key: cord-0266531-2qewcuy3
authors: Manriquez, Valeria; Nivoit, Pierre; Urbina, Tomas; Echenique-Rivera, Hebert; Melican, Keira; Flamant, Patricia; Schmitt, Taliah; Bruneval, Patrick; Obino, Dorian; Duménil, Guillaume
title: Colonization of dermal arterioles by Neisseria meningitidis provides a safe haven from neutrophils
date: 2021-01-07
journal: bioRxiv
DOI: 10.1101/2021.01.07.425689
sha: 151d5fb6900975572fbe97f7c2667ccabc7eae71
doc_id: 266531
cord_uid: 2qewcuy3

Neisseria meningitidis, a human-specific bacterium, is responsible for meningitis and fatal fulminant systemic disease. Bacteria colonize blood vessels, rapidly causing devastating vascular damage despite a neutrophil-rich inflammatory infiltrate. How this pathogen escapes the neutrophil response is unknown. Using a humanized mouse model, we show that vascular colonization leads to the recruitment of neutrophils, partially reducing bacterial burden and vascular damage. This partial effect is due to the ability of bacteria to indiscriminately colonize capillaries, venules and arterioles, as observed in human samples. In venules, potent neutrophil recruitment allows efficient bacterial phagocytosis. In contrast, in infected capillaries and arterioles adhesion molecules such as E-Selectin are not expressed on the endothelium and intravascular neutrophil recruitment is minimal. These results show that colonization of capillaries and arterioles by N. meningitidis create an intravascular niche that preclude the action of neutrophils, resulting in immune escape and subsequent fulminant progression of the infection.

Infections caused by Neisseria meningitidis are characterized by an unusually fast progression of the disease as if bacterial proliferation could not be contained by the innate immune system. Although N. meningitidis is best known for causing cerebrospinal meningitis, the systemic septic form of the infection is responsible for 90% of the mortality attributed to meningococcal infections 1, 2 . In both cases, the infection is characterized by a fulminant evolution and diseases can progress from relatively unspecific "flu-like" symptoms to a life-threatening condition in less than 24 hours 1 . Meningococcal systemic infections are characterized by a typical skin rash called Purpura indicating a perturbation of the vascular function 3, 4 . Clinical studies have revealed major perturbations in vascular function in infected tissues with congestion, coagulation and the loss of vascular integrity 5, 6 . Neisseria meningitidis thus rapidly causes major vascular damages leading to a fatal outcome in absence of treatment as if the first lines of immune defence were not sufficient.

How N. meningitidis evades innate immunity remains largely unknown. More specifically, an intriguing question that remains is how this bacterium escapes the neutrophil response triggered by the infection. Indeed, both clinical and experimental results point to the recruitment of an inflammatory cellular infiltrate 5, 7 including neutrophils, macrophages and monocytes, which are typically seen around infected blood vessels [5] [6] [7] . However, although both neutrophils and monocytic cells frequently contain bacteria 5, 7 , this is not sufficient to clear the infection. The recruitment of neutrophils to infected tissues was also observed in a recently developed humanized mouse model in which grafting human skin allows the formation of a network of human dermal vessels that anastomose with the murine dermal vasculature and are perfused with murine blood 8 . In this model, intravenous bacteria exclusively adhere to the human endothelium, progressively proliferate to occupy the entire vessel lumen, and recapitulate the human disease. Taken together, these studies illustrate the ability of the host to initiate an innate immune response upon meningococcal infections, but raise pivotal questions regarding why neutrophils fail to clear these infections.

In this study we hypothesized that the immune escape abilities of Neisseria meningitidis are linked to its ability to occupy an intravascular niche. This feature is not unique to N. meningitidis but extend to a number of bacterial and viral pathogens that can reach the blood circulation, interact with the endothelium and colonize vessels, including SARS-COV2, as recently described [9] [10] [11] . Of note, intravascular colonization by these pathogens generates an atypical situation in terms of neutrophil recruitment.

