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Vaginal HSV-2 Infection and Tissue Analysis
阴道HSV-2感染和组织分析

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Abstract

The vaginal murine HSV-2 infection model is very useful in studying mucosal immunity against HSV-2 (Overall et al., 1975; Renis et al., 1976; Parr and Parr, 2003). Histologically, the vagina of Depo-Provera-treated mice is lined by a single layer of mucus secreting columnar epithelial cells overlying two to three layers of proliferative cells. Even though this is morphologically different from the human vagina, it closely resembles the endocervical epithelium, which is thought to be the primary site of HSV-2 infection in women (Parr et al., 1994; Kaushic et al., 2011). In the protocol presented here, mice are pre-treated with Depo-Provera before intra-vaginal inoculation with HSV-2. The virus replicates in the mucosal epithelium from where it spreads to and replicates in the CNS including the spinal cord, brain stem, cerebrum and cerebellum. Cytokine responses can be detected in vaginal washings using ELISA or in vaginal tissues using qPCR. Further, the recruitment of leukocytes to the vagina can be determined by flow cytometry. The model is suitable for research of both innate and adaptive immunity to HSV-2 infection.

Keywords: Immunology(免疫学), Vaginal infection(阴道感染), Mucosal immunology(粘膜免疫学), Virus infection(病毒感染), in vivo model(体内模型)

Background

Vaginal infection with HSV-2 has been studied in various animal models, such as rabbits, hamsters, guinea pigs, mice and monkeys with viral replication at the peripheral site and retrograde transport of virus to the neurons (Nahmias et al., 1971; Overall et al., 1975; Renis, 1977; Stanberry et al., 1982; Roizman et al., 2013). There are several pros and cons regarding the different animal models in terms of susceptibility to infection, latency, spontaneous reactivation of HSV-2 and availability of animals, especially with regard to the accessibility of knockout animals, which have been very useful in studies of immune responses to infection. The vaginal epithelium in the genital tract undergoes significant hormonal changes during the menstrual cycles, and both susceptibility to HSV-2 and the nature of the induced immune responses are regulated and affected by sex hormones (Kaushic et al., 2011). Mice are more susceptible to vaginal HSV-2 infection during pregnancy and during the diestrus stage of the murine estrus cycle, when progesterone levels are the highest (Overall et al., 1975; Baker and Plotkin, 1978; Gallichan and Rosenthal, 1996). Pre-treatment of mice with Depo-Provera, a long-lasting commercial progesterone induces the diestrus stage and increases susceptibility to vaginal HSV-2 infection by 100-fold (Parr et al., 1994; Kaushic et al., 2003).
   During the progression of intra-vaginal (i.vag.) HSV-2 infection in mice, the virus initially infects the vaginal epithelial cells in patches that involve the full thickness of the epithelium layer, and the underlying stroma is usually free of infection. The infected epithelial cells are shed off into the vaginal lumen (apical side) and infect the rest of the epithelium. The virus can spread horizontally within the epithelial layers to the epidermis and hair follicles, which results in loss of hair and development of skin lesions (Parr and Parr, 2003; Zhao et al., 2003). Vaginal HSV-2 infection and the resulting replication of the virus seem to be restricted to the vaginal epithelium, with no spread via viremia, as the virus generally cannot be isolated from systemic organs, blood or lymph nodes upon genital HSV infection (Overall et al., 1975; Renis et al., 1976; Podlech et al., 1996; Zhao et al., 2003). HSV-2 reaches dorsal root ganglia (DRG) via sensory neurons that innervate the site of infection, and from there the virus can spread further to the lumbar part of the spinal cord, brain stem and finally the brain (Renis et al., 1976; Georgsson et al., 1987; Podlech et al., 1996; Parr and Parr, 2003). HSV-2 can also spread to parasympathetic neurons via Para-cervical ganglia (major autonomic ganglia) of the bladder and rectum, which can cause retention of urine and feces (Parr and Parr, 2003).

Materials and Reagents

Note: All of the items mentioned in section “Materials and Reagents” can be ordered from any qualified company.

