We thank Peter Staeheli for providing B6.A2G-Mx1 mice and Semra &#214;zcelik for technical advice.
This work was supported by the German Research Foundation through SFB829 and KN536/16, and the K&#246;ln Fortune Program/Faculty of Medicine, University of Cologne.

Ethics statement.
The preparation of epidermal sheets from sacrificed animals is carried out in strict accordance with the recommendations of the Guide of Landesamt f&#252;r 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
<ol>
	<li>Cultivate epidermal sheets in Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)/high glucose containing glutamine dipeptide, 10% FCS, 100 IU/ml penicillin, and 100 &#181;g/ml streptomycin (hereinafter referred to as &#8220;DMEM&#8221;).</li>
	<li>For the preparation of epidermal sheets, carefully dissolve dispase II powder (5 mg/ml) in heated PBS (up to 37 &#176;C). Filter the solution through a 0.22 &#181;m filter and use it directly for treatment.</li>
	<li>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&#160;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.</li>
	<li>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.</li>
</ol>
2. Preparation of Epidermal Sheets from Murine Skin
<ol>
	<li>Preparation of epidermis from newborn back skin for infection studies
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Replace PBS by 1.5 ml dispase II (5 mg/ml PBS) and incubate for either 30 min at 37 &#176;C (for whole mounts) or O/N at 4 &#176;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.</li>
	</ol>
	</li>
	<li>Preparation of epidermis from adult tail skin for infection studies
	<ol>
		<li>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.</li>
		<li>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&#160;at 4 &#176;C without losing significant infection efficiency. If tails are kept up to 48 hr, epidermal sheets could be hardly infected with HSV-1.</li>
	</ol>
	</li>
	<li>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.
	<ol>
		<li>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 &#176;C.</li>
		<li>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.</li>
		<li>Alternatively, incubate the epidermal sheets on enzyme-free cell dissociation solution (CDS) for 15 min at RT and 15 min at 37 &#176;C. Incubate another 15 min at RT to gently flush out the keratinocytes by pipetting CDS up and down.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Dissociation of epidermis to isolate keratinocytes or extract RNA
	<ol>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
3. Infection of Epidermal Sheets with HSV-1
<ol>
	<li>Prepare a solution of HSV-1 wt strain 1711 in not less than 500 &#181;l 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 &#176;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).</li>
	<li>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. &#160; 
	NOTE: In case of epidermal sheets from newborn skin, keep in mind that sheets start to dissociate during incubation.</li>
</ol>
4. Visualization of Infected Cells
<ol>
	<li>Epidermal Whole Mounts13
	<ol>
		<li>Fix epidermal sheets with 3.4% formaldehyde either for 2 hr&#160;at RT or O/N&#160;at 4 &#176;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.</li>
		<li>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).</li>
		<li>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 &#181;g/ml), AF555-conjugated anti-rabbit IgG (1 &#181;g/ml), and DAPI (4&#8217;, 6-diamidino-2-phenylindole) (100 ng/ml) in blocking buffer at RT.</li>
		<li>Wash with PBS-0.2% Tween 20 for 4 hr&#160;at RT. Mount epidermal sheets with their basal side on top of a specimen slide, embed in fluorescent mounting medium and cover with coverslips.</li>
	</ol>
	</li>
	<li>Epidermal Cryosections
	<ol>
		<li>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 &#181;m sections with a cryomicrotome.</li>
		<li>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.</li>
		<li>Then incubate with AF488-conjugated anti-mouse IgG (1 &#181;g/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.&#160;</li>
	</ol>
	</li>
</ol>

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The authors declare that they have no competing financial interests.

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....