Indeed, the textbook description in which bacteria are found in tissues and neutrophils are recruited to that sites following their rolling on the endothelium of postcapillary venules followed by their extravasation cannot apply for pathogens residing inside blood vessels. Whether the intravascular localization of Neisseria meningitidis might account for the incapacity of the innate immune system to clear this infection remains an open question. Therefore, studying the interaction of intravascular bacterial and viral pathogens with endothelial cells is critical to better understand their pathogenesis.

Here, we used the humanized mouse model of meningococcal infection described above, as well as human samples of infected tissues, to explore how neutrophils are recruited to infected vessels and how they target bacteria in this context. We found that Neisseria meningitidis escapes this key cellular component of the innate immune response by infecting arterioles and capillaries where neutrophils are not recruited despite intense infection of these vascular beds. N. meningitidis thus occupies a specific niche that protects them from the neutrophil response.

As previously shown 8, 12 , Neisseria meningitidis specifically colonize human vessels within a few hours ( Fig. 1a and Supplementary Fig. 1a-b) . Intravital time-lapse imaging shows bacteria adhering to the vascular wall (Supplementary Video 1), proliferating in the form of aggregates that become apparent after 2 hours and progressively fuse to finally occupy the vascular space at 6 hours post-infection (p.i.). Absence of colonization of mouse vessels confirms the sharp species specificity of the interaction with the endothelium. Efficient heterotypic interactions between murine neutrophils and the human endothelium in our model were confirmed by subcutaneous injection of human TNFa (Supplementary Video 2), as previously described 13 .

Neutrophil recruitment to infected vessels was assessed by immunofluorescence of histological samples, flow cytometry, and intravital imaging. Immunohistological staining of infected skin tissues revealed that only a few neutrophils could be found within the infected tissue 3h p.i., with numbers of neutrophils accumulating in the vicinity of infected vessels progressively increasing at 6h p.i. onwards (Fig. 1b) .

Quantitative analysis by flow cytometry of dissociated tissue revealed that neutrophils continued to accumulate up to 24h p.i., whereas contralateral mouse skin tissues did not show this neutrophil accumulation ( Fig. 1c and Supplementary Fig. 1c ). Intravital imaging of 56 infected vessels confirmed the recruitment of neutrophils during the early phase of the infection (0 to 6h) but only 46% of the infected vessels exhibited at least one neutrophil in their proximity 6h p.i., pointing to a heterogeneous neutrophil response across the infected tissue ( Supplementary Fig. 1d) . Similarly, the numbers of neutrophils per mm 2 of endothelium progressively increased and reached the mean value of 93±27 neutrophils 6h p.i. (Fig. 1d) .

Reminiscent of human cases 7 , at 16h p.i., the recruitment of LysM GFP -labelled neutrophils visualized by intravital imaging was massive with certain infected vessels being surrounded by large numbers of neutrophils, forming a sheath around the vessel Single cell tracking showed that neutrophils converge from the parenchyma towards the infected vessel ( Fig. 1e and Supplementary Video 3). Together these observations demonstrate that neutrophils progressively accumulate in the vicinity of certain infected vessels following N. meningitidis vascular colonization, starting at 3 hours postinfection and reaching large amounts at 16-24h p.i., as observed in human cases.

We next investigated the bacterial signals involved in neutrophil recruitment. To determine whether neutrophil recruitment relied on vascular colonization by N. meningitidis, we studied mutants that have altered ability to interact along the vascular wall. Grafted mice were first infected with two isogenic bacterial strains, pilC1, a piliated but non-adherent mutant, and pilD, a mutant that does not express any pili on its surface 15 (Fig. 2a) . Both strains cannot adhere to the endothelium and thus cannot colonize vessels. As expected 8 , while mice were inoculated with similar amounts of bacteria ( Fig. 2b) , the numbers of adherent bacteria 24h p.i. were strongly decreased when mice were infected with the mutant bacterial strains (Fig. 2c) . We found that neutrophil recruitment 24h after infection was tightly dependent on bacterial adhesion since their numbers barely increased when mice were infected with either of the mutant bacterial strains (Fig. 2d) .