  1. Pipette tips
  2. Tissue culture plates (58 x 15 mm) (Thermo Fisher Scientific)
  3. Super Frost+ slides (Thermo Fisher Scientific)
  4. TC flask T25 (SARSTEDT)
  5. 70 µm pore size mesh (BD Falcon)
  6. 40 µm pore size mesh (BD Falcon)
  7. 96-well flat bottom plates (NUNC)
  8. 96-well round bottom plates (NUNC)
  9. Safety lock Eppendorf tubes
  10. Stainless steel beads, 5 mm (QIAGEN)
  11. Tissue processing/embedding cassettes (Sigma-Aldrich)
  12. Scalpels (Swann Morton)
  13. Flow-count beads (Beckman Coulter)
  14. Vero cells
  15. L929 cells
  16. Mouse strain: C57BL/6 mice
    Notes:
    1. The genetic background of different mouse strains can influence studies and it has been observed that different mouse strains, C57BL/6, BALB/c, SJL/J, PL/J and A/J have different susceptibilities to HSV. C57BL/6 and BALB/c mice are moderately susceptible to HSV, whereas A/J, PL/J and SJL/J mice strains are highly susceptible (Lopez, 1975; Kastrukoff et al., 2012). It has also been observed that 129Sv background mice produce higher amounts of type I and type III IFN compared to C57BL/6 in response to genital HSV-2 infection, nevertheless no difference in viral titer is observed in the vagina after HSV-2 infection (Ank, 2008).
    2. The vaginal HSV-2 infection model is based on pre-treatment with depo-provera. The pre-treatment with progesterone is an artificial intervention but is required for a reproducible enhanced susceptibility to HSV-2 infection in C57BL/6 mice.
  17. Virus strain: HSV-2 333 strain (laboratory isolate, Stanberry et al., 1982)
  18. Vesicular stomatitis virus (VSV/V10) (Indiana Strain) (ATCC, catalog number: VR-158 )
  19. Phosphate buffered saline solution (PBS) (Sigma-Aldrich)
  20. Depo-Provera (Pfizer)
  21. Isoflourane (Piramal Critical Care)
  22. DMEM (with no supplement of antibiotics or FSC) (Lonza)
  23. Penicillin-streptomycin (Thermo Fisher Scientific)
  24. Fetal calf serum (FCS) (In Vitro Technologies)
  25. 0.2% human immunoglobulin (ZLB Behring)
  26. 0.03% methylene blue (Sigma-Aldrich)
  27. 2% formaldehyde (Sigma-Aldrich)
  28. Ethanol (Sigma-Aldrich)
  29. Xylene (Sigma-Aldrich)
  30. Methanol (Sigma-Aldrich)
  31. 0.5% hydrogen peroxide (H2O2) (Sigma-Aldrich)
  32. 10 mM Tris
  33. 0.5 mM EGTA (pH 9.0)
  34. 50 mM NH4Cl
  35. Polyclonal rabbit anti-HSV-2 (Agilent Technologies, Dako, catalog number: GA521 ) (1:100)
  36. HRP-conjugated secondary antibody (Agilent Technologies, Dako, catalog number: P044801-2 ) (1:200)
  37. 3,3’-diaminobenzidine (Kem-En-Tec)
  38. Mayer’s or Harris’s hematoxylin solution (Sigma-Aldrich)
  39. Eukitt reagent (Eukitt, O. Kindler)
  40. Collagenase/dispase (1 mg/ml) (Roche Diagnostics)
  41. DNase 1 (2 mg/ml) (Roche Diagnostics)
  42. Ethylenediaminetetraacetate acid disodium salt (EDTA) (0.02%)
  43. Anti-mouse CD16/CD32 Ab (eBioscience)
  44. IgG, 1 g/ml (Jackson ImmunoResearch)
  45. Anti-mouse antibodies (BD Pharmingen):
    1. CD45-APC (clone 30-F11)
    2. NK1.1-FITC (clone PK136)
    3. Nk1.1-PE (clone PK136)
    4. CD11b-PE (clone M1/70)
    5. Ly-6g-APC (clone 1A8)
    6. APC RAT IgG2b, κ Isotype
    7. FITC RAT IgG2a, κ Isotype
    8. PE RAT IgG2a, κ Isotype
    9. 7-AAD (Live/dead cell marker)
  46. TRIzol (Invitrogen)
  47. DEPC water
  48. Chloroform (Sigma-Aldrich)
  49. Isopropanol
  50. Invitrogen Ambion DNA free kit
  51. Bovine serum albumin (BSA) (Sigma-Aldrich)
  52. 0.05% saponin (Thermo Fisher Scientific)
  53. 0.2% gelatin (Thermo Fisher Scientific)
  54. 0.3% Triton X-100 (Thermo Fisher Scientific)
  55. ELISA kits (R&D Systems)
  56. RNase free water (Roche Diagnostics)
  57. Oligo (dT) (Roche Diagnostics)
  58. Expand reverse transcriptase (Roche Diagnostics)
  59. Buffer A (see Recipes)
  60. Buffer B (see Recipes)
  61. Buffer C (see Recipes)
  62. FACS buffer (see Recipes)

Equipment

  1. Pipettes
  2. 8 arm multipipette
  3. Microtome (Leica)
  4. Microwave oven
  5. Rocking platform
  6. Humidity chamber
  7. Motar/pestle
  8. Centrifuge
  9. UV-light
  10. Light microscopy
  11. Homogenizer/shaker
  12. Incubator
  13. qPCR platform of choice
  14. NanoDrop
  15. Flow cytometer

Procedure

  1. Vaginal infection
    1. Mice are pretreated by subcutaneous injection of 200 µl (2 mg/mice, diluted in PBS) of Depo-Provera.
    2. Five days later mice are anesthetized with isoflourane and carefully inoculated with a pipette intra vaginally with 20 μl HSV-2 (6.7 x 104 PFU) suspended in PBS, see Figure 1. It is important not to destroy or damage the vaginal epithelial layer during inoculation as this can interfere with the natural infection.


      Figure 1. Inoculation of virus intravaginally with a pipette

    3. Subsequently, the mice are placed on their back and maintained under anesthesia for 10 min.
    4. Vaginal washes/fluids can be collected at desired time points post infection by pipetting a volume of 40 µl PBS in and out of the vagina 12-15 times, repeated twice, and diluted to a final volume of 250 μl also in PBS. For harvest of vaginas mice are euthanized by cervical dislocation (c.d.) and vaginas are removed surgically and used for RNA extraction, flow analysis or immunohistochemistry.
    5. The genitally infected animals should be observed and examined daily and scored for vaginal inflammation, neurological illness, weight loss and death. The severity of disease is scored using the following criteria: 0, healthy; 1, genital erythema; 2, moderate genital inflammation; 3, purulent genital lesion/or generally bad conditions; 4, hind limb paralysis or general very poor conditions. Mice must be sacrificed by c.d. when reaching score 4 according to institutional guidelines.

  2. Collection of vaginal washings for measuring viral titers and cytokine production
    1. At desired days post infection, the vagina of the mice are washed by pipetting 2 x 40 μl of PBS in and out of the vagina, which is then further diluted in PBS to a final volume of 250 μl per mouse. Be careful not to damage vaginal epithelial layer.
    2. Mice are anesthetized with isoflurane during the washing procedure. Viral titers and cytokine concentrations in the vaginal wash are usually highest at day 2 post infection.
    3. After the washing, mice can be sacrificed for harvest of whole vaginal tissue for other analysis or continued for survival or disease score recordings.

