., Düthorn, A., Hickford-Martinez, A., Steinkasserer, A., and Sodeik, B. (2019) Herpes Simplex Virus Type 1 Propagation, Titration and Single-step Growth Curves. Bio-protocol. 9(23): e3441.
  4. Hukkanen, V., Mikola, H., Nykänen, M. and Syrjänen, S. (1999). Herpes simplex virus type 1 infection has two separate modes of spread in three-dimensional keratinocyte culture. J Gen Virol 80 (Pt 8): 2149-2155.
  5. Krummenacher, C., Baribaud, I., Ponce de Leon, M., Whitbeck, J. C., Lou, H., Cohen, G. H. and Eisenberg, R. J. (2000). Localization of a binding site for herpes simplex virus glycoprotein D on herpesvirus entry mediator C by using antireceptor monoclonal antibodies. J Virol 74(23): 10863-10872.
  6. Petermann, P., Thier, K., Rahn, E., Rixon, F. J., Bloch, W., Özcelik, S., Krummenacher, C., Barron, M. J., Dixon, M. J., Scheu, S., et al. (2015). Entry mechanisms of herpes simplex virus 1 into murine epidermis: involvement of nectin-1 and herpesvirus entry mediator as cellular receptors. J Virol 89(1): 262-274.
  7. Rahn, E., Thier, K., Petermann, P. and Knebel-Mörsdorf, D. (2015a). Ex Vivo Infection of Murine Epidermis with Herpes Simplex Virus Type 1. J Vis Exp (102): e53046.
  8. Rahn, E., Petermann, P., Thier, K., Bloch, W., Morgner, J., Wickström, S. A. and Knebel-Mörsdorf, D. (2015b). Invasion of Herpes Simplex Virus Type 1 into Murine Epidermis: An Ex Vivo Infection Study. J Invest Dermatol 135(12): 3009-3016.
  9. Schneider, C. A., Rasband, W. S. and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat Methods 9(7): 671-675.
  10. Syrjänen, S., Mikola, H., Nykänen, M. and Hukkanen, V. (1996). In vitro establishment of lytic and nonproductive infection by herpes simplex virus type 1 in three-dimensional keratinocyte culture. J Virol 70(9): 6524-6528.
  11. Tajpara, P., Mildner, M., Schmidt, R., Vierhapper, M., Matiasek, J., Popow-Kraupp, T., Schuster, C. and Elbe-Bürger, A. (2019). A Preclinical Model for Studying Herpes Simplex Virus Infection. J Invest Dermatol 139(3): 673-682.
  12. Thier, K., Petermann, P., Rahn, E., Rothamel, D., Bloch, W. and Knebel-Mörsdorf, D. (2017). Mechanical Barriers Restrict Invasion of Herpes Simplex Virus 1 into Human Oral Mucosa. J Virol 91(22).
  13. Visalli, R. J., Courtney, R. J. and Meyers, C. (1997). Infection and replication of herpes simplex virus type 1 in an organotypic epithelial culture system. Virology 230(2): 236-243.
  14. Wirtz, L., Möckel, M. and Knebel-Mörsdorf, D. (2020). Invasion of Herpes Simplex Virus 1 into Murine Dermis: Role of Nectin-1 and Herpesvirus Entry Mediator as Cellular Receptors during Aging. J Virol 94(5).

简介

[摘要]虽然单纯疱疹病毒1(HSV-1)是一种经过充分研究的病毒,但该病毒如何通过皮肤和黏膜侵入其人类宿主,到达其受体并引发感染仍然是一个悬而未决的问题。对于皮肤中 HSV-1 感染的研究,小鼠已被用作动物模型。小鼠皮肤感染可在注射或刮擦皮肤后诱发,这提供了对疾病发病机制的深入了解,但明显不同于人体组织的自然进入途径。为了探索 HSV-1 在组织水平上的侵袭途径,我们建立了使用皮肤外植体的离体感染测定。在这里,我们详细介绍了一个协议,该协议允许研究病毒如何克服人体皮肤中的机械屏障以渗透到角质形成细胞和真皮成纤维细胞中。该协议包括制备总皮肤样本、皮肤剃须以及分离的表皮和真皮,然后在病毒悬浮液中孵育。体外感染测定允许在病毒复制和病毒引起的组织损伤之前对表皮和真皮中的单个感染细胞进行可视化、量化和表征。因此,这种实验方法能够识别主要的病毒进入门户。

