Infection-on-Chip: an in vitro human vessel model to study Neisseria meningitidis colonization and vascular damages

  1. Léa Pinon
  2. Mélanie Chabaud
  3. Pierre Nivoit
  4. Jérôme Wong-Ng
  5. Tri Tho Nguyen
  6. Vanessa Paul
  7. Charlotte Bouquerel
  8. Sylvie Goussard
  9. Emmanuel Frachon
  10. Dorian Obino
  11. Samy Gobaa
  12. Guillaume Duménil

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Abstract

Systemic infections leading to sepsis are life-threatening conditions that remain difficult to treat, and the development of innovative therapies is hampered by the limitations of current experimental models. Animal models are constrained by species-specific differences, while 2D cell culture systems fail to capture the complex pathophysiology of infection. To overcome these limitations, we developed a laser photoablation-based, three-dimensional microfluidic model of meningococcal vascular colonization, a human-specific bacterium that causes sepsis and meningitis. We coined our model “Infection-on-Chip”. Laser photoablation-based hydrogel engineering allows the reproduction of vascular networks that are major infection target sites, and this model provides the relevant microenvironment reproducing the physiological endothelial integrity and permeability in vitro . By comparing with human-skin xenograft mouse model, we show that the Infection-on-Chip system not only replicates in vivo key features of the infection, but also enables quantitative assessment with a higher spatio-temporal resolution of bacterial microcolony growth, endothelial cy-toskeleton rearrangement, vascular E-selectin expression, and neutrophil response upon infection. Our device thus provides a robust solution bridging the gap between animal and 2D cellular models, and paving the way for a better understanding of disease progression and the development of innovative therapeutics.

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    Reply to the reviewers

    Reviewer #1

    Evidence, reproducibility, and clarity

    The work by Pinon et al describes the generation of a microvascular model to study Neisseria meningitidis interactions with blood vessels. The model uses a novel and relatively high throughput fabrication method that allows full control over the geometry of the vessels. The model is well characterized. The authors then study different aspects of Neisseria-endothelial interactions and benchmark the bacterial infection model against the best disease model available, a human skin xenograft mouse model, which is one of the great strengths of the paper. The authors show that Neisseria binds to the 3D model in a similar geometry that in the animal xenograft model, induces an increase in permeability short after bacterial perfusion, and induces endothelial cytoskeleton rearrangements. Finally, the authors show neutrophil recruitment to bacterial microcolonies and phagocytosis of Neisseria. The article is overall well written, and it is a great advancement in the bioengineering and sepsis infection field, and I only have a few major comments and some minor.

    Major comments:

    Infection-on-chip. I would recommend the authors to change the terminology of "infection on chip" to better reflect their work. The term is vague and it decreases novelty, as there are multiple infection on chips models that recapitulate other infections (recently reviewed in https://doi.org/10.1038/s41564-024-01645-6) including Ebola, SARS-CoV-2, Plasmodium and Candida. Maybe the term "sepsis on chip" would be more specific and exemplify better the work and novelty. Also, I would suggest that the authors carefully take a look at the text and consider when they use VoC or to current term IoC, as of now sometimes they are used interchangeably, with VoC being used occasionally in bacteria perfused experiments.

    We thank Reviewer #1 for this suggestion. Indeed, we have chosen to replace the term "Infection-on-Chip" by "infected Vessel-on-chip" to avoid any confusion in the title and the text. Also, we have removed all the terms "IoC" which referred to "Infection-on-Chip" and replaced with "VoC" for "Vessel-on-Chip". We think these terms will improve the clarity of the main text.

    Fig 3 and Suppmentary 3: Permeability. The authors suggest that early 3h infection with Neisseria do not show increase in vascular permeability in the animal model, contrary to their findings in the 3D in vitro model. However, they show a non-significant increase in permeability of 70 KDa Dextran in the animal xenograft early infection. This seems to point that if the experiment would have been done with a lower molecular weight tracer, significant increases in permeability could have been detected. I would suggest to do this experiment that could capture early events in vascular disruption.