  3. Virus plaque assay
    1. Place vaginal washes immediately on dry ice and store at -80 °C until analysis.
    2. Virus titers of the vaginal washes are determined on monolayers of Vero cells. Cells are seeded in DMEM supplemented with 5% FCS at a density of 1.2 x 106 cells/tissue culture plate (58 x 15 mm) and left overnight to settle.
    3. The next day the media is discarded and 400 µl fresh DMEM with 5% FCS is added to each tissue culture plate. Subsequently add 100 μl of vaginal washes per culture plate diluted in 10-fold serial dilutions in DMEM with 2% FCS so the total volume is 500 µl in each culture plate. Each culture plate represents one dilution of a vaginal wash. Make duplicates of each dilution.
    4. Incubate cells for 1 h and rock the plates every 15 min to ensure even distribution of the virus. After 1 h 5 ml of DMEM 5% FCS supplemented with 0.2% human immunoglobulin is added.
    5. The tissue culture plates are then further incubated for 2-3 days and stained with 0.03% methylene blue in order to count the plaques.

  4. Immunohistochemistry (IHC)
    Note: Paraffin are preferred in contrast to frozen sections in order to preserve morphology. We do not use perfusion fixation because the blood circulation in the vagina seems to be low. The vaginas are instead surgically removed and fixed by immersion in 2% formaldehyde in PBS for 24 h. Work at room temperature if nothing else is stated.
    1. Vaginas are dissected and placed in 2-4% formaldehyde in PBS and fixed for 24 h.
    2. The fixed tissue is placed in tissue processing/embedding cassettes.
    3. Dehydration of fixed tissue: 70% ethanol for 2 h, 96% ethanol for 2 h and 99% for 2 h.
    4. The dehydrated tissue is then placed in xylene for 2 h.
    5. The tissue is embedded in warm (60 °C) paraffin for 2 h.
    6. Cut two-micrometer sections from tissue blocks on a microtome and mount the sections on Super Frost+ slides.
    7. The slides are the dewaxed in xylene 2 x for 4-5 min.
    8. Sections are rehydrated in a series of ethanol: 3 x 99% ethanol (30 min in total), 2 x 96% ethanol (20 min in total) and finally 10 min in 70% ethanol. Then the sections are rinsed in MilliQ water 3-4 times.
    9. Endogenous peroxidase enzymes are blocked using absolute methanol with 0.5% hydrogen peroxide (H2O2) for 10-15 min.
    10. Sections are boiled in microwave oven in 10 mM Tris, 0.5 mM EGTA (pH 9.0) to reveal antigens. The boiling time depends on the microwave oven ~6 min. full power and then 10 min on half power. This will provide a soft interval-boiling.
    11. Cool down on ice using a rocking platform.
    12. The free aldehyde groups are blocked by 50 mM NH4Cl in PBS for 30 min.
    13. Sections are then washed 3 times (3 x 10 min) in buffer A (see Recipes) and incubated with primary antibody against HSV-2 diluted in buffer B (see Recipes) overnight at 4 °C in a humidity chamber.
    14. The next day take the sections out of the fridge and allow to reach room temp.
    15. Wash with buffer C (see Recipes) (3 x 10 min.) and incubate the sections with HRP-conjugated secondary antibody (P448, 1:200) in buffer B for 1 h.
    16. Rinse in buffer C 3 x (total 30 min).
    17. Antibody binding is visualized by use of 3,3’-diaminobenzidine (DAB) (Kem-En-Tec) in 0.03% H2O2. DAB is freshly made by dissolving 1 tablet in 10 ml MilliQ water and just before use, add 10 µl 35% H2O2.
    18. Rinse in PBS 3 x
    19. The sections are counterstained with Mayer’s or Harris’s hematoxylin solution for 2 min and rinsed for 20 min under running tap water.
    20. The sections are mounted using Eukitt reagent.

  5. Flow cytometry
    1. Single cell suspension of mouse vagina can be obtained by enzymatic and mechanic processing. The dissected vaginas are cut in to small pieces by a scalpel and transferred to a small culture flask (T25). Add 4 ml collagenase/dispase (stock: 1 mg/ml) and 2 ml DNase I (stock: 2 mg/ml) in total 6 ml and incubate under constant stirring at 37 °C for 45 min.
    2. The tissue suspension is then gently mashed using a mortor pestle and filtered through a 70 µm pore size mesh and then passed through a 40 µm pore size mesh (if cells of interest are smaller than 40 µm) and washed with PBS with 0.02% EDTA to stop enzyme activity.
    3. Cells are then centrifuged for 8 min at 300 x g and re-suspended in 200 μl PBS/vagina. The following staining can be done in a round bottom 96-well plate.
    4. Prior to staining the cells are blocked for 10 min on ice with anti-mouse CD16/CD32 Ab (1:100) and mouse and rat IgG (1 µg/ml).
    5. Wash with cold PBS and centrifuge the plate at 300 x g for 4 min at 4 °C.
    6. Stain for LiveDead (7-AAD) (1:1,000 in PBS). Incubate for 15 min on ice.
    7. Wash twice with PBS.
    8. The cells are incubated with primary antibodies in 100 µl for 30 min on ice in the dark and washed in FACS buffer (see Recipies).
    9. The following primary anti-mouse antibodies can be used for flow identification of cell populations: CD45-APC (clone 30-F11), NK1.1-FITC (clone PK136), Nk1.1-PE (clone PK136), CD11b-PE (clone M1/70), Ly-6g-APC (clone 1A8), APC RAT IgG2b, κ Isotype, FITC RAT IgG2a, κ Isotype and PE RAT IgG2a, κ Isotype and 7-AAD (Live/dead cell marker). Flow-count beads can be added to the flow samples just before analysis for determining cell counts.
    10. The cells are resuspended and fixed in 250 μl 1% formaldehyde in PBS.