图形概要:



[背景] 单纯疱疹病毒 1 (HSV-1) 是最普遍的人类病原体之一。在初次感染期间,病毒侵入皮肤和皮肤黏膜部位,随后潜伏感染感觉神经元。重新激活后,HSV-1 会返回到初始感染部位附近,并可能导致病变。由于皮肤和粘膜形成了对感染的保护屏障,一般的假设是病毒通过机械损伤或病理生理改变引起的上皮破裂感染其宿主生物体。病毒复制主要发生在上皮细胞中,产生感染的程度受到免疫反应的限制。 HSV-1如何进入培养物中的单细胞被广泛研究;然而,我们对病毒侵入人体皮肤和黏膜以引发皮肤细胞感染知之甚少。因此,病毒在原发性或复发性感染期间克服上皮屏障并在体内获得其皮肤细胞受体的条件尚待阐明。
为了解决组织中的 HSV-1 感染问题,在 3 维 (3D) 皮肤培养物中进行了初步研究,初步了解病毒如何传播以及如何有效区分人类角质形成细胞 ( Syrjänen) 等。 , 1997;维萨利 等。 , 1997;胡卡宁 等。 , 1999)。最近,人类黏膜和皮肤外植体已被用作体外HSV-1 感染模型,与 3D 培养或动物模型相比,它允许研究病毒感染的各个步骤更接近体内情况。为了分析病毒入侵的决定因素,我们离体感染了人类口腔黏膜,在完整的黏膜样本中未检测到感染细胞,但在去除结缔组织和基底膜后仅侵入基底上皮层( Thier 等。 , 2017)。有趣的是,粘膜样本的机械损伤不足以让 HSV-1 克服上皮屏障( Thier 等。 , 2017)。然而,在体外感染微针预处理的皮肤后,在人体腹部皮肤中观察到成功的 HSV-1 感染,从而为研究抗病毒药物的有效性提供了模型。 等。 , 2018)。 Tajpara及其同事 (2018) 感染了薄皮切开的皮肤,病灶部分穿透表皮和真皮,并在感染 4 天后显示 HSV-1 的发病机制。假设是病毒入侵通过样本边缘和穿过皮肤样本的那些病变处发生,这反过来又会随着时间的推移导致广泛的组织损伤。为了分析 HSV 1 在受伤时侵入人体皮肤的初始部位并解决表皮层和真皮的易感性,我们开发了此处描述的方案(De La Cruz等人,2021)。该程序基于我们对小鼠皮肤和人类口腔黏膜的离体感染研究 ( Rahn 等。 , 2015a, 2015b;梯尔 等。 , 2017;维尔茨等人。 , 2020; De La Cruz 和Knebel-Mörsdorf ,2021 年)。我们的重点是感染的早期事件,以了解病毒如何克服不同的表皮屏障,确定哪些细胞最初被感染,并确定病毒如何在组织中传播。由于小鼠和人类的总皮肤样本受到保护,免受顶端部位的离体感染,从而证实了健康皮肤的保护屏障 ( Rahn 等。 , 2015b;德拉克鲁兹等人。 , 2021),我们解剖了皮肤样本,以研究表皮和真皮的易感性。通过分散酶II处理将真皮与表皮分离后,基底膜被破坏,仅在真皮的顶端部位和表皮的基底层偶尔发现剩余成分。用高剂量病毒感染分离的人表皮会导致基底和基底上角质形成细胞感染,从而导致感染后24 小时( pi )出现细胞病变效应(De La Cruz et al. , 2021)。通过对非常早期表达的病毒基因(如 ICP0 和 ICP4)进行免疫染色,可以观察到单个基底角质形成细胞中病毒感染的开始和基底层上层的病毒传播。或者,可以对病毒颗粒进行染色或标记 HSV-1 毒株可用于感染。挑战是在病毒基因表达之前可视化组织中的单个粒子,区分非感染性和感染性粒子,并将粒子定位在细胞内或细胞外。由于人类表皮对HSV 1 高度敏感,因此可以在 pi 的不同时间确定病毒滴度. , 2021).
此外,在离体感染之前,皮肤样本可能会受到机械损伤或用细胞因子刺激。皮肤表面的进一步修饰可以通过与细菌或病毒病原体进行预孵育来实现。
我们的感染方案最适合研究组织中 HSV-1 感染的早期步骤。这些包括与表皮屏障和细胞外基质成分的相互作用、允许病毒穿透的细胞受体的作用以及对初始感染的细胞反应。人体皮肤在剃须、表皮和真皮中的解剖提供了各种实验工具,可以从不同的角度解决问题。