    Comparing permeability under healthy and infected conditions using Dextran smaller than 70 kDa is challenging. Previous research [1] has shown that molecules below 70 kDa already diffuse freely in healthy tissue. Given this high baseline diffusion, we believe that no significant difference would be observed before and after N. meningitidis infection and these experiments were not carried out. As discussed in the manuscript, bacteria induced permeability in mouse occurs at later time points, 16h post infection as shown previoulsy [2]. As discussed in the manuscript, this difference between the xenograft model and the chip likely reflect the absence in the chip of various cell types present in the tissue parenchyma.

    The authors show the formation of actin of a honeycomb structure beneath the bacterial microcolonies. This only occurred in 65% of the microcolonies. Is this result similar to in vitro 2D endothelial cultures in static and under flow? Also, the group has shown in the past positive staining of other cytoskeletal proteins, such as ezrin in the ERM complex. Does this also occur in the 3D system?

    We thank the Reviewer #1 for this suggestion.

    • According to this recommendation, we imaged monolayers of endothelial cells in the flat regions of the chip (the two lateral channels) using the same microscopy conditions (i.e., Obj. 40X N.A. 1.05) that have been used to detect honeycomb structures in the 3D vessels in vitro. We showed that more than 56% of infected cells present these honeycomb structures in 2D, which is 13% less than in 3D, and is not significant due to the distributions of both populations. Thus, we conclude that under both in vitro conditions, 2D and 3D, the amount of infected cells exhibiting cortical plaques is similar. We have added the graph and the confocal images in Figure S4B and lines 418-419 of the revised manuscript.
    • We recently performed staining of ezrin in the chip and imaged both the 3D and 2D regions. Although ezrin staining was visible in 3D (Fig. 1 of this response), it was not as obvious as other markers under these infected conditions and we did not include it in the main text. Interpretation of this result is not straight forward as for instance the substrate of the cells is different and it would require further studies on the behaviour of ERM proteins in these different contexts.

    One of the most novel things of the manuscript is the use of a relatively quick photoablation system. I would suggest that the authors add a more extensive description of the protocol in methods. Could this technique be applied in other laboratories? If this is a major limitation, it should be listed in the discussion.

    Following the Reviewer's comment, we introduced more detailed explanations regarding the photoablation:

    • L157-163 (Results): "Briefly, the chosen design is digitalized into a list of positions to ablate. A pulsed UV-LASER beam is injected into the microscope and shaped to cover the back aperture of the objective. The laser is then focused on each position that needs ablation. After introducing endothelial cells (HUVEC) in the carved regions,.."
    • L512-516 (Discussion): "The speed capabilities drastically improve with the pulsing repetition rate. Given that our laser source emits pulses at 10kHz, as compared to other photoablation lasers with repetitions around 100 Hz, our solution could potentially gain a factor of 100. Also,..."
    • L1082-1087 (Materials and Methods): "…, and imported in a python code. The control of the various elements is embedded and checked for this specific set of hardware. The code is available upon request."

    Adding these three paragraphs gives more details on how photoablation works thus improving the manuscript.

    Minor comments:

    Supplementary Fig 2. The reference to subpanels H and I is swapped.

    The references to subpanels H and I have been correctly swapped back in the reviewed version.

    Line 203: I would suggest to delete this sentence. Although a strength of the submitted paper is the direct comparison of the VoC model with the animal model to better replicate Neisseria infection, a direct comparison with animal permeability is not needed in all vascular engineering papers, as vascular permeability measurements in animals have been well established in the past.

    The sentence "While previously developed VoC platforms aimed at replicating physiological permeability properties, they often lack direct comparisons with in vivo values." has been removed from the revised text.

    Fig 3: Bacteria binding experiments. I would suggest the addition of more methodological information in the main results text to guarantee a good interpretation of the experiment. First, it would be better that wall shear stress rather than flow rate is described in the main text, as flow rate is dependent on the geometry of the vessel being used. Second, how long was the perfusion of Neisseria in the binding experiment performed to quantify colony doubling or elongation? As per figure 1C, I would guess than 100 min, but it would be better if this information is directly given to the readers.

