Dynamics of macrophage polarization support Salmonella persistence in a whole living organism

  1. Jade Leiba
  2. Tamara Sipka
  3. Christina Begon-Pescia
  4. Matteo Bernardello
  5. Sofiane Tairi
  6. Lionello Bossi
  7. Anne-Alicia Gonzalez
  8. Xavier Mialhe
  9. Emilio J Gualda
  10. Pablo Loza-Alvarez
  11. Anne Blanc-Potard
  12. Georges Lutfalla
  13. Mai E Nguyen-Chi

  • Curated by eLife

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    eLife assessment

    This useful study introduces the development of Salmonella infection model in zebrafish embryos as an important model to study the interaction between macrophages and Salmonella during in vivo infection. Overall, the data presented are convincing and provide an inventory of genes mediating macrophage cell-cell adhesion and interactions that are useful for dissecting tissue macrophage responses and heterogeneity during intracellular bacterial infection. This is important to characterize the infection outcome and the dynamics of the immune response. The work will be of interest to microbiologists.

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Abstract

Numerous intracellular bacterial pathogens interfere with macrophage function, including macrophage polarization, to establish a niche and persist. However, the spatiotemporal dynamics of macrophage polarization during infection within host remain to be investigated. Here, we implement a model of persistent Salmonella Typhimurium infection in zebrafish, which allows visualization of polarized macrophages and bacteria in real time at high resolution. While macrophages polarize toward M1-like phenotype to control early infection, during later stages, Salmonella persists inside non-inflammatory clustered macrophages. Transcriptomic profiling of macrophages showed a highly dynamic signature during infection characterized by a switch from pro-inflammatory to anti-inflammatory/pro-regenerative status and revealed a shift in adhesion program. In agreement with this specific adhesion signature, macrophage trajectory tracking identifies motionless macrophages as a permissive niche for persistent Salmonella . Our results demonstrate that zebrafish model provides a unique platform to explore, in a whole organism, the versatile nature of macrophage functional programs during bacterial acute and persistent infections.

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  1. Version published to 10.7554/elife.89828 on eLife
  2. eLife

    Author Response

    Reviewer #2 (Public Review):

    In this study, Leiba et al. aim at establishing the developing zebrafish embryo as a suitable infection model to study Salmonella persistence in vivo. Under environmental stress (ex: macrophage phagosomes) a proportion of bacteria switch to a slow/arrested growth state conferring increased resistance to antibiotic treatments. Persisters are getting increasingly linked to infection relapses. Understanding how persistent infections emerge and bacteria survive in an organism for long time without replicating before switching back to a replicative state is essential. Zebrafish represents an alternative model to mice offering the possibility to image the whole organism and capture persistency with an amazing spatio-temporal resolution.

    In this paper, the authors demonstrate that persistent infections of Salmonella can be reproduced in the developing zebrafish. The kinetics of infection have been well characterized and shows a very nice heterogeneity between animals demonstrating the complex host-pathogen interactions (Fig 1). From the perspective of persistence, the presence of Salmonella survivors to host clearing is reported until 14dpi demonstrating the possibility to induce persistent infection in this model. Through the manuscript, the authors have used a variety of state-of-the-art technics illustrating the flexibility of this model including microscopy and imaging of specific immune populations, various transgenic animals and selective depletion of macrophages or neutrophils to assess their relative contributions. Overall, the conclusions of the authors are well supported by the presented data. This said, the authors should strengthen the conclusions of the paper by providing a better characterization of the infection.

    Major comments:

    1. Figure 1: What is the general life-spam of the fish?

    The general life-span of the zebrafish is approximately 3 years on average. Persistent infection is determined by the existence of a fraction of bacteria that endure over an extended period (after 96 hpi). Further, we observed Salmonella persistence for 14 days. In figure 1, we don’t think that the information of the general life-span of the zebrafish is critical.

    1. Figure 2: It would be nice to clearly state what infection scenario we are looking at. Have the authors studied "high proliferation", "infected" or "cleared" zebrafish?

    In Figure 2 we have studied the "infected" group. Both "high proliferation" and "cleared" larvae were excluded from the analysis. This is now clearly stated in the legend of Figure 2.

    1. Figure 3 and 4: It would be very informative if the authors can tell us what proportion of Salmonella is associated with macrophages and neutrophils. From panel C and D (Figure 3) and Figure 4 C and D and Suppl Fig 1, it seems that a lot of bacteria are extracellular. Maybe an EM image of the tissue would help to understand if the bacteria is "all" intracellular or intracellular.