关键字



材料和试剂


1.Feather ®无菌一次性手术刀,带塑料手柄的不锈钢刀片(21 号刀片)
2.缝合/手术剪(来自任何合格的供应商)
3.过滤的微量移液器吸头(蓝宝石,各种尺寸,例如10 μL 、300 μL和 1,250 μL ,Greiner,目录号:772363、738265、750265)
4.5和10 mL血清移液管(Greiner,目录号: 606 180)
5.50 mL锥形离心管(Greiner,目录号: 227261 )
6.注射器(各种尺寸,例如,来自任何合格供应商的5 –20 mL )
7.Millex ®一次性注射器过滤器单元,一次性 0.22 μm孔径(Merck Chemicals,目录号: SLGP033RK)
8.40μm细胞过滤器(Corning,目录号: 352340 )
9.Dispase II(Roche,目录号 4942078001),储存于 4°C 
10.组织培养板,24 孔,平底(Corning Incorporated,目录号: 3524) 
11.组织培养板,6孔,平底(Corning Incorporated,目录号: 3516) 
12.高葡萄糖-GlutaMAX ( GIBCO,目录号: 10566016),储存在4°C 
13.胎牛血清(同一批次的 FCS 用于一系列完整的实验,以避免 FCS 成分的可变影响)
14.青霉素/链霉素溶液(来自任何合格的供应商)
15.甲醛溶液(37%)(Sigma-Aldrich,目录号:8187081000),室温储存,新鲜制备工作溶液 
16.Peel-A-Way 嵌入模具,截断-T8( Polysciences ,目录号:18985-1) 
17.组织包埋和处理盒(来自任何合格的供应商)
18.Sakura Finetek TM Tissue - Tek TM OCT 化合物( ThermoFisher Scientific,目录号:12351753)
19.第 3/6/9 号石蜡(Richard-Allan-Scientific, ThermoFisher Scientific,目录号:8335)
20.磷酸盐缓冲盐水(PBS,无菌)(来自任何合格的供应商)
21.Korsolex ® basic(BODE Chemie ,目录号:0022014)用于器械消毒
22.Wt HSV-1(格拉斯哥菌株 17+);通过密度梯度离心(5 – 15% ficoll梯度)从感染的 BHK 细胞的上清液中纯化;通过 Vero-B4 细胞中的噬斑测定确定病毒滴度(参见Grosche 等人,2019)
23.全皮肤分离试剂盒(美天旎 biotec ,目录号:130-101-540)
24.DPX 封固剂(Sigma-Aldrich,目录号: 06522) 
25.TryplE Select(GIBCO,目录号: 12563029) 
26.细胞解离溶液非酶1 × (Sigma-Aldrich,目录号: C5914) 
27.细胞培养基(见食谱) 
28.表皮冷冻切片封闭缓冲液(参见食谱) 
29.真皮和全层(全层)皮肤冷冻切片封闭缓冲液(参见食谱) 
30.Dispase II 溶液(见配方) 
31.Whole Mount Blocking Buffer(见配方) 
32.PBS-Tween(见食谱) 


设备


1.精密镊子(弯的和直的)(来自任何合格的供应商)
2.5% CO 2培养箱,设置在 37°C
3.石蜡切片机
4.低温恒温器
5.II级生物安全柜
6.通风柜
7.FACS机器(Canto II BD Biosciences)


软件


1.ImageJ-win64(开源;Schneider等人,2012)
2.FacsDIVA v6.1.3 (BD)
3.FlowJo v7.6.3 (树星)