    We thank Reviewer #1 for these two suggestions that will improve the text clarity (e.g., L316). (i) Indeed, we have changed the flow rate in terms of shear stress. (ii) Also, we have normalized the quantification of the colony doubling time according to the first time-point where a single bacteria is attached to the vessel wall. Thus, early adhesion bacteria will be defined by a longer curve while late adhesion bacteria by a shorter curve. In total, the experiment lasted for 3 hours (modifications appear in L318 and L321-326).}

    Fig 4: The honeycomb structure is not visible in the 3D rendering of panel D. I would recommend to show the actin staining in the absence of Neisseria staining as well.

    According to this suggestion, a zoom of the 3D rendering of the cortical plaque without colony had been added to the figure 4 of the revised manuscript.

    Line 421: E-selectin is referred as CD62E in this sentence. I would suggest to use the same terminology everywhere.

    We have replaced the "CD62E" term with "E-selectin" to improve clarity.}

    Line 508: "This difference is most likely associated with the presence of other cell types in the in vivo tissues and the onset of intravascular coagulation". Do the authors refer to the presence of perivascular cells, pericytes or fibroblasts? If so, it could be good to mention them, as well as those future iterations of the model could include the presence of these cell types.

    By "other cell types", we refer to pericytes [3], fibroblasts [4], and perivascular macrophages [5], which surround endothelial cells and contribute to vessel stability. The main text was modified to include this information (Lines 548 and 555-570) and their potential roles during infection disussed.

    Discussion: The discussion covers very well the advantages of the model over in vitro 2D endothelial models and the animal xenograft but fails to include limitations. This would include the choice of HUVEC cells, an umbilical vein cell line to study microcirculation, the lack of perivascular cells or limitations on the fabrication technique regarding application in other labs (if any).

    We thank Reviewer #1 for this suggestion. Indeed, our manuscript may lack explaining limitations, and adding them to the text will help improve it:

    • The perspectives of our model include introducing perivascular cells surrounding the vessel and fibroblasts into the collagen gel as discussed previously and added in the discussion part (L555-570).
    • Our choice for HUVEC cells focused on recapitulating the characteristics of venules that respect key features such as the overexpression of CD62E and adhesion of neutrophils during inflammation. Using microvascular endothelial cells originating from different tissues would be very interesting. This possibility is now mentioned in the discussion lines 567-568.
    • Photoablation is a homemade fabrication technique that can be implemented in any lab harboring an epifluorescence microscope. This method has been more detailed in the revised manuscript (L1085-1087).

    Line 576: The authors state that the model could be applied to other systemic infections but failed to mention that some infections have already been modelled in 3D bioengineered vascular models (examples found in https://doi.org/10.1038/s41564-024-01645-6). This includes a capillary photoablated vascular model to study malaria (DOI: 10.1126/sciadv.aay724).

    Thes two important references have been introduced in the main text (L84, 647, 648).}

    Line 1213: Are the 6M neutrophil solution in 10ul under flow. Also, I would suggest to rewrite this sentence in the following line "After, the flow has been then added to the system at 0.7-1 μl/min."

    We now specified that neutrophils are circulated in the chip under flow conditions, lines 1321-1322.

    Significance

    The manuscript is comprehensive, complete and represents the first bioengineered model of sepsis. One of the major strengths is the carful characterization and benchmarking against the animal xenograft model. Its main limitations is the brief description of the photoablation methodology and more clarity is needed in the description of bacteria perfusion experiments, given their complexity. The manuscript will be of interest for the general infection community and to the tissue engineering community if more details on fabrication methods are included. My expertise is on infection bioengineered models.

    Reviewer #2

    Evidence, reproducibility, and clarity

    Summary The authors develop a Vessel-on-Chip model, which has geometrical and physical properties similar to the murine vessels used in the study of systemic infections. The vessel was created via highly controllable laser photoablation in a collagen matrix, subsequent seeding of human endothelial cells and flow perfusion to induce mechanical cues. This vessel could be infected with Neisseria meningitidis, as a model of systemic infection. In this model, microcolony formation and dynamics, and effects on the host were very similar to those described for the human skin xenograft mouse, which is the current gold standard for these studies, and were consistent with observations made in patients. The model could also recapitulate the neutrophil response upon N. meningitidis systemic infection.