Type IV pili also allow bacteria to form three-dimensional viscous aggregates containing thousands of bacteria and participating in vessel occlusion 12 . To test the impact of these bacterial aggregates inside blood vessels on neutrophil recruitment, a strain expressing a sequence variant (SA) of the major pilin unable to generate autoaggregation while still adhering to endothelial cells 16 was compared to the wild type sequence variant (SB) (Fig. 2e) . While both strains showed the same ability to survive inside the circulation, the SA variant showed lower amounts of bacteria colonizing blood vessels as expected ( Fig. 2f-g) . The amounts of neutrophils recruited by the nonaggregative strain was reduced (Fig. 2h) , showing that the formation of bacterial aggregates enhanced neutrophil recruitment. These data show that bacterial adhesion, amplified by bacterial auto-aggregation, both mediated by type IV pili, is essential to initiate a cascade eventually leading to neutrophil recruitment.

To address the role of recruited neutrophils during meningococcal disease, we depleted these cells and evaluated the effect on infection. Grafted mice were pretreated with the monocytes and neutrophil-depleting antibody directed against GR-1 (clone RB6-8C5), 24h prior to infection (Fig. 3a) . This treatment led to an efficient depletion of circulating neutrophils when compared to the mice that received the isotype control antibody (Fig. 3b) . Absence of neutrophils in the circulation did not affect the number of non-adherent circulating bacteria after intravenous injection at any of the tested times post-infection (Fig. 3c ). As expected, the depletion effectively prevented the neutrophil recruitment to infected human tissues (Fig. 3d) . Importantly, the number of adherent bacteria at 24h p.i. was strongly increased when compared to the control condition (Fig. 3e) . Nearly identical results were obtained when the mice were treated with the neutrophil-specific depletion antibody clone 1A8, against Ly-6G, supporting that monocytes likely do not further contribute to bacterial clearance (Supplementary Fig. 2a-d) . These results indicate that neutrophils are the primary cell type controlling the number of bacteria adhering and proliferating along the endothelium, but are unable to completely resolve the infection within this time frame.

As pathological effects of Nm infections are linked to altered vascular function, we also measured the potential contribution of neutrophils to vascular damage. Indeed, neutrophil have been reported to induce vascular damage in other systems 17 , and infection by Neisseria meningitidis in humans have been shown to involve vascular damage 7, 8 . Kinetics of vascular damage after N. meningitidis infection were first determined using histology and intravital imaging to compare with neutrophil recruitment kinetics. Over 500 blood vessels on histological slices were analysed for each time point and classified as healthy, congested (with accumulation of red blood cells), or breached (with perivascular extravasation of red blood cells). Representative images and quantitative results of each vascular condition are shown in Figure 3f . The percentage of healthy vessels gradually declined down to only 4% 24h p.i..

Conversely, congestion and vascular rupture progressively increased with time.

Interestingly, a significant increase in vascular congestion was observed as early as 3h p.i. and thus before recruitment of neutrophils. The percentage of congested vessels continued to increase to reach a maximum percentage of 70% at 16h p.i., which remained stable until 24h p.i. A similar situation occurred with vascular rupture, with a slight increase at 6h p.i. from 2% to 7% that progressively increased, reaching 25% at 16h p.i..

Neutrophil depletion led to a decrease in the number of congested vessels and a concurrent increase in the occurrence of breached vessels with released red blood cells ( Fig. 3g and Supplementary Fig. 2e ). To confirm and extend these results, vessel permeability to serum content was then measured using Evans Blue 18 . This dye was injected intravenously and 10 min post-injection, the circulating dye was removed from blood vessels by a myocardial perfusion of heparin-containing buffer. As expected, a 24h infection led to an increase of vascular permeability evidenced by high Evans blue accumulation in the tissue. In the absence of neutrophils, vascular leakage due to the infection was further increased (Fig. 3h ). Altogether these data are in favour of a protective role for neutrophils during meningococcal infection. After being recruited, neutrophils incompletely control the number of bacteria in the infected vessels and thus partially reduce the vascular damage induced by the bacteria upon vascular colonization.