程序


人体组织的制备和体外感染


A.总(全层)人体皮肤的制备和体外感染(图 1、2)
1.手术后直接在含有 10% FCS 和 1% 青霉素/链霉素的DMEM - 高葡萄糖 - GlutaMAX中储存和运输完整的皮肤活检。避免长时间存放。否则,将活检保存在 4°C 直至准备使用。
2.将皮肤活检置于干净的工作表面 II 级生物安全柜中 并使用镊子和手术剪小心地去除皮下脂肪(图 1a)。
3.清洁脂肪和可见下面的真皮后,使用手术刀 (#21) 将完整的皮肤切成 5 × 5 毫米件(图 1b)。




图 1. (全层) 人体皮肤 (a, b) 和皮肤剃须 (c) 的制备。


4.清洗总(全层)皮肤片 3倍,并放置(每孔 1 个)在 24 孔组织培养板中进行感染。
5.的 1 mL DMEM-高葡萄糖-GlutaMAX 中使用滤嘴制备病毒悬浮液,并带有感兴趣的 MOI(我们建议使用 10–100 PFU/细胞)。病毒剂量的计算可以通过 5 × 5 mm 全层皮肤(约 2.5 × 10 5 个细胞)表面积的近似细胞计数来确定。
6.将总(全层)皮肤浸入含病毒介质(1 mL)中以进行感染。
7.在 37°C的 5% CO 2培养箱中感染所需的时间/时间点(最长 24 小时) 。
8.在所需的时间点,使用过滤嘴去除病毒悬浮液,并用 PBS 清洗一次。
9.立即嵌入含有 Tissue-Tek OCT 化合物的截断 T8 Peel-A-Way 嵌入模具中,并在 -80°C 下冷冻以制备冷冻切片。
10.或者,将 3.4% 甲醛直接固定在组织培养板中,在室温下 2 小时或在 4°C 下过夜,以制备石蜡样品。
11.对于整体安装分析,请执行步骤 C3 – C8。例外:避免在 37°C 下孵育(见注 5)。




总(全层)人体皮肤的制备和体外感染。 
pi比例尺 (50 μm ) 中描述的程序示意图。 (b)冷冻切片的免疫染色仅在 100 PFU/细胞感染后 16 小时在样品边缘显示受感染的 (病毒 ICP0 表达) (绿色) 细胞。胶原蛋白 VII ( colVII )(红色)描绘基底膜,DAPI(蓝色)用作核复染剂。透射光 (TL) 使皮肤样本的边缘和中间部分可视化。比例尺,50 μm 。  


B.制备和体外感染(图 1、3)
1.按照上述步骤 A1 - A3。
2.使用大小至少为 15 cm 2的皮肤样本。
3.将食指周围的皮肤包裹起来以拉伸皮肤,然后使用手术刀将包括表皮在内的顶端部分和真皮的下面顶端部分剃掉。剃须约 1 毫米厚(图 1c)。
4.修剪 剃成 3 × 3 毫米片。
5.继续执行 A4 – A9中描述的步骤。




图 3.剃须的制备和体外感染。 
(a) 感染前 B. HE 染色切片中描述的程序的示意图。比例尺,50 μm 。 (b) 冷冻切片的免疫染色显示皮肤剃须的中间部分。透射光 (TL) 显示形态,胶原蛋白 VII ( colVII )(红色)描绘基底膜,DAPI(蓝色)用作核复染剂。比例尺,50 μm 。


