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 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.
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.
1. Risks and Permissions
2. Skin Graft
3. Infection
4. Sacrifice
5. Organ CFU Counts
6. Histology/Immunohistochemistry
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 105 CFU/ml circulating in the blood (Figure 1A). After 6 hr the counts averaged 4.8 x 104 CFU/ml. By 24 hr the average counts were 2.4 x 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 104 CFU/mg of tissue at 6 hr and 4.4 x 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 (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 'adhesin' 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).
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 Melicanet al.18 Click here to view larger image.
Figure 2. 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 – 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, and 2F are modified from Melican et al.18 Click here to view larger image.
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, 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. 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 'humanization' 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.
The authors have nothing to disclose.
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 Hôpital Européen Georges-Pompidou (HEGP), Dr. David Maladry. 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 médicale) equipment grant, the IBEID Laboratory of excellence consortium, ANR (Agence Nationale pour la Recherche) grant “Bugs-in-flow”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
DMEM | Gibco Invitrogen | 31885-023 | |
Phosphate buffered saline | Gibco Invitrogen | 10010-056 | |
Ketamine 500 | Virbac France | LOT N°VAL4243 | |
Xylazine | Bayer Healthcare | AMM N° FR/8146715 2/1980 | LOT N° KPO809S |
(Rompun 2%) | |||
Optigel | Europhta | Medicament autorisé N°3400933521134 | |
Lacrigel | |||
Tronothane | Lisa Pharma | ||
GC agar Base | Conda | 1106 | |
Human endothelium SFM media | Gibco Invitrogen | 11111 | |
Fetal bovine serum | P A A | A15-101 | |
Human transferrin | Sigma-Aldrich | T3309 | |
UEA lectin – rhodamine | Vector Labs | RL-1062 | |
Hematoxylin | Sigma-Aldrich | H9627 | |
Eosin | Sigma-Aldrich | E4009 | |
Xylene | Sigma-Aldrich | 534056 | |
Humeca BV, Holland | 4.SB01 | ||
Equiptment Name | Company | Catalogue Number | |
Sober Hand Dermatome | Humeca BV, Holland | 4.SB01 | |
Animal housing | Innovive | M-BTM-C8 | |
Biopsy punch (4 mm) | Dominic Dutscher | 30737 | |
Fast-Prep lysing matrix M tubes | MP Bio | 116923050 | |
MagNA Lyzer Green Beads | Roche | 3358941001 | |
MagNA Lyzer | Roche | 3358976001 | |
Vectashield mounting media | Vector Labs | H-1000 | |
Vetbond | 3M | 1469SB | Tissue Glue |
OCT tissue tek | Sakura | 4583 |