  6. Detection of cytokines in vaginal washes
    Note: In order to determine the amount of cytokines in the vaginal washes, we utilized ELISA and Luminex technology and for detection of IFN-α/β we used a L929 cell-based bioassay. Cytokines CXCL10, CXCL9, CCL5 (Rantes), IL-6, IFN-λ and IFN-γ can be easily detected by ELISA kits from any supplier. IFN-α/β bioactivity can be measured by use of a L929 cell-based bioassay in a 96-well plate.
    1. Prior to use HSV-2 in the vaginal samples must be inactivated on ice by UV-light treatment for 6 min.
    2. Add 50 µl DMEM 5% FCS to each well in a flat bottom 96-well plate. Each plate can hold 8 different samples/controls (A2-H2) as the samples are diluted across the plate. Column 1 (A1-H1) serves as a cell control and column 12 (A12-H12) as a virus control.
    3. Add 50 µl of vaginal washes or murine IFN-α/β to each well in row 2 (A2-H2). Make successive two-fold dilutions with an 8 arm multipippette down to row 11 (A11-H11).
    4. L929 cells (2 x 104 cells/well in 100 µl) in DMEM supplemented with 5% FCS are added to each well and the plates incubated over night at 37 °C. L929 cells are grown in DMEM supplemented with 200 IU/ml penicillin, 200 µg/ml streptomycin and 5% FCS.
    5. The next day 50 µl of Vesicular Stomatitis Virus (VSV/V10) (1.6 x 104 PFU/ml) is added to each well EXCEPT row 1 (A1-H1) (serve as a cell control) and the plates are further incubated for 2-3 days.
    6. If there is IFN-α/β in the samples being tested the IFN-α/β will protect the cells against the cytotoxic effect of VSV as observed in the bright field microscope. The dilution mediating 50% protection from virus induced cell death is defined as 1 U of IFN-α/β/ml. The bioassay has a lower detection limit of 6 U/ml.

  7. RNA extraction from tissue and quantitative Real-Time PCR
    Total RNA from isolated vaginas is extracted with TRIzol, according to the recommendations of the manufacturer.
    1. Set the centrifuge with rotor for Eppendorf tubes at 2-3 °C.
    2. Add 100-150 µl DEPC water and a 5 mm steel beads to each sample/vagina and homogenize the tissue using the homogenizer/shaker. Use safety lock Eppendorf tubes. Repeat if needed.
    3. After homogenization immediately place the samples on ice.
    4. Add 500 µl TRIzol to each sample and vortex. (According to Invitrogen use 1 ml TRIzol to 50-100 mg of tissue). Incubate at RT for 5 min.
    5. Add 1:5 chloroform (100 µl in case of 500 µl TRIzol) and vortex until the mixture is ‘candyfloss’ colored. Incubate for 5 min at RT or until two phases can be detected.
    6. Centrifuge for 15 min at 10,000 x g at 4 °C.
    7. Pipet the upper clear phase to new Eppendorf tubes. Avoid the white protein film on the side of the tubes.
    8. Add isopropanol (1:1). Vortex
    9. Place the tubes at -20 °C for 2 h or at RT for 10 min.
    10. Centrifuge for 12 min at 10,000 x g at 4 °C.
    11. Discard the supernatant. The RNA precipitate forms a white gel-like pellet at the bottom of the tube.
    12. Add 0.5 ml ice-cold ethanol (80 %) and vortex briefly. Centrifuge for 4 min at 10,000 x g. Repeat wash step.
    13. Discard the supernatant. Next, air-dry the pellet. If needed use a heating block at 80 °C.
      Note: It can be difficult to dissolve the RNA pellet.
    14. Pellet is re-suspended in 20-50 µl DEPC-water. Vortex.
    15. DNase-free treatment: (Invitrogen Ambion DNA free kit)
      1. Add 0.1 volume 10x DNase I buffer and 1 µl rDNase I to the RNA, and mix gently. Incubate at 37 °C for 20-30 min.
      2. Re-suspend DNase Inactivation Reagent and add 0.1 volume (min. 2 µl) to each sample and mix.
      3. Incubate the samples for 2 min at RT. Mix occasionally. Prepare new Eppendof tubes.
      4. Centrifuge for 1.5 min at 10,000 x g at 4 °C.
      5. Transfer the suspensions to new Eppendorf tubes. Store the samples at -80 °C.

        Notes:

        1. The RNA concentration can be measured by use of a NanoDrop, A260 for nucleic acid and 280 for protein.
        2. For RNA the 260/280 ratio ≈ 2 is considered pure.

Recipes

  1. DMEM supplemented with 200 IU/ml penicillin, 200 µg/ml streptomycin and FCS
  2. DMEM supplemented with 2% FCS and 0.2% human immunoglobulin
  3. Buffer A
    PBS with 1% BSA, 0.05% Saponin, 0.2% gelatin
  4. Buffer B
    PBS with 0.1% BSA and 0.3% Triton X-100
  5. Buffer C
    PBS with 0.1% BSA, 0.2% gelatin and 0.05% Saponin
  6. FACS buffer
    2 % BSA in PBS (0.09% Azide if necessary)

Acknowledgments

We thank K.S. Petersen and I.M. Poulsen for technical assistance and the AU FACS Core facility for technical help. Supported by The Danish Medical Research Council (12-124330 to S.R.P.; 1331-xg00133B to H.H.W.), the Novo Nordisk Foundation (S.R.P.), the Lundbeck Foundation (R34-3855 to S.R.P.), Aarhus University Research Foundation (S.R.P), Faculty of Health Sciences, AU (M.B.I.), the Danish National Research Foundation (DNRF107 to H.H.W.), the Excellence Program for Interdisciplinary Research (CDO2016 to H.H.W.).

