Name: humanized mouse model to study bacterial infections targeting the microvasculature
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The authors would like to thank all members of the Dumenil lab, particularly Silke Silva for critical reading of the manuscript. The surgery department at H&#244;pital Europ&#233;en Georges-Pompidou (HEGP), Dr. David Maladry.&#160; Michael Hivelin and Dr. Patrick Bruneval, Pathology Department at HEGP. The animal facility at PARCC, headed by Elizabeth Huc. This work was supported by the following grant agencies: Marie Curie IEF fellowship no. 273223 (KM), ATIP-Avenir Grant from INSERM, CODDIM equipment grant (Ile de France Region), FRM (fondation pour la recherche m&#233;dicale) equipment grant, the IBEID Laboratory of excellence consortium, ANR (Agence Nationale pour la Recherche) grant &#8220;Bugs-in-flow&#8221;. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

1. Risks and Permissions
<ol>
	<li>Local ethics committees must approve all animal protocols. This protocol was conducted in accordance with guidelines established by the French and European regulations for the care and use of laboratory animals (D&#233;crets 87-848, 2001-464, 2001-486 and 2001-131 and European Directive 2010/63/UE) and approved by the local ethical committee Comit&#233; d'Ethique en mati&#232;re d'Exp&#233;rimentation Animale, Universite Paris Descartes, Paris, France. No: CEEA34.GD.002.11.</li>
	<li>For human skin, written informed patient consent was obtained and all procedures were performed according to French national guidelines and approved by the local ethical committee, Comit&#233; d'Evaluation Ethique de l'INSERM IRB 00003888 FWA 00005881, Paris, France Opinion: 11-048.</li>
	<li>N. meningitidis is a potentially harmful human pathogen and all necessary precautions must be taken when handling the organism. These precautions include vaccination and working under Biosafety level 2 conditions.</li>
</ol>
2. Skin Graft
<ol>
	<li>Collect human skin as close to grafting time as possible. The majority of skin in this work comes from plastic surgery operations involving the abdominal area. Other sources such as breast, leg and even face have been used with success.</li>
	<li>Prepare the human skin sample by cleaning skin surface with 70% EtOH and lying it flat on a well-cleaned surface, preferably under a flow hood.</li>
	<li>Slice 200-400 &#181;m slices from the top of the skin using a dermatome with preset thickness.</li>
	<li>Check the skin slices for integrity and cut them into 2 cm x 2 cm squares, then place in PBS (without divalent cations) if grafting immediately or at 4 &#176;C in DMEM with 10% serum for short-term storage (3-12 hr).</li>
	<li>Anaesthetize 6-8 week old SCID.bg mice (approximately 20 g) with Ketamine (100 mg/kg) and Xylazine (10 mg/kg) injected i.p.</li>
	<li>Once the mice are anaesthetized, check for pain response by performing a toe pinch, a standard method for establishing pain reflex. Apply gel to eyes to prevent corneal drying and shave the left side of the animal behind the shoulder, where the graft will be placed. Keep the mouse warm on a heat pad throughout the procedure.</li>
	<li>After prepping mouse for surgery, clean the shaved area with 70% EtOH.</li>
	<li>Apply local anesthetic topically to the graft site (Tronothane) and prepare the graft site by carefully removing the superficial layer of mouse skin with a pair of curved scissors. Be careful not to disrupt the underlying vasculature.</li>
	<li>Remove skin in an area a bit smaller than the area of the human skin slice.</li>
	<li>Remove skin slice from PBS (or DMEM) and place over graft bed, ensuring the epidermal side is facing up. Steps 2.8-2.10 should be performed as quickly as possible, ensuring the graft bed does not dry out.</li>
	<li>Align one corner or edge and fix with tissue glue. Carefully fit the rest of the skin slice to the graft bed, placing small amounts of tissue glue around the edges. Always trim the human skin to size rather than make the graft bed larger.</li>
	<li>Do not pull the skin too tight over the graft bed as the mouse skin is very flexible.</li>
	<li>Make sure the graft is fixed with tissue glue in each corner.</li>
	<li>Clean the area with an iodine solution.</li>
	<li>Apply a Band-Aid with the cushion area over the graft. Make sure the Band-Aid is firm but not tight and that the animal's breathing is not affected.</li>
	<li>Fix the Band-Aid with dressing tape.</li>
	<li>Allow the animal to recover on a warm surface.</li>
	<li>Once awake, return the animal to its cage. Animals are housed 2-3 per cage.</li>
	<li>Check mice regularly during recovery for signs of pain or distress. 10-14 days after grafting, remove the Band-Aid, being careful not to disrupt the graft. In our experience postoperative pain is minimal. Nevertheless animals should be monitored every day in particular in the few days following operation. Criteria for pain or distress are the following.
	<ul>
		<li>Loss of weight over 10%</li>
		<li>Physical appearance, fur, hunchback position, eyes</li>
		<li>Increased respiration or heart rates</li>
		<li>Decreased or unusual movements.</li>
		<li>Rise in body temperature</li>
		<li>In case signs of pain are detected paracetamole is administered orally (300 mg/kg, every 4 hr). Humane endpoints are strictly adhered to if the pain persists.</li>
	</ul>
	</li>
	<li>If the graft looks dry apply baby oil every 2-3 days to keep the skin supple.</li>
	<li>The graft is ready for experimentation 21 days post graft.</li>
</ol>
3. Infection
<ol>
	<li>The day before infection, prepare bacterial strain by streaking onto GCB agar plates with appropriate antibiotics. Grow overnight at 37 &#176;C in 5% CO2.</li>
	<li>Bacteria preparation
	<ol>
		<li>Reinoculate bacteria into 5 ml of Human endothelium SFM media with 10% serum to an OD600 0.05.</li>
		<li>Incubate while shaking for 2 hr in incubator (37 &#176;C in 5% CO2) with loose lid, until a bacterial density of approximately OD600 0.1.</li>
		<li>Centrifuge the bacteria for 3 min 15,000 rpm.</li>
		<li>Remove supernatant and resuspend in PBS, then centrifuge again.</li>
		<li>Repeat the wash step (2.2.4-2.2.5) twice.</li>
		<li>Resuspend bacterial pellet in PBS.</li>
		<li>Measure OD600.</li>
		<li>Make a culture of either OD600 0.1 (108 CFU/ml), or OD600 1.0 (109 CFU/ml). From this known concentration, dilute to 107 CFU/ml. Try to prepare bacteria as close to injection time as possible.</li>
	</ol>
	</li>
	<li>Mice
	<ol>
		<li>Weigh mice.</li>
		<li>Anaesthetize mice with Ketamine (100 mg/kg) and Xylazine (10 mg/kg) injected i.p.</li>