    Major comments:

    I have no major comments. The claims and the conclusions are supported by the data, the methods are properly presented and the data is analyzed adequately. Furthermore, I would like to propose an optional experiment could improve the manuscript. In the discussion it is stated that the vascular geometry might contribute to bacterial colonization in areas of lower velocity. It would be interesting to recapitulate this experimentally. It is of course optional but it would be of great interest, since this is something that can only be proven in the organ-on-chip (where flow speed can be tuned) and not as much in animal models. Besides, it would increase impact, demonstrating the superiority of the chip in this area rather than proving to be equal to current models.

    We have conducted additional experiments on infection in different vascular geometries now added these results figure 3/S3 and lines 288-305. We compared sheared stress levels as determined by Comsol simulation and experimentally determined bacterial adhesion sites. In the conditions used, the range of shear generated by the tested geometries do not appear to change the efficiency of bacterial adhesion. These results are consistent with a previous study from our group which show that in this range of shear stresses the effect on adhesion is limited [6] . Furthermore, qualitative observations in the animal model indicate that bacteria do not have an obvious preference in terms of binding site.

    Minor comments:

    I have a series of suggestions which, in my opinion, would improve the discussion. They are further elaborated in the following section, in the context of the limitations.

    How to recapitulate the vessels in the context of a specific organ or tissue? If the pathogen is often found in the luminal space of other organs after disseminating from the blood, how can this process be recapitulated with this mode, if at all?

    • For reasons that are not fully understood, postmortem histological studies reveal bacteria only inside blood vessels but rarely if ever in the organ parenchyma. The presence of intravascular bacteria could nevertheless alter cells in the tissue parenchyma. The notable exception is the brain where bacteria exit the bacterial lumen to access the cerebrospinal fluid. The chip we describe is fully adapted to develop a blood brain barrier model and more specific organ environments. This implies the addition of more cell types in the hydrogel. A paragraph on this topic has been added (Lines 548 and 552-570).

    Similarly, could other immune responses related to systemic infection be recapitulated? The authors could discuss the potential of including other immune cells that might be found in the interstitial space, for example.

    • This important discussion point has been added to the manuscript (L623-636). As suggested by Reviewer #2, other immune cells respond to N. meningitis and can be explored using our model. For instance, macrophages and dendritic cells are activated upon N. meningitis infection, eliminate the bacteria through phagocytosis, produce pro-inflammatory cytokines and chemokines potentially activating lymphocytes [7]. Such an immune response, yet complex, would be interesting to study in our model as skin-xenograft mice are deprived of B and T lymphocytes to ensure acceptance of human skin grafts.

    A minor correction: in line 467 it should probably be "aspects" instead of "aspect", and the authors could consider rephrasing that sentence slightly for increased clarity.

    • We have corrected the sentence with "we demonstrated that our VoC strongly replicates key aspects of the in vivo human skin xenograft mouse model, the gold standard for studying meningococcal disease under physiological conditions." in lines 499-503.

    Strengths and limitations

    The most important strength of this manuscript is the technology they developed to build this model, which is impressive and very innovative. The Vessel-on-Chip can be tuned to acquire complex shapes and, according to the authors, the process has been optimized to produce models very quickly. This is a great advancement compared with the technologies used to produce other equivalent models. This model proves to be equivalent to the most advanced model used to date, but allows to perform microscopy with higher resolution and ease, which can in turn allow more complex and precise image-based analysis. However, the authors do not seem to present any new mechanistic insights obtained using this model. All the findings obtained in the infection-on-chip demonstrate that the model is equivalent to the human skin xenograft mouse model, and can offer superior resolution for microscopy. However, the advantages of the model do not seem to be exploited to obtain more insights on the pathogenicity mechanisms of N. meningitidis, host-pathogen interactions or potential applications in the discovery of potential treatments. For example, experiments to elucidate the role of certain N. meningiditis genes on infection could enrich the manuscript and prove the superiority of the model. However, I understand these experiments are time-consuming and out of the scope of the current manuscript. In addition, the model lacks the multicellularity that characterizes other similar models. The authors mention that the pathogen can be found in the luminal space of several organs, however, this luminal space has not been recapitulated in the model. Even though this would be a new project, it would be interesting that the authors hypothesize about the possibilities of combining this model with other organ models. The inclusion of circulating neutrophils is a great asset; however it would also be interesting to hypothesize about how to recapitulate other immune responses related to systemic infection.