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 published3-6.
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&#160;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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>DMEM/high glucose/GlutaMAX</td>
			<td>Life Technologies</td>
			<td align="right">31966047</td>
			<td>needed for cultivation of epidermal sheets</td>
		</tr>
		<tr>
			<td>dispase II powder</td>
			<td>Roche</td>
			<td align="right">4942078001</td>
			<td>has to be solved in heated PBS</td>
		</tr>
		<tr>
			<td>enzyme-free cell dissociation solution</td>
			<td>Sigma</td>
			<td>C5914</td>
			<td>needed for very gentle dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>TrypLE select cell dissociation solution</td>
			<td>Life Technologies</td>
			<td>12563-029</td>
			<td>needed for dissociation of epidermal sheets</td>
		</tr>
		<tr>
			<td>chelex 100 resin</td>
			<td>Bio-Rad</td>
			<td>142-2832</td>
			<td>needed for chelation of polyvalent metal ions from the fetal calf serum</td>
		</tr>
		<tr>
			<td>gelatin from cold water fish skin&#160;</td>
			<td>Sigma</td>
			<td>G7765</td>
			<td>needed for minimization of non-specific antibody binding</td>
		</tr>
		<tr>
			<td>Keratin 14 Polyclonal Antibody (AF64) (conc.: 1 mg/ml)</td>
			<td>Covance</td>
			<td>PRB-155P</td>
			<td>used to visualize the intermediate filament keratin 14 which is a marker of the basal layer of the epidermis</td>
		</tr>
		<tr>
			<td>Mouse IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 488 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-11029</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor&#174; 555 conjugate (conc.: 2 mg/ml)</td>
			<td>Life Technologies</td>
			<td>A-21429</td>
			<td>used as secondary antibodies</td>
		</tr>
		<tr>
			<td>4&#8242;,6-Diamidino-2-phenylindole dihydrochloride (DAPI dihydrochloride)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; (conc.: 0.1 mg/ml)</td>
			<td>Sigma</td>
			<td align="right">36670</td>
			<td>used to counterstain the nucleus</td>
		</tr>
	</tbody>
</table>

ex vivo infection of murine epidermis with herpes simplex virus type 1

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.

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...

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

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.

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

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 ...

ex-vivo-infection-of-murine-epidermis-with-herpes-simplex-virus-type-1

Research

JoVE Journal

Immunology and Infection

11.0K Views.  University of Cologne. 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.

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

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration of T cells into lymph nodes is a complex process which involves both cellular and chemical factors including chemokines. Recently, the use of two-photon microscopy has permitted to track T cells in intact lymph nodes and to derive some quantitative information on their behavior and their interactions with other cells. While there are obvious advantages to an in vivo system, this approach requires a complex and expensive instrumentation and provides limited access to the tissue. To analyze the behavior of T cells within murine lymph nodes, we have developed a slice assay 
1, originally set up by neurobiologists and transposed recently to murine thymus 2. In this technique, fluorescently labeled T cells are plated on top of an acutely prepared lymph node slice. In this video-article, the localization and migration of T cells into the tissue are analyzed in real-time with a widefield and a confocal microscope. The technique which complements in vivo two-photon microscopy offers an effective approach to image T cells in their natural environment and to elucidate mechanisms underlying T cell migration.

Ex vivo Imaging of T Cells in Murine Lymph Node Slices with Widefield and Confocal Microscopes

This protocol describes a method to image fluorescent T cells introduced into lymph node slices. The technique permits real-time analyses of T cell migration with traditional widefield fluorescence or confocal microscopes.

Na&iuml;ve T cells continuously traffic to secondary lymphoid organs, including peripheral lymph nodes, to detect rare expressed antigens. The migration ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies

Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13. Importantly, through ex vivo infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh) cell population and are separated by fluorescence activated cell sorting 3.
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4.

We describe the rapid isolation of primary murine type II alveolar epithelial cells (AECII) by flow cytometric negative selection. These AECII show high viability and purity and are suitable for a wide range of functional and molecular studies regarding their role in respiratory conditions such as autoimmune or infectious diseases.

Throughout  the last years, the contribution of alveolar type II epithelial cells (AECII)  to various aspects of immune regulation in the lung has been ...

Trans-vivo Delayed Type Hypersensitivity Assay for Antigen Specific Regulation

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections.
Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse "trans-vivo" DTH assay was developed 1,2. This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a na&iuml;ve mouse and DTH-like swelling is measured after 18-24 hr 3. The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens.
The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts 2,4-14. The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function.
A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity15,16 and also in tumor immunology 17.

We describe a valuable diagnostic assay that could potentially be used to decide the withdrawal of immunosuppression after transplant without elevated risk of graft rejection. The assay uses the principles of Delayed Type Hypersensitivity and provides accurate assessment of both donor specific effector and regulatory immune responses mounted by recipients.

Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, mortality, and immunosuppression in infected birds. In this study, we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with IBDV, and the quantification of viral replication. The addition of chicken CD40 ligand significantly increased cell proliferation fourfold over six days of culture and significantly enhanced cell viability. Two strains of IBDV, a cell-culture adapted strain, D78, and a very virulent strain, UK661, replicated well in the ex vivo cell cultures. This model will be of use in determining how cells respond to IBDV infection and will permit a reduction in the number of infected birds used in IBDV pathogenesis studies. The model can also be expanded to include other viruses and could be applied to different species of birds.

