Date Published: October 22, 2018
Publisher: Public Library of Science
Author(s): Sandrina Heyde, Lars Philipsen, Pauline Formaglio, Yan Fu, Iris Baars, Guido Höbbel, Corinna L. Kleinholz, Elena A. Seiß, Juliane Stettin, Patricia Gintschel, Anne Dudeck, Philippe Bousso, Burkhart Schraven, Andreas J. Müller, Nathan C. Peters.
The virulence of intracellular pathogens such as Leishmania major (L. major) relies largely on their ability to undergo cycles of replication within phagocytes, release, and uptake into new host cells. While all these steps are critical for successful establishment of infection, neither the cellular niche of efficient proliferation, nor the spread to new host cells have been characterized in vivo. Here, using a biosensor for measuring pathogen proliferation in the living tissue, we found that monocyte-derived Ly6C+CCR2+ phagocytes expressing CD11c constituted the main cell type harboring rapidly proliferating L. major in the ongoing infection. Synchronization of host cell recruitment and intravital 2-photon imaging showed that these high proliferating parasites preferentially underwent cell-to-cell spread. However, newly recruited host cells were infected irrespectively of their cell type or maturation state. We propose that among these cells, CD11c-expressing monocytes are most permissive for pathogen proliferation, and thus mainly fuel the cycle of intracellular proliferation and cell-to-cell transfer during the acute infection. Thus, besides the well-described function for priming and activating T cell effector functions against L. major, CD11c-expressing monocyte-derived cells provide a reservoir for rapidly proliferating parasites that disseminate at the site of infection.
Many pathogens have developed strategies to hijack host phagocytes and withstand their intracellular defense mechanisms. The ability to undergo cycles of replication within these phagocytes, release, and uptake into new host cells is central to the intracellular lifestyle, but has hardly been studied in the ongoing infection . The parasite Leishmania major (L. major) represents such a well-adapted pathogen which can survive and replicate under the harsh microenvironmental conditions of endocytic compartments of professional phagocytes [2–4]. Although adaptive T cell responses increase the capability of phagocytes to control pathogens and limit further infection [5–7], L. major can prevail at the site of infection for extended periods of time. This can result in chronic infections of several months duration, often accompanied with disfiguring and disabling pathologies .
To understand the interaction between the immune system and the pathogen, it is indispensable to extract data on the proliferation states of infectious microorganisms as well as to define the niches in which differentially proliferating populations are located. This information is especially critical for intracellular pathogens persisting for long periods of time at an infection site, such as L. major. Several recent approaches have addressed this question in the ongoing infection [22,30]. These elegant experiments have defined slow overall proliferation rates and postulated high proliferating subpopulations of parasites in the established infection, but neither approach was compatible with intravital or multiparameter host cell-resolved analysis of pathogen proliferation. This has severely hampered the unambiguous assignment of surface marker expression levels to individual host cells harboring high versus low proliferating parasites. However, this information would be indispensable for elucidating the link between L. major proliferation and host cell tropism. We achieved exactly this side-by-side analysis of pathogen proliferation and cellular surface markers by using the mKikume reporter system. Specifically, our experiments show that fluorescence recovery after photoconversion-based measurement of LmSWITCH infected cells and tissues is compatible not only with intravital 2-photon microscopy, but also with immunofluorescence-based analysis approaches such as flow cytometry as well as confocal and multiparameter microscopy (MELC).