    We thank Reviewer #2 for his/her comment on the strengths and limitations of our work. The difficulty is that our study opens many futur research directions and applications and we hope that the work serves as the basis for many future studies but one can only address a limited set of experiments in a single manuscript.

    • Experiments investigating the role of N. meningitidis genes require significant optimization of the system. Multiplexing is a potential avenue for future development, which would allow the testing of many mutants. The fast photoablation approach is particularly amenable to such adaptation.
    • Cells and bacteria inside the chambers could be isolated and analyzed at the transcriptomic level or by flow cytometry. This would imply optimizing a protocol for collecting cells from the device via collagenase digestion, for instance. This type of approach would also benefit from multiplexing to enhance the number of cells.
    • As mentioned above, the revised manuscript discusses the multicellular capabilities of our model, including the integration of additional immune cells and potential connections to other organ systems. We believe that these approaches are feasible and valuable for studying various aspects of N. meningitidis infection.

    Advance

    The most important advance of this manuscript is technical: the development of a model that proves to be equivalent to the most complex model used to date to study meningococcal systemic infections. The human skin xenograft mouse model requires complex surgical techniques and has the practical and ethical limitations associated with the use of animals. However, the Infection-on-chip model is completely in vitro, can be produced quickly, and allows to precisely tune the vessel's geometry and to perform higher resolution microscopy. Both models were comparable in terms of the hallmarks defining the disease, suggesting that the presented model can be an effective replacement of the animal use in this area.

    Other vessel-on-chip models can recapitulate an endothelial barrier in a tube-like morphology, but do not recapitulate other complex geometries, that are more physiologically relevant and could impact infection (in addition to other non-infectious diseases). However, in the manuscript it is not clear whether the different morphologies are necessary to study or recapitulate N. meningitidis infection, or if the tubular morphologies achieved in other similar models would suffice.

    We thank Reviewer #2 for his/her comment, also raised by reviewer 1. To answer this question, we have now infected vessel-on-chips of different geometries, to dissect the impact of flow distribution in N. meningitidis infection (Figures 3 and S3, explained in lines 288-307). In this range of shear stress, we show that bacterial infection is not strongly affected by geometry-induced shear stress variation. These observations are constistent with observations in flow chambers and qualitative observations of human cases and in the xenograft model [6].

    Audience

    This manuscript might be of interest for a specialized audience focusing on the development of microphysiological models. The technology presented here can be of great interest to researchers whose main area of interest is the endothelium and the blood vessels, for example, researchers on the study of systemic infections, atherosclerosis, angiogenesis, etc. Thus, the tool presented (vessel-on-chip) can have great applications for a broad audience. However, even when the method might be faster and easier to use than other equivalent methods, it could still be difficult to implement in another laboratory, especially if it lacks expertise in bioengineering. Therefore, the method could be more of interest for laboratories with expertise in bioengineering looking to expand or optimize their toolbox. Alternatively, this paper present itself as an opportunity to begin collaborations, since the model could be used to test other pathogen or conditions.

    Field of expertise: Infection biology, organ-on-chip, fungal pathogens.

    I lack the expertise to evaluate the image-based analysis.

    References:

    1. Gyohei Egawa, Satoshi Nakamizo, Yohei Natsuaki, Hiromi Doi, Yoshiki Miyachi, and Kenji Kabashima. Intravital analysis of vascular permeability in mice using two-photon microscopy. Scientific Reports, 3(1):1932, Jun 2013. ISSN 2045-2322. doi: 10.1038/srep01932.

    2. Valeria Manriquez, Pierre Nivoit, Tomas Urbina, Hebert Echenique-Rivera, Keira Melican, Marie-Paule Fernandez-Gerlinger, Patricia Flamant, Taliah Schmitt, Patrick Bruneval, Dorian Obino, and Guillaume Duménil. Colonization of dermal arterioles by neisseria meningitidis provides a safe haven from neutrophils. Nature Communications, 12(1):4547, Jul 2021. ISSN 2041-1723. doi:10.1038/s41467-021-24797-z.