    We apologize for any misunderstanding regarding the presence of intra- and extracellular bacteria depicted in Figure 3 C and D, Figure 4 C and D and Figure 3 -Suppl Fig 1. These figures illustrate infection experiments conducted in single-reporter larvae, limiting our analysis to bacteria associated with a single cell type. Figure 3G and Figure 4E-G, the panels depict infection experiments carried out in dual-reporter larvae, showing bacteria associated or not with macrophages and neutrophils. The present study aimed to establish the role of neutrophils and macrophages in the control of early and persistent Salmonella infection but further studies will focus on the exact localization of Salmonella during the course of the infection and, despite being a challenging technique for zebrafish, electron microscopy could be of great interest, allowing to visualize any type of cells (to determine if all bacteria are intracellular) at high resolution.

    1. Figure 3 and 4: It would be very useful if the authors can tell us if the intracellular bacteria are mainly found individually (like in Figure 3C) or does host cells harbor many intracellular bacteria. Looking at figure 4G: it is not clear to me how many intracellular bacteria can be counted on this image.

    This is an interesting suggestion. At present, an accurate quantification of the intracellular bacteria on microscopy 3D-datasets is challenging because bacteria aggregate inside the cells. At 4 hpi, single bacteria can occasionally be observed outside leukocytes, while most of infected macrophages harbored several intracellular bacteria (bacteria aggregates). To compare the levels of intracellular bacterial between acute and persistent stages, we measured the size of E2Crimson-positive (E2Crimson+) events. At 5 hpi, the median volume of E2Crimson+ events was lower than that at 4 dpi. The size distribution analysis of E2Crimson+ events indicated a higher representation of smaller volumes (0.5-1.5 m3 and 1.5-10 m3) at 5 hpi compared to 4 dpi, a stage during which very large E2Crimson+ events were observed (between 100-1000 m3, with some exceeding 1000 m3). This observation suggests an elevated presence of intracellular bacteria within the cells during persistent stages and that intracellular bacteria are predominantly observed as multiple rather than as solitary entities. This analysis has been incorporated in new Figure 5.

    1. Figure 3 and 4: The authors should also perform an experiment with a Salmonella strain harboring a growth reporter to quantify the amount of replicating and non-replicating bacteria. This experiment is not absolutely necessary for the story, but if possible, it would provide a very nice add-up to the story and impact to the paper.

    We welcome the reviewers’ suggestion, which we have indeed considered and planning to carry on in the future, along with experimented more oriented on the bacterial side.

    1. Figure 6: The authors should provide in suppl. the flow cytometry scatter plots used to delineate the different subpopulations.

    We agree with the reviewer that the flow cytometry scatter plots used to delineate the different subpopulations were missing and are now incorporated in new Fig 7 - figure supplement 2.

    1. Figure 6: A specific characterization of macrophages harboring Salmonella persisters at 4dpi is missing. As shown by the authors in Figure 6, the tnfa- populations of macrophages at 4dpi are very similar for both infected and non-infected larvae. Persisters should indeed reside within tnfa- macrophages but they should also induce a specific signature through the actions of Salmonella effectors. Measuring this signature will allow a direct comparison with published data in mice and assess how accurately the zebrafish model recapitulates the manipulation of macrophages by Salmonella

    We agree with the reviewer that a specific characterization of macrophages harboring persistent Salmonella at 4 dpi is missing. However due to the technical limitation inherent to the model (limited recovery of infected cells following FACS sorting), we were not able to specifically sort infected macrophages at 4 dpi.

  3. eLife

    eLife assessment

    This useful study introduces the development of Salmonella infection model in zebrafish embryos as an important model to study the interaction between macrophages and Salmonella during in vivo infection. Overall, the data presented are convincing and provide an inventory of genes mediating macrophage cell-cell adhesion and interactions that are useful for dissecting tissue macrophage responses and heterogeneity during intracellular bacterial infection. This is important to characterize the infection outcome and the dynamics of the immune response. The work will be of interest to microbiologists.