		<li>Once asleep, check that the pain response is absent by a toe-pinch, keep the mice warm with a heat pad and put eye ointment on to prevent drying.</li>
		<li>Give each mouse 8 mg of human transferrin, resuspended in saline or PBS, injected i.p.</li>
		<li>Take a small drop of blood from the tail and spread onto an agar plate to check blood is sterile prior to infection.</li>
		<li>Inject 100 &#181;l 107 CFU/ml bacterial culture (106 CFU total) intravenously via the retro-orbital or tail vein. Measurements of CFU taken 5 min post injection are consistent regardless of injection site.</li>
		<li>After a few minutes, snip the tail end and spread blood sample onto agar plates with appropriate antibiotics with one 10-fold dilution to establish blood CFU immediately following infection.</li>
		<li>Seal the tail with tissue glue.</li>
		<li>After injection, dilute the bacterial inoculum and spread onto agar plates with appropriate antibiotics to establish CFU.</li>
		<li>Allow the mice to recover on a heat pad until they are up and moving</li>
		<li>At 6 hr post infection draw a small amount of blood from the tail, dilute and plate to establish CFU at 6 hr.</li>
		<li>Incubate all agar plates at 37 &#176;C in 5% CO2 overnight.</li>
	</ol>
	</li>
</ol>
4. Sacrifice
<ol>
	<li>At the chosen time of infection, anaesthetize mice with Ketamine (100 mg/kg) and Xylazine (10 mg/kg) injected i.p.</li>
	<li>Once asleep, keep the mouse warm with a heat pad.</li>
	<li>When the pain reflex has gone (toe-pinch), snip end of tail and draw blood.</li>
	<li>Spread blood onto agar plates with appropriate antibiotics; 5 &#956;l direct and 10 &#181;l 1:10 dilution to establish CFU.</li>
	<li>Sacrifice the mouse by cervical dislocation.</li>
	<li>After sacrificing the mouse according to all relevant institutional and ethical guidelines, confirm death by lack of heart beat. Make a midline incision, cut through rib cage, and make an incision in the heart.</li>
	<li>Collect as much blood as possible from the heart/chest cavity and place in a sterile tube.</li>
	<li>Remove organs (e.g. liver, spleen, brain) into a sterile plate.</li>
	<li>Remove the skin graft and a section of mouse skin into the sterile plate.</li>
	<li>Dispose of the mouse body.</li>
	<li>After the collected blood has been allowed to clot for 15 min, centrifuge it 15 min at 3,000 rpm.</li>
	<li>Remove plasma from blood and store at -80 &#176;C for later analysis, for example for cytokine detection by ELISA or FACS based methods.</li>
</ol>
5. Organ CFU Counts
<ol>
	<li>Weigh homogenizing tubes containing 500 &#956;l of PBS each prior to taking biopsy samples.</li>
	<li>Take 4 mm biopsy samples from each tissue using a sterile biopsy punch.</li>
	<li>Place biopsies into preweighed homogenizing tubes containing 500 &#181;l PBS. Homogenize skin samples with 2 mm beads, use smaller beads for other organs.</li>
	<li>Place the remaining tissues in 4% Paraformaldehyde (PFA) and store at 4 &#176;C for microscopy (see below).</li>
	<li>Weigh homogenizing tubes containing the biopsies to establish biopsy weight (for later calculation of CFU/mg tissue).</li>
	<li>Homogenize samples.
	<ol>
		<li>Homogenize liver, spleen and brain, at 6,000 rpm for 30 sec.</li>
		<li>Homogenize skin samples at 6,000 rpm for 30 sec and repeat if necessary.</li>
	</ol>
	</li>
	<li>Spread dilutions from each homogenizing tube onto agar plates and grow overnight at 37 &#176;C in 5% CO2 to establish organ CFU counts.</li>
</ol>
6. Histology/Immunohistochemistry
<ol>
	<li>Fixation
	<ol>
		<li>Fix the tissues in 4% PFA overnight at 4 &#176;C. The fixation step may be longer; if storing for longer periods place the tissues in 0.25% PFA.</li>
		<li>Rinse the tissues in PBS and then place in 20% sucrose solution and return to 4 &#176;C overnight or until the tissues have sunk in the solution.</li>
		<li>Wash the tissues in PBS, cut to size and place in OCT solution (either in a tissue mold or adhered to a base board).</li>
		<li>Quickly freeze the tissues on a Petri dish floating in liquid nitrogen.</li>
		<li>After freezing, put the tissues onto dry ice before transfer to -80 &#176;C for storage.</li>
	</ol>
	</li>
	<li>Slicing
	<ol>
		<li>Remove tissues from -80 &#176;C and allow to equilibrate to -20 &#176;C in a cryostat.</li>
		<li>Slice tissues with a thickness of approximately 10-15 &#181;m and place onto coated microscopy slides.</li>
		<li>Store slides at -20 &#176;C until needed.</li>
	</ol>
	</li>
	<li>Staining
	<ol>
		<li>Allow slides to come to room temperature.</li>
		<li>Draw around the slice on the slide with a hydrophobic pen. Wait 5-10 min.</li>
		<li>Rehydrate/wash slice in PBS for 5 min, repeat twice.</li>
		<li>Permeabilize slice in 0.1% Triton in PBS for 10 min.</li>
		<li>Wash in PBS 2-3x for 5 min.</li>
		<li>Block in PBS 0.1% Triton 1% BSA for between 10-60 min depending on the antibody or stain to be used.</li>
		<li>Remove blocking buffer and add primary antibody diluted as required in block buffer. Incubate for determined time. (Often 1 hr at room temperature or overnight at 4 &#176;C).</li>
		<li>Wash in PBS 2-3x for 5 min.</li>
		<li>Apply secondary antibody diluted as appropriate in bock buffer; DAPI is typically included in this step. Incubate for determined time, often 1-2 hr room temperature, in the dark.</li>
		<li>Wash in PBS 2-3x for 5 min.</li>
		<li>Mount in fade protecting mounting media with a cover glass and seal with nail varnish.</li>
		<li>Image stained slides immediately or store at 4 &#176;C.</li>
		<li>Perform imaging with a confocal or fluorescent microscopy set up with filters/lasers appropriate to fluorophores used.</li>
	</ol>
	</li>
	<li>Histology
	<ol>
		<li>Allow slides to be used for histology to come to room temperature.</li>
		<li>Stain slides using a standard hematoxylin /eosin staining protocol.
		<ol>
			<li>Place the slides in slide rack.</li>
			<li>Transfer rack with slides between solutions in the following order:</li>
			<li>2 sec into running tap water.</li>
			<li>3 min in filtered hematoxylin solution.</li>
			<li>10 sec into running water.</li>
			<li>10 sec into distilled water.</li>
			<li>10 sec into ethanol 100%.</li>
			<li>2 sec into filtered eosin.</li>
			<li>10 sec into running water.</li>
			<li>10 sec into ethanol 100%</li>
			<li>2 min into xylene x 3 (bath I, II, III).</li>
			<li>Transfer immediately and add few drops of fixation glue onto each slides, place cover slip on top, remove air bubbles, let dry for a few hours.</li>
			<li>Image slides under a standard white light microscope.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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The authors declare no competing financial interests.