References

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简介

阴道鼠HSV-2感染模型在研究针对HSV-2的粘膜免疫中是非常有用的(总体等人,1975; Renis等人,1976; Parr和Parr,2003)。在组织学上,Depo-Provera治疗的小鼠的阴道由覆盖两到三层增殖细胞的单层粘液分泌柱状上皮细胞排列。尽管这在形态上与人体阴道不同,但是它非常类似于宫颈上皮,被认为是妇女HSV-2感染的主要部位(Parr等人,1994; Kaushic et al。,2011)。在本文提出的方案中,在用HSV-2进行阴道内接种之前,用Depo-Provera预处理小鼠。该病毒在粘膜上皮中复制,其中它扩散到包含脊髓,脑干,大脑和小脑的CNS中并在其中复制。可以使用ELISA或使用qPCR在阴道组织中检测阴道洗液中的细胞因子反应。此外,可以通过流式细胞术测定白细胞向阴道的募集。该模型适用于对HSV-2感染的先天性和适应性免疫力的研究。
【背景】已经在各种动物模型中研究了具有HSV-2的阴道感染,例如在周边部位具有病毒复制的兔子,仓鼠,豚鼠,小鼠和猴子,并将病毒逆行转运到神经元(Nahmias et al。 ,1971;总体等人,1975; Renis,1977; Stanberry等人,1982; Roizman等人, 2013)。关于感染易感性,潜伏期,HSV-2的自发再激活和动物的可用性,特别是关于敲除动物的可及性的不同动物模型,有几个优点和缺点,这在免疫研究中非常有用对感染的反应。生殖道中的阴道上皮在月经周期期间经历显着的激素变化,并且HSV-2的易感性和诱导的免疫应答的性质受性激素的调节和影响(Kaushic等人, ,2011)。当孕酮水平最高时,小鼠在怀孕期间和小鼠发情周期的发情期间更容易受到阴道HSV-2感染(总体而言,1975年; Baker和Plotkin,1978; Gallichan和Rosenthal,1996)。 Depo-Provera(一种持久的商业黄体酮)预处理小鼠诱发妊娠期并增加阴道HSV-2感染的易感性(100%)(Parr等人,1994; Kaushic et al。,2003)。
 在小鼠阴道内(i.vag。)HSV-2感染进展期间,病毒最初感染包含上皮层全层的贴片中的阴道上皮细胞,下面的基质通常没有感染。感染的上皮细胞被排出到阴道腔(顶端)并感染上皮的其余部分。病毒可以在上皮层内水平扩散到表皮和毛囊,导致头发的损失和皮肤损伤的发展(Parr和Parr,2003; Zhao等人,2003)。阴道HSV-2感染和所产生的病毒复制似乎限于阴道上皮,没有通过病毒血症传播,因为病毒通常不能从生殖器HSV感染的系统器官,血液或淋巴结中分离出来1975年; Renis等人,1976; Podlech等人,1996; Zhao等人。 ,2003)。 HSV-2通过支配感染部位的感觉神经元到达背根神经节(DRG),从那里病毒可以进一步扩散到脊髓,脑干和最后大脑的腰部(Renis等人1976年; Georgsson等人,1987; Podlech等人,1996; Parr和Parr,2003)。 HSV-2也可以通过膀胱和直肠的副神经节(主要自主神经节)扩散到副交感神经元,这可能导致尿液和粪便的滞留(Parr和Parr,2003)。