  4. eLife

    Reviewer #1 (Public Review):

    In this manuscript, the authors seek to investigate the spatiotemporal dynamics of macrophage polarization during Salmonella infection. They undertake intravital microscopy of Salmonella Typhimurium infection in the hindbrain ventricle of zebrafish larvae and couple this with transcriptomic analysis of macrophages from infected tissues. They find that macrophages and neutrophils are rapidly recruited to the site of infection within hours after inoculation. Macrophage abundance is significantly increased in the persistent infection stage at 4 days post-inoculation (dpi), compared to in the early stage, hours post-inoculation. The authors observe that Salmonella bacilli selectively co-localize with aggregates of macrophages, but not neutrophils, during persistent infection. Furthermore, they show that in early infection stage, a markedly higher fraction of macrophages at the site of infection expressed tnfa and exhibits stronger transcriptional signature of pro-inflammatory, M1-like phenotype, compared to macrophages in persistent infection stage. Additionally, the authors find that genes involved in cell-cell adhesion are down-regulated in persistent stage macrophages and these cells have reduced motility. This study's approach, further developing and employing a zebrafish S. Typhimuirum infection model and intravital microscopy of whole living animals, presents an exciting strategy to investigate macrophage responses and their roles during vacuolar intracellular bacterial infection in vivo, complementary to the more commonly utilized murine infection models. The study's findings are useful and largely observational. The data presented have the potential but additional analyses and experiments are needed to clarify and support the conclusions.

  5. eLife

    Reviewer #2 (Public Review):

    In this study, Leiba et al. aim at establishing the developing zebrafish embryo as a suitable infection model to study Salmonella persistence in vivo. Under environmental stress (ex: macrophage phagosomes) a proportion of bacteria switch to a slow/arrested growth state confering increased resistance to antibiotic treatments. Persisters are getting increasingly linked to infection relapses. Understanding how persistent infections emerge and bacteria survive in an organism for long time without replicating before switching back to a replicative state is essential. Zebrafish represents an alternative model to mice offering the possibility to image the whole organism and capture persistency with an amazing spatio-temporal resolution.

    In this paper, the authors demonstrate that persistent infections of Salmonella can be reproduced in the developing zebrafish. The kinetics of infection have been well characterized and shows a very nice heterogeneity between animals demonstrating the complex host-pathogen interactions (Fig 1). From the perspective of persistence, the presence of Salmonella survivors to host clearing is reported until 14dpi demonstrating the possibility to induce persistent infection in this model. Through the manuscript, the authors have used a variety of state-of-the-art technics illustrating the flexibility of this model including microscopy and imaging of specific immune populations, various transgenic animals and selective depletion of macrophages or neutrophils to assess their relative contributions. Overall, the conclusions of the authors are well supported by the presented data. This said, the authors should strengthen the conclusions of the paper by providing a better characterization of the infection.

    Major comments:
    1- Figure 1: What is the general life-spam of the fish?

    2- Figure 2: It would be nice to clearly state what infection scenario we are looking at. Have the authors studied "high proliferation", "infected" or "cleared" zebrafish?

    3- Figure 3 and 4: It would be very informative if the authors can tell us what proportion of Salmonella is associated with macrophages and neutrophils. From panel C and D (Figure 3) and Figure 4 C and D and Suppl Fig 1, it seems that a lot of bacteria are extracellular. Maybe an EM image of the tissue would help to understand if the bacteria is "all" intracellular or intracellular.

    4- Figure 3 and 4: It would be very useful if the authors can tell us if the intracellular bacteria are mainly found individually (like in Figure 3C) or does host cells harbor many intracellular bacteria. Looking at figure 4G: it is not clear to me how many intracellular bacteria can be counted on this image.

    5- Figure 3 and 4: The authors should also perform an experiment with a Salmonella strain harboring a growth reporter to quantify the amount of replicating and non-replicating bacteria. This experiment is not absolutely necessary for the story, but if possible, it would provide a very nice add-up to the story and impact to the paper.

    6- Figure 6: The authors should provide in suppl. the flow cytometry scatter plots used to delineate the different subpopulations.

    7- Figure 6: A specific characterization of macrophages harboring Salmonella persisters at 4dpi is missing. As shown by the authors in Figure 6, the tnfa- populations of macrophages at 4dpi are very similar for both infected and non-infected larvae. Persisters should indeed reside within tnfa- macrophages but they should also induce a specific signature through the actions of Salmonella effectors. Measuring this signature will allow a direct comparison with published data in mice and assess how accurately the zebrafish model recapitulates the manipulation of macrophages by Salmonella

  6. Version published to 10.1101/2023.05.09.539693v1 on bioRxiv