Animal models are critically important to bacterial pathogenesis research. It is impossible to fully mimic the in vivo environment in cell culture and it is becoming apparent that host-pathogen interaction is influenced by many dynamic factors. The human specificity of some clinically important pathogens, such as N. meningitidis, HIV, HCV, Plasmodium falciparum, Listeria monocytogenes,&#160;and Salmonella typhi has limited the use of in vivo models for these infections. However, as we begin to understand which infectious steps are involved in the specificity, humanized models are being developed. The protocol described here is a demonstration of this with the introduction of human microvessels into mice, allowing for extensive in vivo infection with N. meningitidis, resulting in vascular damage and occasionally the development of purpuric rashes.
Using this model, we have been able to define that the adhesive properties of Tfp are involved in vascular colonization in vivo by using bacterial mutants and that the vascular damage is reduced in the absence of adhesion18. Previously, circulating bacterial products have been implicated in this damage but our results suggest a decisive role for local adhesion and vascular colonization. This opens up new possibilities for the development of novel treatment targets. If adhesion of pathogenic bacteria could be blocked pharmaceutically it could possibly prevent the development of dermal lesions and lead to better outcomes for meningococcal survivors in terms of tissue necrosis, debridement and amputations.&#160;The work has also demonstrated the complexity of the infection and the involvement of the immune response and coagulation cascade. We identified human cytokine signaling in the serum of infected mice despite the relatively small amount of human endothelium present18. This indicated a significant cytokine response, along with the infiltration of mouse immune cell populations into the area.
Animal models can of course never fully replicate human disease and all results garnered from them must be considered with this in mind. For instance, in this model the blood and circulating cells are of mouse origin and we cannot discount that they may behave differently to human cells. An advantage of this however, as demonstrated in our recent publication18, is the ability to differentiate signaling originating from the human endothelium from that of the circulating mouse cells. The immunocompromised background of the mice used in this model would also allow for the allogenic transfer of human immune cell populations, adding a further &#39;humanization&#39; aspect. The immunocompromised background of the mice may however mask a role for NK, T or B cells, which are all lacking or defective in this model. The relatively short timeframe (24 hr) used in this model mainly concerns the innate response, but for longer-term infections and the development of immunity other options may need to be explored.
The skin is an important infection site for N. meningitidis but having a relatively small amount of human vessels also means that extrapolating the data to a systemic infection involving numerous organs is difficult. While this model allows for the study of dermal lesion development, important steps of meningococcal infection such as epithelial and blood-brain crossing are not included. Further development of these humanized models is needed to address these other aspects of infection. Nevertheless this model offers great potential for numerous human specific pathogens, particularly those targeting the blood vessels.