关键字:免疫学, 阴道感染, 粘膜免疫学, 病毒感染, 体内模型

材料和试剂

注意:“材料和试剂”一节中提到的所有项目都可以从任何合格的公司订购。

  1. 移液器提示
  2. 组织培养板(58 x 15 mm)(Thermo Fisher Scientific)
  3. 超级冰霜+幻灯片(Thermo Fisher Scientific)
  4. TC烧瓶T25(SARSTEDT)
  5. 70μm孔径网(BD Falcon)
  6. 40μm孔径网(BD Falcon)
  7. 96孔平底板(NUNC)
  8. 96孔圆底板(NUNC)
  9. Eppendorf管安全锁
  10. 不锈钢珠,5毫米(QIAGEN)
  11. 组织加工/嵌入盒(Sigma-Aldrich)
  12. 手镯(Swann Morton)
  13. 流量珠(Beckman Coulter)
  14. Vero细胞
  15. L929细胞
  16. 小鼠株:C57BL / 6小鼠
    注意:
    1. 不同小鼠品种的遗传背景可以影响研究,已经观察到不同的小鼠品系C57BL / 6,BALB / c,SJL / J,PL / J和A / J对HSV具有不同的敏感性。 C57BL / 6和BALB / c小鼠对HSV中度敏感,而A / J,PL / J和SJL / J小鼠株高度敏感(Lopez,1975; Kastrukoff等,2012)。还观察到,对于生殖器HSV-2感染,129Sv背景小鼠与C57BL / 6相比产生更高量的I型和III型IFN,然而在HSV-2感染后阴道中没有观察到病毒滴度的差异( Ank,2008)。
    2. 阴道HSV-2感染模型基于用depo-provera预处理。孕激素的预处理是一种人为干预,但对于C57BL / 6小鼠中对HSV-2感染的再现性增强的敏感性是必需的。
  17. 病毒株:HSV-2 333株(实验室分离株,Stanberry等人,1982)
  18. 水泡性口炎病毒(VSV / V10)(印第安那菌株)(ATCC,目录号:VR-158)
  19. 磷酸盐缓冲盐水溶液(PBS)(Sigma-Aldrich)
  20. Depo-Provera(辉瑞)
  21. 异氟烷(Piramal Critical Care)
  22. DMEM(不含抗生素或FSC)(Lonza)
  23. 青霉素 - 链霉素(Thermo Fisher Scientific)
  24. 胎牛血清(FCS)(体外技术)
  25. 0.2%人免疫球蛋白(ZLB Behring)
  26. 0.03%亚甲基蓝(Sigma-Aldrich)
  27. 2%甲醛(Sigma-Aldrich)
  28. 乙醇(Sigma-Aldrich)
  29. 二甲苯(Sigma-Aldrich)
  30. 甲醇(Sigma-Aldrich)
  31. 0.5%过氧化氢(H 2 O 2 O 2)(Sigma-Aldrich)
  32. 10 mM Tris
  33. 0.5mM EGTA(pH9.0)
  34. 50mM NH 4 Cl
  35. 多克隆兔抗HSV-2(Agilent Technologies,Dako,目录号:GA521)(1:100)
  36. HRP共轭二抗(Agilent Technologies,Dako,目录号:P044801-2)(1:200)
  37. 3,3'-二氨基联苯胺(Kem-En-Tec)
  38. Mayer's或Harris's苏木精溶液(Sigma-Aldrich)
  39. Eukitt试剂(Eukitt,O.Kindler)
  40. 胶原酶/分散素(1 mg / ml)(Roche Diagnostics)
  41. DNase 1(2 mg / ml)(Roche Diagnostics)
  42. 乙二胺四乙酸二钠盐(EDTA)(0.02%)
  43. 抗小鼠CD16 / CD32 Ab(eBioscience)
  44. IgG,1μg/ ml(Jackson ImmunoResearch)
  45. 抗小鼠抗体(BD Pharmingen):
    1. CD45-APC(克隆30-F11)
    2. NK1.1-FITC(克隆PK136)
    3. Nk1.1-PE(克隆PK136)
    4. CD11b-PE(克隆M1 / 70)
    5. Ly-6g-APC(克隆1A8)
    6. APC RAT IgG2b,κ同种型
    7. FITC RAT IgG2a,κ同种型
    8. PE RAT IgG2a,κ同种型
    9. 7-AAD(活/死细胞标记)
  46. TRIzol(Invitrogen)
  47. DEPC水
  48. 氯仿(Sigma-Aldrich)
  49. 异丙醇
  50. Invitrogen Ambion DNA免费试剂盒
  51. 牛血清白蛋白(BSA)(Sigma-Aldrich)
  52. 0.05%皂角蛋白(Thermo Fisher Scientific)
  53. 0.2%明胶(Thermo Fisher Scientific)
  54. 0.3%Triton X-100(Thermo Fisher Scientific)
  55. ELISA试剂盒(R& D Systems)
  56. 无RNA酶水(Roche Diagnostics)
  57. Oligo(dT)(Roche Diagnostics)
  58. 展开逆转录酶(Roche Diagnostics)
  59. 缓冲液A(参见食谱)
  60. 缓冲液B(参见食谱)
  61. 缓冲区C(见配方)
  62. FACS缓冲区(见配方)

设备

  1. 移液器
  2. 8臂多功能一体机
  3. 切片机(Leica)
  4. 微波炉
  5. 摇摆平台
  6. 湿度室
  7. Motar /杵
  8. 离心机
  9. 紫外线灯
  10. 光学显微镜
  11. 均质器/振动筛
  12. 孵化器
  13. qPCR选择平台
  14. NanoDrop
  15. 流式细胞仪

程序

  1. 阴道感染
    1. 通过皮下注射Depo-Provera的200μl(2mg /小鼠,在PBS中稀释的PBS)预处理小鼠。
    2. 五天后,用异氟烷麻醉小鼠,并用悬浮在PBS中的20μlHSV-2(6.7×10 4 /μl)PFU阴道内用移液管仔细接种,见图1.重要的是不破坏或在接种过程中损伤阴道上皮层,因为这可能会干扰自然感染

      图1.用移液管阴道内接种病毒

    3. 随后,将小鼠放置在背部并在麻醉下保持10分钟。
    4. 可以在感染后的所需时间点通过将体积为40μl的PBS吸入和移出阴道12-15次来收集阴道洗液/液体,重复两次,并且还在PBS中稀释至最终体积为250μl。对于阴道收获,通过颈椎脱位(c.d.)对小鼠进行安乐死,通过手术取出阴道,用于RNA提取,流式分析或免疫组织化学。
    5. 每天应观察和检查感染原殖的动物,并评分阴道炎症,神经系统疾病,体重减轻和死亡。使用以下标准评估疾病的严重程度:0,健康; 1,生殖器红斑; 2,中度生殖器炎症; 3,脓性生殖器损伤/一般条件差; 4,后肢麻痹或一般情况非常差。小鼠必须由c.d.牺牲根据制度指导达到4级。

  2. 收集用于测量病毒滴度和细胞因子产生的阴道清洗液
    1. 在感染后的期望天,通过将2×40μlPBS吸入和移出阴道来洗涤小鼠的阴道,然后将其进一步在PBS中稀释至每只小鼠250μl的最终体积。注意不要损伤阴道上皮层。
    2. 在洗涤过程中,用异氟烷麻醉小鼠。阴道洗液中的病毒滴度和细胞因子浓度通常在感染后第2天最高。
    3. 洗完后,可以将小鼠处死,以收集整个阴道组织进行其他分析,或继续进行生存或疾病评分记录。