An Ex Vivo Chicken Primary Bursal-cell Culture Model to Study Infectious Bursal Disease Virus Pathogenesis

Here we describe the isolation of chicken primary bursal cells from the bursa of Fabricius, the culture and infection of the cells with infectious bursal disease virus, and the quantification of viral replication.

Infectious bursal disease virus (IBDV) is a birnavirus of economic importance to the poultry industry. The virus infects B cells, causing morbidity, ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

An Advanced Murine Model for Nonalcoholic Steatohepatitis in Association with Type 2 Diabetes

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, such as type 2 diabetes and nonalcoholic steatohepatitis (NASH). Recent research has established that pro-inflammatory immune cells infiltrate obese hypertrophic adipose tissue and liver. Given the emerging importance of immune cells in the context of metabolic homeostasis, there is a critical need to quantify and characterize their modification during the development of type 2 diabetes and NASH. However, animal models that induce pathophysiological features typical of human NASH are sparse.
In this article, we provide a detailed protocol to identify immune cell subsets isolated from liver and adipose tissue in a reliable mouse model of NASH, established by housing high-fat diet (HFD) mice under non-specific pathogen-free (SPF) conditions without a barrier for at least seven weeks. We demonstrate the handling of mice in non-SPF conditions, digestion of the tissues and identification of macrophages, natural killer (NK) cells, dendritic cells, B and T cell subsets by flow cytometry. Representative flow cytometry plots from SPF HFD mice and non-SPF mice are provided. To obtain reliable and interpretable data, the use of antibodies, accurate and precise methods for tissue digestion and proper gating in flow cytometry experiments are critical elements.
The intervention to restore physiological antigen exposure in mice by housing them in non-SPF conditions and unspecific exposure to microbial antigens could provide a relevant tool for investigating the link between immunological alterations, diet-induced obesity and related long term complications.

A simple and reliable diet-induced rodent animal model for nonalcoholic steatohepatitis (NASH) is described, achieved through non-SPF housing of the animals and administration of a specific high-fat diet. We describe identification of hepatic and adipose immune cell subsets to recapitulate human immunological conditions by exposing mice to environmental germs.

Obesity is associated with chronic low-grade inflammation and insulin resistance, contributing to an increasing prevalence of chronic metabolic diseases, ...

In Vitro ELISA Test to Evaluate Rabies Vaccine Potency

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy for the Replacement, Reduction, and Refinement of the laboratory animal testing. The development of in vitro approaches is recommended at the WHO and European levels as alternatives to the NIH test for evaluating the rabies vaccine potency. At the surface of the rabies virus (RABV) particle, trimers of glycoprotein constitute the major immunogen to induce Viral Neutralizing Antibodies (VNAbs). An ELISA test, where Neutralizing Monoclonal Antibodies (mAb-D1) recognize the trimeric form of the glycoprotein, has been developed to determine the contents of the native folded trimeric glycoprotein along with the production of the vaccine batches. This in vitro potency test demonstrated a good concordance with the NIH test and has been found suitable in collaborative trials by RABV vaccine manufacturers and OMCLs. Avoidance of animal use is an achievable objective in the near future.
The method presented is based on an indirect ELISA sandwich immunocapture using the mAb-D1 which recognizes the antigenic sites III (aa 330 to 338) of the trimeric RABV glycoprotein, i.e., the immunogenic RABV antigen. mAb-D1 is used for both coating and detection of glycoprotein trimers present in the vaccine batch. Since the epitope is recognized because of its conformational properties, the potentially denatured glycoprotein (less immunogenic) cannot be captured and detected by the mAb-D1. The vaccine to be tested is incubated in a plate sensitized with the mAb-D1. Bound trimeric RABV glycoproteins are identified by adding the mAb-D1 again, labeled with peroxidase and then revealed in the presence of substrate and chromogen. Comparison of the absorbance measured for the tested vaccine and the reference vaccine allows for the determination of the immunogenic glycoprotein content.

Here we describe an indirect ELISA sandwich immunocapture to determine the immunogenic glycoprotein contents in rabies vaccines. This test uses a neutralizing Monoclonal Antibody (mAb-D1) recognizing glycoprotein trimers. It is an alternative to the in vivo NIH test to follow the consistency of vaccine potency during production.

The growing global concern for the animal welfare is encouraging manufacturers and the National Control Laboratories (OMCLs) to follow the 3Rs strategy ...