C.制备和体外感染 (图 4)
1.按照上述步骤 A1 - A3。
2.将全部(全层)皮肤片放入 6 孔板中(大约10-15片可以放入一个孔中)并用 PBS洗涤 3次。
3.使用精密秤,在 50 mL 锥形离心管中称量 4 U/mL分散酶II,并通过涡旋溶解在 PBS 中。
4.准备一个新的 50 mL 离心管并使用 0.20 μm注射器过滤器对分散酶II 溶液进行过滤。
5.将每孔 2 mL 的分散酶II 溶液放入含有总皮肤片的 6 孔板中,使皮肤片漂浮在溶液中(确保表皮朝上)。
6.在 4°C 下孵育过夜。
7.用 PBS清洗分散酶II 处理的皮肤片 3 × 。
8.使用两个镊子,从皮肤的角落开始,轻轻地从真皮剥离表皮。如果遇到阻力,将样品在分散酶II 溶液中在 37°C 下再孵育 15 分钟,必要时重复(参见视频Rahn 等人,2015a)。
9.制备病毒悬浮液时,将分离的表皮样品放入24 孔组织培养板中的 1 mL PBS中。
10.的 DMEM-高葡萄糖-GlutaMAX中制备病毒悬浮液,并带有感兴趣的 MOI。病毒剂量的计算可以通过 5 × 5 mm 表皮样本(约 3 × 10 5 个细胞)的基底层的近似细胞计数来确定。
11.用含病毒介质 (1 mL) 替换含有表皮样本的井中的 PBS。表皮片应漂浮,基底面朝下。
12.在 37°C的 5% CO 2培养箱中感染所需的时间/时间点。
13.在所需的时间点,去除病毒悬浮液并用 PBS 清洗一次。
14.立即嵌入Tissue-Tek OCT 化合物中并在 -80°C 冷冻以制备冷冻切片。
15.或者,在 3.4% 甲醛中在室温下固定 2 小时或在 4°C 下过夜,以制备石蜡样品或整个安装。




图 4.分离的人表皮的制备和体外感染。 
感染前 C. HE 染色切片和 24 小时pi比例尺 (25 μm ) 中所述程序的示意图。 (b) 以 100 PFU/细胞感染 6 小时后,表皮整体免疫染色可显示基底层中具有核 ICP0 点(绿色)的感染细胞和一些具有细胞质 ICP0(绿色)的细胞。冷冻切片的免疫染色显示表皮在 6 小时后具有表达 ICP0(绿色)的细胞,并且在模拟感染后没有感染细胞。 DAPI(蓝色)用作核复染剂,loricrin(lor)(红色)主要描绘颗粒层。 比例尺,50 μm 。


D.制备和体外感染(图 5)
1.按照上述步骤 C1 - C8。
2.制备病毒悬浮液时,将分离的皮肤样本放入24 孔组织培养板中的 1 mL PBS中。
3.在含有 10% FCS 和 1% 青霉素/链霉素的 DMEM-高葡萄糖-GlutaMAX中制备 1 mL 病毒悬浮液,并带有感兴趣的 MOI。病毒剂量的计算可以通过 5 × 5 mm 真皮(约 2 × 10 5 个细胞)表面积中的近似细胞计数来确定。
4.在 37°C的 5% CO 2培养箱中感染所需的时间/时间点。
5.在所需的时间点,去除病毒悬浮液并用 PBS 清洗一次。
6.立即嵌入Tissue-Tek OCT 化合物中,并在 -80°C 下冷冻,以制备冷冻切片或整个贴片。
7.或者,在 3.4% 甲醛中在室温下固定 2 小时或在 4°C 下过夜,以制备石蜡样品。




图 5.制备和体外感染分离人体真皮。 
pi比例尺 (50 μm ) 中描述的程序示意图。 (b) 在 50 PFU/细胞感染后,冷冻切片的免疫染色在 24 小时后仅显示一些表达 ICP0 的细胞(绿色),用DAPI(蓝色)作为核复染剂。透射光 (TL) 显示形态。比例尺,50 μm 。


感染后程序
组织学分析
A.石蜡包埋组织样品的制备用于石蜡切片的苏木精和伊红 (HE) 染色
1.将组织固定在 3.4% 的甲醛中后,用 PBS 清洗样品,并储存在 70% 乙醇溶液中的组织包埋和处理盒中。
2.将所有感兴趣的样品固定并储存在 70% 乙醇溶液中后,继续将样品浸入石蜡中。
3.将样品嵌入嵌入模具中。
4.– 45°C的水浴在切片机上制备 8 µm 厚的切片。
5.在染色之前,对样品进行脱蜡并相应地染色 HE。
6.使用 DPX 安装介质嵌入带有玻璃盖玻片的样品,并允许在通风橱下过夜干燥。
7.在光学显微镜中获取 HE 染色样品的透射光图像。