  3. 病毒斑块测定
    1. 在干冰上立即将阴道洗涤,并储存在-80°C直到分析。
    2. 在Vero细胞的单层上测定阴道洗液的病毒滴度。将细胞接种在补充有5%FCS的DMEM中,密度为1.2×10 6细胞/组织培养板(58×15mm),并放置过夜以沉降。
    3. 第二天丢弃培养基,并向每个组织培养板中加入400μl具有5%FCS的新鲜DMEM。随后,每个培养板中加入100μl阴道洗液,稀释于10%连续稀释于含有2%FCS的DMEM中,因此每个培养板中的总体积为500μl。每个培养皿代表阴道洗涤液的一种稀释液。重复每次稀释。
    4. 孵育细胞1小时,每15分钟摇一下板,以确保病毒的均匀分布。 1小时后,加入5ml补充有0.2%人免疫球蛋白的DMEM 5%FCS。
    5. 然后将组织培养板进一步温育2-3天,用0.03%亚甲蓝染色,以计数斑块。

  4. 免疫组织化学(IHC)
    注意:与冷冻切片相比,石蜡是优选的,以保持形态。我们不使用灌注固定,因为阴道的血液循环似乎很低。将阴道通过浸入2%甲醛的PBS中24小时而手术取出并固定。如果没有其他的话,在室温下工作。
    1. 将阴道切开并置于PBS中的2-4%甲醛中并固定24h
    2. 将固定的组织放置在组织处理/嵌入盒中
    3. 固定组织脱水:70%乙醇2小时,96%乙醇2小时,99%2小时。
    4. 然后将脱水组织置于二甲苯中2小时。
    5. 将组织嵌入温热(60℃)石蜡中2小时。
    6. 从切片机上的组织块切割两千米的部分,并将部分安装在超级霜+幻灯片上。
    7. 载玻片在二甲苯中脱蜡4-5分钟。
    8. 切片在一系列乙醇中再水化:3×99%乙醇(总共30分钟),2×96%乙醇(共20分钟),最后在70%乙醇中10分钟。然后将切片在MilliQ水中漂洗3-4次。
    9. 使用具有0.5%过氧化氢(H 2 O 2 O 2 2)的绝对甲醇将内源性过氧化物酶阻断10-15分钟。
    10. 将切片在10mM Tris,0.5mM EGTA(pH 9.0)的微波炉中煮沸以显示抗原。沸腾时间取决于微波炉〜6分钟。全功率,然后10分钟半功率。这将提供软间隔沸腾。
    11. 使用摇摆平台在冰上冷却。
    12. 游离醛基被PBS中50mM NH 4 Cl封闭30分钟。
    13. 然后将切片在缓冲液A中洗涤3次(3×10分钟)(参见食谱)并与在缓冲液B中稀释的HSV-2的一抗(参见食谱)一起在4℃在湿度室中过夜。
    14. 第二天,把这些部分从冰箱里拿出来,允许达到室温。
    15. 用缓冲液C(参见食谱)(3×10分钟)洗涤,并将缓冲液B中HRP-缀合的二抗(P448,1:200)的切片孵育1小时。
    16. 冲洗缓冲液C 3 x(共30分钟)。
    17. 通过在0.03%H 2 O 2 O 2中使用3,3'-二氨基联苯胺(DAB)(Kem-En-Tec)显现抗体结合。 DAB通过将1片溶解在10ml MilliQ水中并刚好在使用之前新添加10μl35%H 2 O 2 O 2。
    18. 在PBS中冲洗3 x
    19. 切片用Mayer's或Harris's苏木精溶液复染2分钟,并在自来水冲洗20分钟。
    20. 这些部分使用Eukitt试剂安装。

  5. 流式细胞仪
    1. 小鼠阴道的单细胞悬液可以通过酶和机械加工获得。将解剖的阴道通过解剖刀切成小块,并转移到小型培养瓶(T25)中。加入4ml胶原酶/分散酶(储备:1mg / ml)和2ml DNase I(储备:2mg / ml),总共6ml,并在37℃恒温搅拌下孵育45分钟。
    2. 然后使用杵杵将组织悬浮液轻轻捣碎,并通过70μm孔径筛网过滤,然后通过40μm孔径的网孔(如果感兴趣的细胞小于40μm),并用0.02%EDTA的PBS洗涤至停止酶活性。
    3. 然后将细胞以300×g离心8分钟并重新悬浮于200μlPBS /阴道中。可以在圆底96孔板中进行以下染色。
    4. 在染色之前,用抗小鼠CD16 / CD32Ab(1:100)和小鼠和大鼠IgG(1μg/ ml)在冰上封闭细胞10分钟。
    5. 用冷PBS洗涤,并在4℃下以300×g离心板4分钟。
    6. LiveDead(7-AAD)染色(PBS中1:1,000)。在冰上孵育15分钟。
    7. 用PBS洗两次。
    8. 将细胞与100μl的一抗在黑暗中冰上孵育30分钟,并在FACS缓冲液中洗涤(参见Recipies)。
    9. 以下主要抗小鼠抗体可用于细胞群体的流动鉴定:CD45-APC(克隆30-F11),NK1.1-FITC(克隆PK136),Nk1.1-PE(克隆PK136),CD11b-PE (克隆M1 / 70),Ly-6g-APC(克隆1A8),APC RAT IgG2b,κ同种型,FITC RAT IgG2a,κ同种型和PE RAT IgG2a,κ同种型和7-AAD(活/死细胞标记)。流量计数珠可以在分析前用于确定细胞计数的流量样本。
    10. 将细胞重新悬浮并固定在PBS中的250μl1%甲醛中。