Our results at this stage reveal a paradox, neutrophils are recruited upon vascular colonization and provide protection but fail to prevent the initiation and progression of the infection. A possible explanation of this incomplete neutrophil response is that the recruitment is insufficient in terms of number. This hypothesis is supported by the relatively low overall numbers of neutrophils (93±27 neutrophils.mm -2 at 6h p.i.) that are observed following N. meningitidis infection. As a comparison, TNFa stimulation or similar inflammatory situations can lead to about 5-fold this value 19 . We thus hypothesized that the unusual intraluminal location of the bacteria, rather than the bacterium itself, is responsible for this moderate recruitment. To test this hypothesis, bacteria were injected directly into the xenograft intradermally and the inflammatory infiltrate evaluated. The amounts of injected bacteria were adjusted to reach the amount found following intravascular infection (Fig. 4a ). In these conditions, histological analysis of the human skin graft 3h post-intradermal infection showed a considerable infiltration of inflammatory cells in contrast to intravascular infection at the same time point (Fig. 4b ). This was confirmed by flow cytometry analysis that highlighted a 10-fold increase in neutrophil numbers as early as 3h post-intradermal infection (Fig. 4c ). Even at 6h post intravascular infection such numbers were not reached (Fig. 1c) . These results point to an unexpectedly slow kinetics of neutrophil recruitment during vascular colonization and a particular impact of the vessel intraluminal location of the bacteria during infection.

It is important to note that numbers given above represent average values throughout the tissue that could be highly heterogeneous and mask specific locations. We thus analysed in depth whether bacteria equally infected distinct types of vessels. For this, infected vessels were categorized into three classes: capillaries, venules and arterioles. The distinction between venules and arterioles was based on a combination of parameters: morphology, intensity and direction of blood flow as well as an arteriolespecific staining approach, as previously described 20 (Supplementary Fig. 3a-b ).

Capillaries were characterized by a luminal diameter below or equal to 10 µm.

Remarkably, this classification revealed that N. meningitidis could infect all vessel types (Fig. 4d) . Observed infected venules and arterioles had a similar diameter, 43.73±7.52 µm and 49.13±3.77 µm, respectively (Fig. 4e) . In order to validate these observations made in an experimental mouse model, evidence of colonization of venules and arterioles in a human case were sought. Post-mortem samples from a case of purpura fulminans were immuno-stained to visualize bacteria and tissue organization. Evidence of colonization of both venules and arterioles could be found in different tissues, such as in liver and choroid plexus, in which aggregates of bacteria were associated to the endothelium (Fig. 4f , red arrows). N. meningitidis is thus able to colonize all types of vascular beds both in an experimental system and during human infections.

Neutrophil rolling was shown to preferentially occur on the venular endothelium but to a much lesser extent on arterioles and capillaries 21 . We therefore hypothesized that they might not be efficiently recruited to bacteria colonies localized in arterioles and capillaries. To test this hypothesis, the recruitment of neutrophils to the different vessel beds was evaluated. Neutrophils were rarely recruited to infected capillaries (Fig. 4g) .

The kinetics and percentage of infected vessels recruiting at least a single neutrophil was similar between venules and arterioles showing that some level of recruitment occurred in both of these vessel types (Fig. 4g) . Importantly, however, the number of neutrophils recruited was much higher in venules than in arterioles, with 248±115 versus 83±25 neutrophils per mm 2 of endothelium, respectively at 6h p.i. (Fig. 4h ). The number of neutrophils recruited to capillaries was even lower than in arterioles (22±18 neutrophils per mm 2 of endothelium). The ability of meningococci to colonize arterioles could thus provide an environment with lower numbers of neutrophils and thus an edge over the innate immune system.

Results described above show a preferential recruitment of neutrophils to infected venules. We thus wondered whether neutrophils could have efficient bacterial killing properties in this context. The recruitment of neutrophils to infected venules was therefore explored in further detail in terms of location, kinetics and phagocytic activity. Collectively, these experimental and clinical observations suggest that meningococci can adhere to venules and that neutrophils are readily recruited to these sites where they can efficiently phagocytose bacteria adhering to the endothelial wall.

Our results show that in the context of arterioles only few neutrophils were recruited following infection, leading to the hypothesis that the neutrophil response would be inefficient in this vessel type. The recruitment of neutrophils to arterioles was then examined in detail. Intravital imaging revealed that the few neutrophils recruited to arterioles were either located in an intra-or extra-luminal location, occasionally both Therefore, the ability of meningococci to adhere to different vascular beds and more specifically arterioles and capillaries allow them to rapidly colonize these vessels before an efficient innate immune response can be mounted.