Meningococcal sepsis is a frequently fatal blood born infection caused by the bacterial pathogen Neisseria meningitidis. Meningococcal sepsis patients often present with a petechial or purpuric rash on their skin that has previously been associated with vascular destruction caused by circulating bacteria and bacterial products1. Skin biopsies from clinical patients show bacteria associated with microvessels, often filling the vessels2. Apart from the bacteria, extensive thrombosis, coagulation, congestion and vascular leak is seen in the purpuric regions3-5. This vascular damage can lead to extensive necrosis of the skin and surrounding tissues, resulting in debridement and even amputation in meningococcal survivors. Understanding how infection causes this vascular damage is important to optimize prevention and treatment strategies. The majority of research on meningococcal sepsis has been performed in vitro on human cell lines due to the human specificity of N. meningitidis. Many aspects of infection have been studied in vitro including bacterial adhesion, host cell response as well as cytokine response6-9. Type IV pili (Tfp) have been implicated as the major adhesion organelle for N. meningitidis on both epithelial and endothelial cells10. It has also been shown that adhesion of N. meningitidis to host cells is shear stress dependent and is therefore thought to be related to blood flow rates in the microvasculature11. This suggests the dynamic stresses the bacteria face in vivo are crucial to pathogenesis. It is however very difficult to model the microenvironment of small vessels in vitro.
The adhesion receptor for Neisseria Tfp is still unknown and therefore knock-in strategies to achieve bacterial adhesion in an animal model cannot be envisaged at this time. CD46 was suggested to be the Tfp receptor and transgenic animals were produced to act as mouse models. However, infection in these animals does not lead to extensive infection or to rash development12,13. Other animal models that have been described for the bacteremia aspect of Neisseria infection take into account the bacterial preference for human transferrin as an iron source14,15. Either supplementing human transferrin or expressing it from a transgene results in an increased bacterial load in the blood stream over an extended time period, but this model shows no bacterial adhesion or rash development16,17.
In this protocol, we describe a humanized mouse model in which human skin, including the dermal microvasculature, is transplanted onto immunocompromised mice18,19. This results in functional human vessels, perfused with the mouse circulation. Combined with human transferrin supplementation, this model accounts for at least two of the human specific aspects of N. meningitidis, i.e. human endothelium and human transferrin, in an in vivo environment. N. meningitidis introduced intravenously into this model adhere specifically to the human endothelium, producing a pathology that is similar to what is reported in clinical patients, including vascular damage and purpuric rash development18.

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			<td height="21" style="height:21px;width:247px;">MagNA Lyzer</td>
			<td style="width:161px;">Roche</td>
			<td style="width:119px;">3358976001</td>
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			<td style="width:161px;">Sakura</td>
			<td style="width:119px;">4583</td>
			<td></td>
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humanized mouse model to study bacterial infections targeting the microvasculature

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood vessels resulting in vascular leak, the development of purpuric rashes and eventual tissue necrosis. Studying the pathogenesis of this infection was previously limited by the human specificity of the bacteria, which makes in vivo models difficult. In this protocol, we describe a humanized model for this infection in which human skin, containing dermal microvessels, is grafted onto immunocompromised mice. These vessels anastomose with the mouse circulation while maintaining their human characteristics. Once introduced into this model, N. meningitidis adhere exclusively to the human vessels, resulting in extensive vascular damage, inflammation and in some cases the development of purpuric rash. This protocol describes the grafting, infection and evaluation steps of this model in the context of N. meningitidis infection. The technique may be applied to numerous human specific pathogens that infect the blood stream.