    3. Mats Hellström, Holger Gerhardt, Mattias Kalén, Xuri Li, Ulf Eriksson, Hartwig Wolburg, and Christer Betsholtz. Lack of pericytes leads to endothelial hyperplasia and abnormal vascular morphogenesis. Journal of Cell Biology, 153(3):543–554, Apr 2001. ISSN 0021-9525. doi: 10.1083/jcb.153.3.543.

    4. Arsheen M. Rajan, Roger C. Ma, Katrinka M. Kocha, Dan J. Zhang, and Peng Huang. Dual function of perivascular fibroblasts in vascular stabilization in zebrafish. PLOS Genetics, 16(10):1–31, 10 2020. doi: 10.1371/journal.pgen.1008800.

    5. Huanhuan He, Julia J. Mack, Esra Güç, Carmen M. Warren, Mario Leonardo Squadrito, Witold W. Kilarski, Caroline Baer, Ryan D. Freshman, Austin I. McDonald, Safiyyah Ziyad, Melody A. Swartz, Michele De Palma, and M. Luisa Iruela-Arispe. Perivascular macrophages limit permeability. Arteriosclerosis, Thrombosis, and Vascular Biology, 36(11):2203–2212, 2016. doi: 10.1161/ATVBAHA. 116.307592.

    6. Emilie Mairey, Auguste Genovesio, Emmanuel Donnadieu, Christine Bernard, Francis Jaubert, Elisabeth Pinard, Jacques Seylaz, Jean-Christophe Olivo-Marin, Xavier Nassif, and Guillaume Dumenil. Cerebral microcirculation shear stress levels determine Neisseria meningitidis attachment sites along the blood–brain barrier . Journal of Experimental Medicine, 203(8):1939–1950, 07 2006. ISSN 0022-1007. doi: 10.1084/jem.20060482.

    7. Riya Joshi and Sunil D. Saroj. Survival and evasion of neisseria meningitidis from macrophages. Medicine in Microecology, 17:100087, 2023. ISSN 2590-0978. doi: https://doi.org/10.1016/j.medmic.2023.100087.

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    The authors develop a Vessel-on-Chip model, which has geometrical and physical properties similar to the murine vessels used in the study of systemic infections. The vessel was created via highly controllable laser photoablation in a collagen matrix, subsequent seeding of human endothelial cells and flow perfusion to induce mechanical cues. This vessel could be infected with Neisseria meningitidis, as a model of systemic infection. In this model, microcolony formation and dynamics, and effects on the host were very similar to those described for the human skin xenograft mouse, which is the current gold standard for these studies, and were consistent with observations made in patients. The model could also recapitulate the neutrophil response upon N. meningitidis systemic infection.

    Major comments

    I have no major comments. The claims and the conclusions are supported by the data, the methods are properly presented and the data is analyzed adequately. Furthermore, I would like to propose an optional experiment could improve the manuscript. In the discussion it is stated that the vascular geometry might contribute to bacterial colonization in areas of lower velocity. It would be interesting to recapitulate this experimentally. It is of course optional but it would be of great interest, since this is something that can only be proven in the organ-on-chip (where flow speed can be tuned) and not as much in animal models. Besides, it would increase impact, demonstrating the superiority of the chip in this area rather than proving to be equal to current models.

    Minor comments

    I have a series of suggestions which, in my opinion, would improve the discussion. They are further elaborated in the following section, in the context of the limitations.

    • How to recapitulate the vessels in the context of a specific organ or tissue? If the pathogen is often found in the luminal space of other organs after disseminating from the blood, how can this process be recapitulated with this mode, if at all?
    • Similarly, could other immune responses related to systemic infection be recapitulated? The authors could discuss the potential of including other immune cells that might be found in the interstitial space, for example. A minor correction: in line 467 it should probably be "aspects" instead of "aspect", and the authors could consider rephrasing that sentence slightly for increased clarity.

    Referee cross-commenting

    I agree with the rest of the comments, and also agree that the manuscript is already complete and could be published as it is.