感染细胞的可视化
B.免疫组织化学——冷冻切片
1.注意:冷冻样品不是固定的!
2.在 -21°C 的低温恒温器 3050 (Leica) 上制备 8 µm 厚的切片。 
3.每个显微镜载玻片至少准备三个切片,每个样品至少 100 μm的距离。
早期病毒蛋白的可视化(例如,ICP0):
4.在室温下固定在 1 – 2% 甲醛中 10 分钟。
5.用表皮冷冻切片缓冲液(用于表皮样品;参见食谱)或真皮/总皮肤阻断缓冲液(用于真皮、总皮肤和剃须样品;参见食谱)在室温下孵育冷冻切片长达 6 小时。
与目标一抗(例如, ICP0,单克隆抗体 11060;1:60;Everett等人,1993)在 4°C 下孵育过夜(De La Cruz等人,2021)。
6.清洗 3 × 5 分钟。
7.与二级抗体和 4 ',6-二脒基-2-苯基吲哚 (DAPI) 在 RT 中孵育 45 分钟。
8.通过用盖玻片覆盖部分嵌入Dako荧光介质。
9.在共聚焦显微镜(带 TCS-SP5 设备的 Leica DM IRBE)上获取图像。


C.免疫组织化学-整个坐骑
1.用 PBS 清洗 FA 固定的表皮整体样品。
2.参见配方 5)孵育表皮片(基底面朝下) 1 小时。
3.与感兴趣的一抗(例如, ICP0 1:60 )在室温下孵育过夜,摇动。
4.清洗至少 4 × 45 分钟(参见配方 6)。
5.与二抗和 4',6-diamidino-2-phenylindole (DAPI) 在 4°C 下孵育过夜。
6.再次清洗至少 4 × 45 分钟(见配方 6) 
7.对于嵌入,将基面朝上的表皮片放在载玻片上,并通过用盖玻片覆盖表皮来嵌入Dako荧光介质。如果难以区分基部和顶端,则在安装过程中使用解剖显微镜。


D.通过流式细胞术测定受体表面表达(感染前)的细胞解离
1.Nectin-1 表面检测 - 表皮细胞:
a.将表皮片(准备参见 C)放在TrypLE Select 溶液上,在室温下放置 30 分钟。
b.用两倍体积的 DMEM 停止反应,并用镊子将细胞从组织中机械分离。
c.通过 40 μm细胞过滤器过滤细胞并用计数室计数。
2.HVEM 表面检测 – 表皮细胞:
将表皮片(制备参见 C)置于无酶解离溶液中,在室温下放置 30 分钟(参见Petermann 等人,2015)。
3.受体表面表达——真皮细胞:
a.用全皮肤分离试剂盒 ( Miltenyi ) 在 37°C 下摇动 (180 rpm) 消化真皮薄片 2.5 小时。
b.通过 40 μm细胞过滤器过滤细胞并用计数室计数。
4.抗体:
a.抗 nectin-1(单克隆抗体 CK41;1:100; Krummenacher 等。 , 2000)。用抗小鼠 IgG-Cy5 (1:100; Jackson) 观察 Nectin-1。
b.与藻红蛋白 (PE) 缀合的抗 HVEM 抗体 (CD270-PE, REA247, 1:11; Miltenyi )。


数据分析


1.由于来自不同患者和/或不同身体区域的皮肤样本之间观察到的高度差异,单个样本的数量不得少于三个。根据样本大小,最好将实验重复进行(如果有足够的样本允许,则重复三次)。
2.图像采集:
a.使用共聚焦显微镜(带 TCS-SP5 设备的 Leica DM IRBE)获取图像。
b.模拟感染样本用作染色对照。
c.查看每个样本的三个部分,并将代表性区域捕获为 z 堆栈(0.17-0.5 µm 堆栈)或部分。
3.感染效率分析:使用 ImageJ-win64 通过细胞计数或平均荧光强度测量 (MFI) 确定。为 MFI 测量分析的图像应在同一时间使用相同的显微镜设置获取。