CFU counts
The N. meningitidis strain used in these representative results is N. meningitidis 8013 clone 12, a serogroup C clinical isolate, expressing a class I SB pilin, Opa-, Opc-, PilC1+/PilC2+ 20. The strain had been engineered to express green fluorescent protein (GFP) from a chromosomal insertion18. Bacterial colony forming unit counts are established by counting the number of colonies on the agar plates and calculating either CFU/ml of blood or CFU/mg of tissue from the known volumes plated. Blood counts showed that 5 min after i.v. injection of 106 CFU bacteria there was an average of 1.5 x&#160;105 CFU/ml circulating in the blood (Figure 1A). After 6 hr the counts averaged 4.8 x&#160;104 CFU/ml. By 24 hr the average counts were 2.4 x&#160;104 CFU/ml but in this group of 10 mice, 5 had no detectable circulating bacteria while the other 5 had relatively high counts (Figure 1A). CFU counts taken from the skin samples show the majority of mice having considerable counts in the human skin, averaging 2.1 x&#160;104 CFU/mg of tissue at 6 hr and 4.4 x&#160;102 CFU/mg of tissue at 24 hr (Figure 1B). The contralateral mouse skin samples showed no bacterial counts at 24 hr and only a very low number at 6 hr, demonstrating the strong preference for N. meningitidis to the human vessels in the grafted skin (Figure 1B). In general bacterial counts were very low to nondetectable in the other organs sampled although 2 animals did show counts which may be attributed to contamination from the blood, as they correlated with high circulating bacterial numbers&#160;(Figure 1C). The model can also be used to determine the role of virulence factors in vivo, for example the type IV pili. Bacterial strains with defined mutations in the pilD gene21, resulting in a strain lacking Tfp , as well as the pilC1 gene8, lacking the proposed Tfp &#39;adhesin&#39; were introduced into the model. These mutations resulted in no bacterial adhesion in the human skin graft, confirming the crucial role Tfp is playing in adhesion in vivo (Figure 1D).
Immunohistochemistry/Histology
In these experiments we used N. meningitidis strains that express green fluorescence protein (GFP) to enable fluorescent detection without the need for secondary staining. Human vessels were stained using a marker for human endothelium, either CD31 (PECAM-1) or the lectin Ulex europaeus agglutinin (UEA)18,22. The UEA was conjugated to rhodamine allowing one-step staining (Figures 2A-C). Cell nuclei can be stained with DAPI to help identify tissue structures (Figures 2A-B). Histology was performed using a standard hematoxylin/eosin staining. The epidermal/dermal border of the skin was clearly identifiable. Inflammation, thrombosis and vascular leak were concentrated to vessels close to this border 24 hr after infection (Figure 2D). Thrombosis was visible in several small dermal vessels, often accompanied by congestion and inflammation. Leakage of red blood cells out into the tissues could be seen, indicating an extensive level of vessel damage. The histopathology of grafted skin in noninfected mice appeared normal with no distinguishable inflammation18. In about 30% of the infections bacterial adhesion in the skin leads to the development of macroscopically detectable purpura (Figures 2E and 2F).
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/51134/51134fig1highres.jpg" src="/files/ftp_upload/51134/51134fig1.jpg" /> 
Figure 1. CFU counts. (A) Bacterial CFU counts per ml of blood at 5 min, 6 hr, and 24 hr post infection. (B) Bacterial CFU counts from skin samples comparing human skin (HS) and mouse skin (MS) at both 6 hr and 24 hr post infection. (C) Bacterial CFU counts from other organs taken 24 hr post infection. (D) Comparing bacterial CFU counts from human and mouse skin samples taken at 24 hr post infection from mice infected with the wild type N. meningitidis 2C43 stain (WT), a strain with a mutation in the pilD gene (pilD) or a strain with a mutation in the pilC1 gene (pilC1). All graphs are shown as raw data with median. Figure is modified from&#160;Melicanet al.18&#160;<a href="https://www.jove.com/files/ftp_upload/51134/51134fig1highres.jpg" target="_blank">Click here to view larger image</a>.