    Significance

    Strengths and limitations

    The most important strength of this manuscript is the technology they developed to build this model, which is impressive and very innovative. The Vessel-on-Chip can be tuned to acquire complex shapes and, according to the authors, the process has been optimized to produce models very quickly. This is a great advancement compared with the technologies used to produce other equivalent models. This model proves to be equivalent to the most advanced model used to date, but allows to perform microscopy with higher resolution and ease, which can in turn allow more complex and precise image-based analysis. However, the authors do not seem to present any new mechanistic insights obtained using this model. All the findings obtained in the infection-on-chip demonstrate that the model is equivalent to the human skin xenograft mouse model, and can offer superior resolution for microscopy. However, the advantages of the model do not seem to be exploited to obtain more insights on the pathogenicity mechanisms of N. meningitidis, host-pathogen interactions or potential applications in the discovery of potential treatments. For example, experiments to elucidate the role of certain N. meningiditis genes on infection could enrich the manuscript and prove the superiority of the model. However, I understand these experiments are time consuming and out of the scope of the current manuscript. In addition, the model lacks the multicellularity that characterizes other similar models. The authors mention that the pathogen can be found in the luminal space of several organs, however, this luminal space has not been recapitulated in the model. Even though this would be a new project, it would be interesting that the authors hypothesize about the possibilities of combining this model with other organ models. The inclusion of circulating neutrophils is a great asset; however it would also be interesting to hypothesize about how to recapitulate other immune responses related to systemic infection.

    Advance

    The most important advance of this manuscript is technical: the development of a model that proves to be equivalent to the most complex model used to date to study meningococcal systemic infections. The human skin xenograft mouse model requires complex surgical techniques and has the practical and ethical limitations associated with the use of animals. However, the Infection-on-chip model is completely in vitro, can be produced quickly, and allows to precisely tune the vessel's geometry and to perform higher resolution microscopy. Both models were comparable in terms of the hallmarks defining the disease, suggesting that the presented model can be an effective replacement of the animal use in this area. Other vessel-on-chip models can recapitulate an endothelial barrier in a tube-like morphology, but do not recapitulate other complex geometries, that are more physiologically relevant and could impact infection (in addition to other non-infectious diseases). However, in the manuscript it is not clear whether the different morphologies are necessary to study or recapitulate N. meningitidis infection, or if the tubular morphologies achieved in other similar models would suffice.

    Audience

    This manuscript might be of interest for a specialized audience focusing on the development of microphysiological models. The technology presented here can be of great interest to researchers whose main area of interest is the endothelium and the blood vessels, for example, researchers on the study of systemic infections, atherosclerosis, angiogenesis, etc. Thus, the tool presented (vessel-on-chip) can have great applications for a broad audience. However, even when the method might be faster and easier to use than other equivalent methods, it could still be difficult to implement in another laboratory, especially if it lacks expertise in bioengineering. Therefore, the method could be more of interest for laboratories with expertise in bioengineering looking to expand or optimize their toolbox. Alternatively, this paper present itself as an opportunity to begin collaborations, since the model could be used to test other pathogen or conditions.

    Field of expertise: infection biology, organ-on-chip, fungal pathogens

    I lack the expertise to evaluate the image-based analysis.

  3. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    The work by Pinon et al describes the generation of a microvascular model to study Neisseria meningitidis interactions with blood vessels. The model uses a novel and relatively high throughput fabrication method that allows full control over the geometry of the vessels. The model is well characterized. The authors then study different aspects of Neisseria-endothelial interactions and benchmark the bacterial infection model against the best disease model available, a human skin xenograft mouse model, which is one of the great strengths of the paper. The authors show that Neisseria binds to the 3D model in a similar geometry that in the animal xenograft model, induces an increase in permeability short after bacterial perfusion, and induces endothelial cytoskeleton rearrangements. Finally, the authors show neutrophil recruitment to bacterial microcolonies and phagocytosis of Neisseria. The article is overall well written, and it is a great advancement in the bioengineering and sepsis infection field, and I only have a few major comments and some minor.

    Major comments:

    Infection-on-chip. I would recommend the authors to change the terminology of "infection on chip" to better reflect their work. The term is vague and it decreases novelty, as there are multiple infection on chips models that recapitulate other infections (recently reviewed in https://doi.org/10.1038/s41564-024-01645-6) including Ebola, SARS-CoV-2, Plasmodium and Candida. Maybe the term "sepsis on chip" would be more specific and exemplify better the work and novelty. Also, I would suggest that the authors carefully take a look at the text and consider when they use VoC or to current term IoC, as of now sometimes they are used interchangeably, with VoC being used occasionally in bacteria perfused experiments.