笔记


1.与培养细胞的同步 HSV-1 感染相比,含有病毒的培养基在感染后 1 小时不更换为新鲜培养基,而是一直保持到达到结束时间点。根据皮肤厚度、其延伸或其他影响因素,病毒到达其靶细胞可能需要不同的时间。此外,组织损伤似乎先于病毒附着。因此,将样本保持在病毒悬浮液中可以使病毒在以后的时间点接触皮肤细胞。
2.我们对分离表皮的感染研究仅限于24小时,样品与真皮分离后直接感染。根据我们的经验,较长的孵育时间可能会以多种方式影响组织。这包括但不限于紧密连接复合物的重排、组织解体和细胞损失(De La Cruz等人,2021)。需要模拟感染样本来确定这些变化对 HSV-1 感染分析的贡献。为了分析毒素/细胞因子可能引起的影响,我们将完整的皮肤样本预孵育长达 3 天,然后感染 24 或 48 小时pi
3.为了关联感染的结果和组织的状态,我们总是在感染前和感染后的不同时间进行 HE染色。此外,感染前的 HE染色允许从患者材料中排除表现出异常皮肤特征(例如,太薄或太厚的表皮等)的皮肤样本,并确保我们的分析使用至少具有可比性的样本进行。
4.分散酶II 处理过夜后没有分离的情况。在这些情况下,我们建议将样品在 37°C 下孵育更长的时间。每 15 分钟检查一次,直到表皮和真皮容易分离并且感觉它们之间没有阻力。
5.为了分析总皮肤离体感染后表皮的感染模式,感染后可通过分散酶II 处理将表皮与真皮分离,并准备整个支架用于基底细胞的染色和可视化 (A11)。对于这种方法,应避免在 37°C 下与分散酶II 一起孵育,以防止感染进展。
6.此外,在冰上用 1 PFU/细胞感染表皮层 1 小时,然后在 37°C (5% CO 2 )下孵育后,可以确定病毒滴度。感染后 1 小时更新培养基,感染后 3小时收集上清液,代表输入病毒。样品再次用新鲜、温热的培养基覆盖,并在感染后 48 小时收集上清液以确定产生的病毒。可以获得额外的更长或更短的时间点;但是,必须为每个所需的时间点准备单个表皮样品(不要使用一张表皮片来获得多个时间点的上清液)。为了确定产生的病毒滴度,然后可以对收集的上清液进行传统的噬斑测定。
7.使用 3% Korsolex 60 分钟对任何可重复使用的实验室设备进行消毒。


食谱


1.细胞培养基(也用作病毒悬浮培养基)
DMEM (1 × ) + GlutaMAX辅以
10% 胎牛血清
100微克/毫升链霉素
100 U/mL 青霉素


2.表皮冷冻切片封闭缓冲液
试剂最终浓度数量
普通山羊血清5%500微升_
吐温-200.2%20 微升_
PBS (1 × )广告10 毫升


3.真皮和全层(全层)皮肤冷冻切片封闭缓冲液
试剂最终浓度数量
牛奶粉0.5%0.25 克
冷水鱼皮明胶(预热至40°C)0.25%125 微升_
海卫 X-1000.5%250微升_
普通山羊血清5%2500 µL
牛血清白蛋白 (10% w/v)0.1%500 µL
PBS广告50 毫升


4.Dispase II 溶液
× PBS中新鲜制备 4 U/mL 。
过滤灭菌溶液。


5.整装封闭缓冲液
试剂最终浓度数量
牛奶粉0.5%0.25 克
冷水鱼皮明胶(预热至40°C)0.25%125 微升_
海卫 X-1000.5%250微升_
TBS (1 × )广告50 毫升


6.PBS-吐温
试剂最终浓度数量
吐温-200.2%50微升_
PBS (1 × )广告25 毫升


致谢


这项研究由德国研究基金会 (KN536/16-3)、科隆大学科隆财富计划/医学院和 Maria- Pesch基金会资助。我们感谢 Wolfram Malter (科隆大学医院妇科)和 Max Zinser (科隆大学医院整形、重建和美容外科)提供人体皮肤样本。该协议用于我们之前发表的作品(De La Cruz 等人, 2021)。