<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/51134/51134fig2highres.jpg" src="/files/ftp_upload/51134/51134fig2.jpg" /> 
Figure 2.&#160;Microscopy. (A) Projection of a confocal stack showing a human microvessel stained with UEA lectin (red) close to the dermal (d) epidermal (e) border. The vessel is infected with N. meningitidis (green) 2 hr post infection. (B) An optical slice and a slice projection (C) showing N. meningitidis microcolonies (green) infection a human vessel (UEA &#8211; red) within a skin graft 2 hr post infection. (D) Haematoxylin/Eosin staining of human skin graft infected for 24 hr with N. meningitidis. The epidermal (e), dermal (d) border is clearly visible and extensive thrombosis and congestion of microvessels (arrows) is seen in the dermis. Inflammation and some vascular leak (arrowhead) can also be identified. (E) Human skin graft prior to infection. (F) The same skin graft as (E) 24 hr post infection with N. meningitidis showing purpuric regions (arrows). Figures 2B, 2D, 2E,&#160;and 2F are modified from&#160;Melican&#160;et al.18&#160;<a href="https://www.jove.com/files/ftp_upload/51134/51134fig2highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature at JoVE.com

Humanized Mouse Model to Study Bacterial Infections Targeting the Microvasculature

Neisseria meningitidis is a human specific pathogen that infects blood vessels. In this protocol human microvessels are introduced into a mouse by grafting human skin onto immunocompromised mice. Bacteria adhere extensively to the human vessels, leading to vascular damage and development of the purpuric rash typically observed in human cases.

Neisseria meningitidis causes a severe, frequently fatal sepsis when it enters the human blood stream. Infection leads to extensive damage of the blood ...

humanized-mouse-model-to-study-bacterial-infections-targeting

Research

JoVE Journal

Immunology and Infection

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Introducing Shear Stress in the Study of Bacterial Adhesion

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the initial and determining step of the pathogenesis. Although experimentally adhesion is mostly studied in static conditions adhesion actually takes place in the presence of flowing liquid. First encounters between bacteria and their host often occur at the mucosal level, mouth, lung, gut, eye, etc. where mucus flows along the surface of epithelial cells. Later in infection, pathogens occasionally access the blood circulation causing life-threatening illnesses such as septicemia, sepsis and meningitis. A defining feature of these infections is the ability of these pathogens to interact with endothelial cells in presence of circulating blood. The presence of flowing liquid, mucus or blood for instance, determines adhesion because it generates a mechanical force on the pathogen. To characterize the effect of flowing liquid one usually refers to the notion of shear stress, which is the tangential force exerted per unit area by a fluid moving near a stationary wall, expressed in dynes/cm2. Intensities of shear stress vary widely according to the different vessels type, size, organ, location etc. (0-100 dynes/cm2). Circulation in capillaries can reach very low shear stress values and even temporarily stop during periods ranging between a few seconds to several minutes 1. On the other end of the spectrum shear stress in arterioles can reach 100 dynes/cm2 2. The impact of shear stress on different biological processes has been clearly demonstrated as for instance during the interaction of leukocytes with the endothelium 3. To take into account this mechanical parameter in the process of bacterial adhesion we took advantage of an experimental procedure based on the use of a disposable flow chamber 4. Host cells are grown in the flow chamber and fluorescent bacteria are introduced in the flow controlled by a syringe pump. We initially focused our investigations on the bacterial pathogen Neisseria meningitidis, a Gram-negative bacterium responsible for septicemia and meningitis. The procedure described here allowed us to study the impact of shear stress on the ability of the bacteria to: adhere to cells 1, to proliferate on the cell surface 5and to detach to colonize new sites 6 (Figure 1). Complementary technical information can be found in reference 7. Shear stress values presented here were chosen based on our previous experience1 and to represent values found in the literature. The protocol should be applicable to a wide range of pathogens with specific adjustments depending on the objectives of the study.

During the infection process, a key step is the adhesion of pathogens with host cells. In most instances this adhesion step occurs in the presence of mechanical stress generated by flowing liquid. We describe a technique that introduces shear stress as an important parameter in the study of bacterial adhesion.

During bacterial infections a sequence of interactions occur between the pathogen and its host. Bacterial adhesion to the host cell surface is often the ...

Using the BLT Humanized Mouse as a Stem Cell based Gene Therapy Tumor Model

Small animal models such as mice have been extensively used to study human disease and to develop new therapeutic interventions. Despite the wealth of information gained from these studies, the unique characteristics of mouse immunity as well as the species specificity of viral diseases such as human immunodeficiency virus (HIV) infection led to the development of humanized mouse models. The earlier models involved the use of C. B 17 scid/scid mice and the transplantation of human fetal thymus and fetal liver termed thy/liv (SCID-hu) 1, 2 or the adoptive transfer of human peripheral blood leukocytes (SCID-huPBL) 3. Both models were mainly utilized for the study of HIV infection.
One of the main limitations of both of these models was the lack of stable reconstitution of human immune cells in the periphery to make them a more physiologically relevant model to study HIV disease. To this end, the BLT humanized mouse model was developed. BLT stands for bone marrow/liver/thymus. In this model, 6 to 8 week old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) immunocompromised mice receive the thy/liv implant as in the SCID-hu mouse model only to be followed by a second human hematopoietic stem cell transplant 4. The advantage of this system is the full reconstitution of the human immune system in the periphery. This model has been used to study HIV infection and latency 5-8.
We have generated a modified version of this model in which we use genetically modified human hematopoietic stem cells (hHSC) to construct the thy/liv implant followed by injection of transduced autologous hHSC 7, 9. This approach results in the generation of genetically modified lineages. More importantly, we adapted this system to examine the potential of generating functional cytotoxic T cells (CTL) expressing a melanoma specific T cell receptor. Using this model we were able to assess the functionality of our transgenic CTL utilizing live positron emission tomography (PET) imaging to determine tumor regression (9).
The goal of this protocol is to describe the process of generating these transgenic mice and assessing in vivo efficacy using live PET imaging. As a note, since we use human tissues and lentiviral vectors, our facilities conform to CDC NIH guidelines for Biosafety Level 2 (BSL2) with special precautions (BSL2+). In addition, the NSG mice are severely immunocompromised thus, their housing and maintenance must conform to the highest health standards (<a href="http://jaxmice.jax.org/research/immunology/005557-housing.html" target="_blank">http://jaxmice.jax.org/research/immunology/005557-housing.html</a>).