    Fig 3 and Suppmentary 3: Permeability. The authors suggest that early 3h infection with Neisseria do not show increase in vascular permeability in the animal model, contrary to their findings in the 3D in vitro model. However, they show a non-significant increase in permeability of 70 KDa Dextran in the animal xenograft early infection. This seems to point that if the experiment would have been done with a lower molecular weight tracer, significant increases in permeability could have been detected. I would suggest to do this experiment that could capture early events in vascular disruption.

    The authors show the formation of actin of a honeycomb structure beneath the bacterial microcolonies. This only occurred in 65% of the microcolonies. Is this result similar to in vitro 2D endothelial cultures in static and under flow? Also, the group has shown in the past positive staining of other cytoskeletal proteins, such as ezrin in the ERM complex. Does this also occur in the 3D system?

    One of the most novel things of the manuscript is the use of a relatively quick photoablation system. I would suggest that the authors add a more extensive description of the protocol in methods. Could this technique be applied in other laboratories? If this is a major limitation, it should be listed in the discussion.

    Minor comments:

    Supplementary Fig 2. The reference to subpanels H and I is swapped.

    Line 203: I would suggest to delete this sentence. Although a strength of the submitted paper is the direct comparison of the VoC model with the animal model to better replicate Neisseria infection, a direct comparison with animal permeability is not needed in all vascular engineering papers, as vascular permeability measurements in animals have been well established in the past.

    Fig 3: Bacteria binding experiments. I would suggest the addition of more methodological information in the main results text to guarantee a good interpretation of the experiment. First, it would be better that wall shear stress rather than flow rate is described in the main text, as flow rate is dependent on the geometry of the vessel being used. Second, how long was the perfusion of Neisseria in the binding experiment performed to quantify colony doubling or elongation? As per figure 1C, I would guess than 100 min, but it would be better if this information is directly given to the readers.

    Fig 4: The honeycomb structure is not visible in the 3D rendering of panel D. I would recommend to show the actin staining in the absence of Neisseria staining as well.

    Line 421: E-selectin is referred as CD62E in this sentence. I would suggest to use the same terminology everywhere.

    Line 508: "This difference is most likely associated with the presence of other cell types in the in vivo tissues and the onset of intravascular coagulation". Do the authors refer to the presence of perivascular cells, pericytes or fibroblasts? If so, it could be good to mention them, as well as those future iterations of the model could include the presence of these cell types.

    Discussion: The discussion covers very well the advantages of the model over in vitro 2D endothelial models and the animal xenograft but fails to include limitations. This would include the choice of HUVEC cells, an umbilical vein cell line to study microcirculation, the lack of perivascular cells or limitations on the fabrication technique regarding application in other labs (if any).

    Line 576: The authors state that the model could be applied to other systemic infections but failed to mention that some infections have already been modelled in 3D bioengineered vascular models (examples found in https://doi.org/10.1038/s41564-024-01645-6). This includes a capillary photoablated vascular model to study malaria ( DOI: 10.1126/sciadv.aay724).

    Line 1213: Are the 6M neutrophil solution in 10ul under flow. Also, I would suggest to rewrite this sentence in the following line "After, the flow has been then added to the system at 0.7-1 μl/min."

    Referee cross-commenting

    I agree with the other reviewer's comments. The manuscript is already very complete could be published without the addition of other experiments, but the ones I proposed could validate even more the in vitro model. For example the permeability with lower molecular weight tracers, could show that the changes in vessel permeability might already exist at early timepoints in the xenograft model, similarly than in the in vitro model.

    Significance

    The manuscript is comprehensive, complete and represents the first bioengineered model of sepsis. One of the major strengths is the carful characterization and benchmarking against the animal xenograft model. Its main limitations is the brief description of the photoablation methodology and more clarity is needed in the description of bacteria perfusion experiments, given their complexity. The manuscript will be of interest for the general infection community and to the tissue engineering community if more details on fabrication methods are included.

    My expertise is on infection bioengineered models.

  4. Version published to 10.1101/2024.02.09.579276v2 on bioRxiv
  5. Version published to 10.1101/2024.02.09.579276v1 on bioRxiv