利益争夺


作者声明没有竞争利益。


伦理


在所有患者知情同意后获得人体皮肤标本。该研究得到了伦理委员会的批准 科隆大学医学院(批准号 17-481)。


参考


1.De La Cruz, NC, Möckel , M., Wirtz, L., Sunaoglu , K., Malter , W., Zinser , M. 和Knebel-Mörsdorf , D. (2021)。单纯疱疹病毒 1 对人体皮肤的体外感染显示机械伤口作为通过皮肤表面的入口不足。 病毒学杂志 95(21):e0133821 。
2.Everett, RD, Cross, A. 和 Orr, A. (1993)。一种截短形式的单纯疱疹病毒 1 型即早蛋白 Vmw110 以细胞类型依赖性方式表达。 病毒学197(2):751-756。
3.Grosche , L.、 Döhner , K.、 Düthorn , A.、 Hickford -Martinez, A.、 Steinkasserer , A. 和Sodeik , B. (2019) 1 型单纯疱疹病毒传播、滴定和单步生长曲线。 生物协议。 _ 9(23):e3441。
4.Hukkanen , V.、 Mikola , H.、 Nykänen , M. 和Syrjänen , S. (1999)。单纯疱疹病毒 1 型感染在三维角质形成细胞培养物中有两种不同的传播方式。 J Gen Virol 80(第 8 部分):2149-2155。
5.Krummenacher , C., Baribaud , I., Ponce de Leon, M., Whitbeck, JC, Lou, H., Cohen, GH 和 Eisenberg, RJ (2000)。通过使用抗受体单克隆抗体定位单纯疱疹病毒糖蛋白 D 在疱疹病毒进入介质 C 上的结合位点。 病毒学杂志 74(23):10863-10872 。
6.Petermann , P., Thier , K., Rahn , E., Rixon , FJ, Bloch, W., Özcelik , S., Krummenacher , C., Barron, MJ, Dixon, MJ, Scheu, S.等。 (2015 年)。单纯疱疹病毒 1 进入小鼠表皮的机制:nectin-1 和疱疹病毒进入介质作为细胞受体的参与。 病毒学杂志 89(1):262-274。
7.Rahn , E., Thier , K., Petermann , P. 和Knebel-Mörsdorf , D. (2015a)。用单纯疱疹病毒 1 型体外感染小鼠表皮。 J Vis Exp (102):e53046。
8.Rahn , E., Petermann , P., Thier , K., Bloch, W., Morgner , J., Wickström, SA 和Knebel-Mörsdorf , D. (2015b)。单纯疱疹病毒 1 型侵入小鼠表皮:离体感染研究。 J Invest Dermatol 135(12):3009-3016。
9.Schneider, CA, Rasband , WS 和Eliceiri , KW (2012)。 NIH Image to ImageJ:25 年的图像分析。 Nat 方法9(7):671-675。
10.Syrjänen , S.、 Mikola , H.、 Nykänen , M. 和Hukkanen , V. (1996)。在三维角质形成细胞培养中体外建立单纯疱疹病毒 1 型溶解性和非生产性感染。 病毒学杂志 70(9):6524-6528 。
11.Tajpara , P., Mildner , M., Schmidt, R., Vierhapper , M., Matiasek , J., Popow-Kraupp , T., Schuster, C. 和 Elbe- Bürger , A. (2019)。研究单纯疱疹病毒感染的临床前模型。 J Invest Dermatol 139(3):673-682。
12.Thier , K., Petermann , P., Rahn , E., Rothamel , D., Bloch, W. 和Knebel-Mörsdorf , D. (2017)。机械屏障限制单纯疱疹病毒 1 侵入人类口腔粘膜。 病毒学杂志91(22)。
13.Visalli , RJ, Courtney, RJ 和 Meyers, C. (1997)。 1 型单纯疱疹病毒在器官型上皮培养系统中的感染和复制。 病毒学230(2):236-243。
14.Wirtz, L.、 Möckel , M. 和Knebel-Mörsdorf , D. (2020)。单纯疱疹病毒 1 侵入小鼠真皮:Nectin-1 和疱疹病毒进入介质在衰老过程中作为细胞受体的作用。 病毒学杂志94(5)。

登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2022 The Authors; exclusive licensee Bio-protocol LLC.
引用:De La Cruz, N. C., Möckel, M., Wirtz, L. and Knebel-Mörsdorf, D. (2022). Ex vivo Human Skin Infection with Herpes Simplex Virus 1. Bio-protocol 12(9): e4411. DOI: 10.21769/BioProtoc.4411.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。