The generation and characterization of tumor specific T cells using humanized mice is described here. Human thymic tissue and genetically modified human hematopoietic stem cells are transplanted into immunocompromised mice. This results in the reconstitution of an engineered human immune system allowing for in vivo examination of anti-tumor immune responses.

Small animal models such as mice have been extensively used to study human  disease and to develop new therapeutic interventions. Despite the wealth of  ...

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system1. Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides2,3. In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals1. Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections.
Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis4-7. Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model8-11. Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality.
G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 &deg;C to 30 &deg;C) and infection studies can be conducted between 15 &deg;C to above 37 &deg;C12,13, allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD5014, measurement of bacterial survival15,16 and examination of the infection process17. Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections18,19.

The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis

Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of ...

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the respective immune response to each single pathogen and may thereby affect pathogenesis and disease outcome. Coinfected patients may also respond differentially to anti-infective interventions. Coinfection between tuberculosis as caused by mycobacteria and the malaria parasite Plasmodium, both of which are coendemic in many parts of sub-Saharan Africa, has not been studied in detail. In order to approach the challenging but scientifically and clinically highly relevant question how malaria-tuberculosis coinfection modulate host immunity and the course of each disease, we established an experimental mouse model that allows us to dissect the elicited immune responses to both pathogens in the coinfected host. Of note, in order to most precisely mimic naturally acquired human infections, we perform experimental infections of mice with both pathogens by their natural routes of infection, i.e. aerosol and mosquito bite, respectively.

An Experimental Model to Study Tuberculosis-Malaria Coinfection upon Natural Transmission of Mycobacterium tuberculosis and Plasmodium berghei

Tuberculosis and malaria are two of the most prevalent infections in humans and major causes of morbidity and mortality in impoverished populations in the tropics. We established an experimental model system to study outcome of malaria-tuberculosis coinfection in mice after challenge with both pathogens via their natural route of infection.

Coinfections naturally occur due to the geographic overlap of distinct types of pathogenic organisms. Concurrent infections most likely modulate the ...

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence factors, which can subvert and manipulate key host functions. The study of host/pathogen interactions is therefore extremely important to understand bacterial infections and develop alternative strategies to counter infectious diseases. This approach however, requires the development of new high-throughput assays for the unbiased, automated identification and characterization of bacterial virulence determinants. Here, we describe a method for the generation of a GFP-tagged mutant library by transposon mutagenesis and the development of high-content screening approaches for the simultaneous identification of multiple transposon-associated phenotypes. Our working model is the intracellular bacterial pathogen Coxiellaburnetii, the etiological agent of the zoonosis Q fever, which is associated with severe outbreaks with a consequent health and economic burden. The obligate intracellular nature of this pathogen has, until recently, severely hampered the identification of bacterial factors involved in host pathogen interactions, making of Coxiella the ideal model for the implementation of high-throughput/high-content approaches.

Generation and Multi-phenotypic High-content Screening of Coxiella burnetii Transposon Mutants

Coxiella burnetii is an obligate intracellular Gram-negative bacterium responsible for the zoonotic disease Q fever. Here we describe methods for the generation of Coxiella fluorescent transposon mutants as well as the automated identification and analysis of the resulting internalization, replication and cytotoxic phenotypes.

Invasion and colonization of host cells by bacterial pathogens depend on the activity of a large number of prokaryotic proteins, defined as virulence ...

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the shear stress of flowing blood.
To identify the bacterial and host factors that contribute to vascular adhesion of microorganisms, appropriate models that study these interactions under physiological shear conditions are needed. Here, we describe an in vitro flow chamber model that allows to investigate bacterial adhesion to different components of the extracellular matrix or to endothelial cells, and an intravital microscopy model that was developed to directly visualize the initial adhesion of bacteria to the splanchnic circulation in vivo. These methods can be used to identify the bacterial and host factors required for the adhesion of bacteria under flow. We illustrate the relevance of shear stress and the role of von Willebrand factor for the adhesion of Staphylococcus aureus using both the in vitro and in vivo model.