  6. 检测阴道清洗中的细胞因子
    注意:为了确定阴道洗液中细胞因子的量,我们使用ELISA和Luminex技术,并检测IFN-α/β,我们使用了基于L929细胞的生物测定法。细胞因子CXCL10,CXCL9,CCL5(Rantes),IL-6,IFN-λ和IFN-γ可以通过任何供应商的ELISA试剂盒轻松检测。 IFN-α/β生物活性可以通过在96孔板中使用基于L929细胞的生物测定来测量。
    1. 在阴道样品中使用HSV-2之前,必须通过紫外线治疗6小时将其在冰上灭活。
    2. 在平底96孔板中的每个孔中加入50μlDMEM 5%FCS。每个板可以容纳8个不同的样品/对照(A2-H2),因为样品在板上稀释。第1列(A1-H1)作为细胞对照,列12(A12-H12)作为病毒对照。
    3. 向行2(A2-H2)中的每个孔中加入50μl阴道洗涤液或鼠IFN-α/β。使用8臂多面体连续两次稀释至第11排(A11-H11)。
    4. 在每个孔中加入补充有5%FCS的DMEM中的L929细胞(2×10 4个细胞/100μl中的孔),并将板在37℃下孵育过夜。 L929细胞在补充有200IU / ml青霉素,200μg/ ml链霉素和5%FCS的DMEM中生长。
    5. 第二天将50μl水泡性口炎病毒(VSV / V10)(1.6×10 4 /μl)/ PFU / ml加入每个孔,并将板再孵育2-3天。
    6. 如果在测试样品中存在IFN-α/β,IFN-α/β将保护细胞免受VSV的细胞毒作用,如在明场显微镜中观察到的。介导50%保护免受病毒诱导的细胞死亡的稀释液定义为1U IFN-α/β/ ml。生物测定具有较低的检测限,为6U / ml
  7. 来自组织的RNA提取和定量实时PCR
    根据制造商的建议,用TRIzol提取来自分离的阴道的总RNA。
    1. 将离心机用于Eppendorf管的转子在2-3°C。
    2. 向每个样品/阴道添加100-150μlDEPC水和一个5mm钢珠,并使用匀浆器/振荡器匀浆组织。使用安全锁Eppendorf管。如果需要,重复。
    3. 均化后立即将样品置于冰上
    4. 向每个样品中加入500μlTRIzol并涡旋。 (根据Invitrogen,使用1ml TRIzol至50-100mg组织)。在室温下孵育5分钟。
    5. 加入1:5氯仿(100μl,500μlTRIzol),涡旋直到混合物为“糖果”。在室温下孵育5分钟,或直到检测到两个相
    6. 在4℃下以10,000×g离心15分钟。
    7. 将上清液吸入新的Eppendorf管。避免管子侧面的白色蛋白质膜。
    8. 加入异丙醇(1:1)。漩涡
    9. 将试管置于-20℃2小时或室温10分钟
    10. 在4℃下以10,000×g离心12分钟。
    11. 丢弃上清液。 RNA沉淀物在管的底部形成白色凝胶状颗粒。
    12. 加入0.5ml冰冷乙醇(80%)并短暂涡旋。以10,000 x g离心4分钟。重复洗涤步骤。
    13. 丢弃上清液。接下来,将颗粒空气干燥。如果需要,在80°C下使用加热块。
      注意:可能难以溶解RNA颗粒。
    14. 将颗粒重新悬浮于20-50μlDEPC-水中。涡流。
    15. 无DNA酶处理:(Invitrogen Ambion DNA免费试剂盒)
      1. 向RNA中加入0.1体积的10×DNase I缓冲液和1μlrDNaseⅠ,并轻轻混合。在37℃下孵育20-30分钟
      2. 重新中止DNase灭活试剂,并向每个样品添加0.1体积(最少2μl)并混合。
      3. 在室温下孵育样品2分钟。偶尔混合准备新的Eppendof管。
      4. 在4℃下以10,000xg离心1.5分钟。
      5. 将悬浮液转移到新的Eppendorf管中。将样品储存在-80°C。

        注意:

        1. RNA浓度可以通过使用NanoDrop,A260用于核酸和280作为蛋白质来测量。
        2. 对于RNA,260/280比例≈2被认为是纯的。

食谱

  1. DMEM补充200 IU / ml青霉素,200μg/ ml链霉素和FCS
  2. 补充有2%FCS和0.2%人免疫球蛋白的DMEM
  3. 缓冲区A
    PBS含1%BSA,0.05%皂角蛋白,0.2%明胶
  4. 缓冲区B
    含有0.1%BSA和0.3%Triton X-100的PBS
  5. 缓冲区C
    含有0.1%BSA,0.2%明胶和0.05%皂苷的PBS
  6. FACS缓冲区
    PBS中的2%BSA(如果需要,为0.09%叠氮化合物)

致谢

我们感谢K.S. Petersen和I.M.Pulsen提供技术援助和AU FACS核心设施的技术帮助。由丹麦医学研究理事会(12-124330至SRP; 1331-xg00133B至HHW),诺和诺德基金会(SRP),隆德贝克基金会(R34-3855至SRP),奥胡斯大学研究基金会(SRP),健康科学,澳大利亚(MBI),丹麦国家研究基金会(DNRF107至HHW),跨学科研究优秀计划(CDO2016至HHW)。

参考

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引用:Iversen, M. B., Paludan, S. R. and Holm, C. K. (2017). Vaginal HSV-2 Infection and Tissue Analysis. Bio-protocol 7(13): e2383. DOI: 10.21769/BioProtoc.2383.
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