The above results raised the question of why neutrophils were efficiently recruited to venules but not to capillaries and arterioles despite large accumulation of bacteria in all these vessel types. During infections caused by other pathogens, occurring inside tissues, neutrophils specifically exit the circulation through venules to reach the infection site. In this case, the specificity for venules is linked to the expression of a set of adhesion receptors on the endothelium surface, which are triggered by inflammatory signals coming from the infection site 21 . Although the intravascular location of the N. meningitidis infection generates a different situation, the difference in neutrophil response could be due to a differential expression of adhesion receptors. We first . 7b) . Interestingly, the signal was heterogeneous along the human endothelium surface and strongly colocalized with bacterial colonies (Fig. 7c) . Signals leading to Eselectin expression thus had a local component linked to bacterial adhesion. Together, these results show that despite massive infection and in contrast to the venular endothelium, endothelia from arterioles and capillaries fail to express E-selectin on their surface upon infection, thus preferentially targeting neutrophil recruitment towards infected venules over arterioles and capillaries.

To mount a protective innate immune response, the inflammatory cascade needs to trigger the recruitment of numerous neutrophils to the precise site of infection, leading to the efficient clearance of the pathogen. In the case of fulminant infections caused by meningococci, the innate immune response is not successful at keeping the pathogen in check. Experimental results provided here, confirmed by observations in human cases, explain how this is due to the particular proliferation niche of this bacterium. While meningococci have the ability to colonize different vascular beds; capillaries, venules, and arterioles; only infected venules allow efficient neutrophil recruitment.

The ability of bacteria to adhere to the endothelium wall is the starting point for the innate immune response as this allows the concentration of bacteria at a specific focal point rather than their systemic distribution in the blood. In absence of adhesion, neutrophil recruitment is barely detectable. Bacterial accumulation on the endothelial surface is thus a trigger for neutrophil recruitment. The ability of bacteria to autoaggregate also enhances neutrophil recruitment. This is likely explained by the amplification of bacterial numbers at the site of infection due to the three-dimensional accumulation of bacteria. It could also be envisioned that the formation of aggregates leads to more perturbations in blood flow and subsequently more inflammatory signals.

Nevertheless, type IV pili are central players in triggering the inflammatory response by allowing adhesion and auto-aggregation. Bacterial adhesion also shapes the innate immune response by leading to the colonization of different vessel types including capillaries. In a previous study focusing on meningitis and infection of the brain, we had shown a preferential colonization of capillaries in this organ 23 comprising the human epidermis and the papillary dermis was immediately placed over the graft bed. Grafts were fixed in place with surgical glue (Vetbond, 3M, USA) and dressings were applied for 2 weeks. Grafted mice were used for experimentation 3-6 weeks post-surgery when the human dermal microvasculature is anastomosed to the mouse circulation without evidence of local inflammation, as previously described 8 . All efforts were made to minimize suffering.

All N. meningitidis strains described in this study were derived from the recently sequenced 8013 serogroup C strain (http://www.genoscope.cns.fr/agc/nemesys) 31 . Mutations in PilD and PilC1 genes have been previously described 31, 32 . Wildtype (SB), SA, pilD and pilC1 bacterial strains were genetically modified to constitutively express either the green fluorescent protein (GFP) 8 Imaging heterotypic interactions between murine neutrophils and human endothelium. 

Data supporting this work are available in the paper. Further information and materials related to the findings of this study are available from the corresponding authors upon request. n³4 mice per group, in total, pooled from N=2 independent experiments. ns, not significant; *p<0.05; **p<0.005; ***p<0.0005 and ****p<0.0001.