In Vitro and In Vivo Model to Study Bacterial Adhesion to the Vessel Wall Under Flow Conditions

To study the interaction of bacteria with the blood vessels under shear stress, a flow chamber and an in vivo mesenteric intravital microscopy model are described that allow to dissect the bacterial and host factors contributing to vascular adhesion.

In order to cause endovascular infections and infective endocarditis, bacteria need to be able to adhere to the vessel wall while being exposed to the ...

Human Placental and Decidual Organ Cultures to Study Infections at the Maternal-fetal Interface

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is imperative to design experiments using human cells and tissues. An advantage of organ culture is maintenance of three-dimensional (3D) structural organization and extracellular matrix. The goal of the method described here is successful establishment of ex vivo human gestational tissue organ cultures and their healthy culture maintenance for 72-96 hr. The protocol details the immediate processing of research-consented, placental and decidual specimens fresh from the operating suite. These are abundant specimens that would otherwise be discarded. Detailed instructions on the sterile collection of these samples, including morphologic details on how to select appropriate tissues to establish 3D organ cultures, is provided. Placental villous and decidual tissues are microdissected into 2-3 mm3 pieces and placed separately on matrix-lined transwell filters and cultured for several days. Villous and decidual organ cultures are well suited for the study of human host-pathogen interaction. As compared to other model organisms, these human cultures are particularly advantageous to examine mechanism of infection for pathogens that demonstrate variable patterns of host specificity. As an example, we demonstrate infection of placental and decidual organ cultures with the clinically relevant, facultative intracellular bacterial pathogen Listeria monocytogenes.

A simple method to establish primary human placental (villous) and decidual organ cultures is described. Villous and decidual organ cultures are invaluable tools for studying pathogenesis at the human maternal-fetal interface. Infection with the facultative intracellular bacterium Listeria monocytogenes is demonstrated.

The placenta shows a large degree of interspecies anatomic variability. To best understand biology and pathophysiology of the human placenta, it is ...

Methods to Study Lipid Alterations in Neutrophils and the Subsequent Formation of Neutrophil Extracellular Traps

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of lipids. The function of lipids (e.g., in host-pathogen interactions or host entry) has been reported to play a crucial role in cellular processes. Here, we show a method to determine lipid composition, with a focus on the cholesterol level of primary blood-derived neutrophils, by HPTLC in comparison to high performance liquid chromatography (HPLC). The aim was to investigate the role of lipid/cholesterol alterations in the formation of neutrophil extracellular traps (NETs). NET release is known as a host defense mechanism to prevent pathogens from spreading within the host. Therefore, blood-derived human neutrophils were treated with methyl-&#946;-cyclodextrin (M&#946;CD) to induce lipid alterations in the cells. Using HPTLC and HPLC, we have shown that M&#946;CD treatment of the cells leads to lipid alterations associated with a significant reduction in the cholesterol content of the cell. At the same time, M&#946;CD treatment of the neutrophils led to the formation of NETs, as shown by immunofluorescence microscopy. In summary, here we present a detailed method to study lipid alterations in neutrophils and the formation of NETs.

Lipids are known to play an important role in cellular functions. Here, we describe a method to determine the lipid composition of neutrophils, with emphasis on the cholesterol level, by using both HPTLC and HPLC to gain a better understanding of the underlying mechanisms of neutrophil extracellular trap formation.

Lipid analysis performed by high performance thin layer chromatography (HPTLC) is a relatively simple, cost-effective method of analyzing a broad range of ...

The WinCF Model - An Inexpensive and Tractable Microcosm of a Mucus Plugged Bronchiole to Study the Microbiology of Lung Infections

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their mucus-plugged bronchioles create a favorable habitat for microbial colonization. Various pathogens thrive in this environment interacting with each other and driving many of the symptoms associated with CF disease. Like any microbial community, the chemical conditions of their habitat have a significant impact on the community structure and dynamics. For example, different microorganisms thrive in differing levels of oxygen or other solute concentrations. This is also true in the CF lung, where oxygen concentrations are believed to drive community physiology and structure. The methods described here are designed to mimic the lung environment and grow pathogens in a manner more similar to that from which they cause disease. Manipulation of the chemical surroundings of these microbes is then used to study how the chemistry of lung infections governs its microbial ecology. The method, called the WinCF system, is based on artificial sputum medium and narrow capillary tubes meant to provide an oxygen gradient similar to that which exists in mucus-plugged bronchioles. Manipulating chemical conditions, such as the media pH of the sputum or antibiotics pressure, allows for visualization of the microbiological differences in those samples using colored indicators, watching for gas or biofilm production, or extracting and sequencing the nucleic acid contents of each sample.

The mucus plugged airways of cystic fibrosis (CF) patients are an ideal environment for microbial pathogens to thrive. The manuscript describes a novel method for studying the CF lung microbiome in an environment that mimics where they cause disease and how alterations of chemical conditions can drive microbial dynamics.

Many chronic airway diseases result in mucus plugging of the airways. Lungs of an individual with cystic fibrosis are an exemplary case where their ...