damages. a, Schematic of the experimental approach used: neutrophil depletion was achieved by intravenous injection of the neutrophil-depleting antibody (anti-GR-1, clone RB6-8C5) 24h prior to mouse infection. Mice were sacrificed 24h post-infection and analyses were carried out. b, Numbers of blood circulating neutrophils in noninfected mice pre-treated with either the isotype control antibody (-) or the neutrophildepleting antibody (+). c, Bacterial colony forming unit (CFU) counts from blood (circulating bacteria) of mice pre-treated with either the isotype control antibody (-) or the neutrophil-depleting antibody (+) and infected for 5 minutes or 24 hours. d, Neutrophil numbers in human xenografts of mice pre-treated with either the isotype control antibody (-) or the neutrophil-depleting antibody (+) and infected for 24 hours. neutrophil-depleting antibody (+) and infected for 24 hours. Two-tailed Man-Whitney test. n³15 mice per group, in total, pooled from N=5 independent experiments. d, Bacterial colony forming unit (CFU) counts from dissociated human xenografts (adherent bacteria) collected from mice pre-treated with either the isotype control antibody (-) or the neutrophil-depleting antibody (+) and infected for the indicated times. Kruskal-Wallis test with Dunn's correction for multiple comparisons. n³4 mice per group, in total, pooled from N=2 (3h) and 3 (24h) independent experiments. e, Quantification of vascular damage upon mouse infection for the indicated time points following neutrophil depletion by intravenous injection of the neutrophil-specific depletion antibody (anti-Ly-6G, clone 1A8) 24h prior to mouse infection. Quantification were performed on n³200 vessels, in total, pooled from N=3 mice per time point. ns, not significant; *p<0.05; **p<0.005; ***p<0.0005 and ****p<0.0001.

Supplementary Figure 3 . Identification of the different vessel types based on hydrazide AlexaFluor633 labelling upon infection. a, Representative images of vessel labelling using Hydrazide AlexaFluor633 (AF633) in a chimeric human/murine arteriole, confirming that Hydrazide AF633 equally stains human and murine arterioles. b, Representative images of vascular colonization of the different human vascular beds 1h post-infection by GFP-expressing Neisseria meningitidis (green) as revealed by the UEA-1 lectin (human endothelium, grey) and Hydrazide AF633 (arterioles, cyan) double labelling. Scale bar, 20 µm.

UtechS Photonic BioImaging (Imagopole, C2RT, Institut Pasteur

UMR-970, PARCC) for the analysis of human cases pathology. This work was supported by the Integrative Biology of Emerging Infectious Diseases (IBEID) laboratory of excellence (ANR-10-LABX-62), and the VIP European Research Council-starting grant (310790-VIP

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Videos obtained from intravital imaging were used to quantify the percentage of vessels effectively recruiting neutrophils during the first 6 hours of the infection. Data are shown as percentage of vessels. Quantifications were performed on n=56 vessels, in total, pooled from N=7 infected mice imaged independently. ns, not significant

Two-tailed Unpaired t test. n³9 mice per group, in total, pooled from N=4 independent experiments. b, Bacterial colony forming unit (CFU) counts from blood (circulating bacteria) of mice pre-treated with either the isotype control antibody (-) or the neutrophil-depleting antibody (+) and infected for the indicated times. Kruskal-Wallis test with Dunn's correction for multiple comparisons. n³4 mice per group

The authors declare no competing interests.

Intravital imaging of iRFP-expressing Neisseria meningitidis vascular colonization. Bacteria rapidly bind to the human endothelium (UEA-1 lectin, grey) and locally proliferate. At 6 hours post-infection, the 3D-rendering shows the complete colonization of the human endothelium whereas no bacteria were detected within adjacent mouse vessels (mouse CD31, red). Time, hh:min:sec. Scale bar, 50 µm.

Intravital visualization of the interaction between mouse neutrophils (Ly-6G, magenta) and TNFa-mediated inflamed human (UEA-1 lectin, grey) and/or mouse (mouse CD31, red) endothelia. A chimeric human/mouse vessel is shown in the first field of view. Supplementary Video 6. Reduced neutrophil dynamics following vascular colonization of human arterioles. Intravital visualization of neutrophil (Ly-6G, magenta) dynamics in an infected human arteriole (UEA-1 lectin, grey) 7h00 postinfection. Neutrophils containing engulfed iRFP-expressing bacteria (green) display a very low motility compared to neutrophils present in the adjacent non-infected mouse venules (mouse CD31, red). Time, hh:min:sec. Scale bar, 20 µm.