Periodic Reporting for period 4 - ImmProDynamics (Dissecting the interplay between the dynamics of immune responses and pathogen proliferation in vivo)
Période du rapport: 2021-09-01 au 2022-02-28
Many infectious microorganisms can invade into cells of the body in order to hide from the defence mechanisms of the immune system. Some microbes can not only survive, but even grow to high numbers within infected cells. It is unknown if, or by which mechanism, the immune system is able to specifically detect microbes which are growing, which would be a very important information to identify a threat by such intracellular microorganisms. Vice versa, not every cell represents an equally well-suited niche for pathogen proliferation, and the interplay between the host immune system with the pathogen that affects the pathogen’s proliferation rate is unclear.
The question whether and how the immune system perceives growth of a microorganism is important regarding the fact that vaccines containing live microbes exhibit dramatically different outcomes of immune protection. Also, the growth rate of a pathogen might have an important impact on its resilience towards antimicrobial defences. Therefore, understanding how the growth rate of infectious microbes impacts on the activation, dynamics and plasticity of immune cells, and how this interaction in turn affects the pathogen’s physiology, could provide critical information on how infections can be controlled, and how microbial persistence in the body could be counteracted. This knowledge can on the one hand help to improve the prevention and treatment of infectious diseases. On the other hand, by revealing how the immune system’s plasticity and dynamics is affected by the interaction with the pathogen, the findings obtained in the project will open new possibilities to modulate immune responses in the context of a variety of diseases ranging from cancer through autoimmunity.
In order to elucidate the interplay between pathogen proliferation and the dynamics and plasticity of the immune system, used a biosensor that permits measuring the growth rate of the microbe Leishmania major in real-time and in the ongoing infection. This enabled us to determine (1) whether fast versus slow growing pathogens occupy different cellular niches in the infected individual, (2) whether immune cells show a differential behavior when confronted with microbes of different growth rates, and (3) how molecular activation signals and the metabolic state of immune cells are connected to the growth activity of the pathogen.
The question whether and how the immune system perceives growth of a microorganism is important regarding the fact that vaccines containing live microbes exhibit dramatically different outcomes of immune protection. Also, the growth rate of a pathogen might have an important impact on its resilience towards antimicrobial defences. Therefore, understanding how the growth rate of infectious microbes impacts on the activation, dynamics and plasticity of immune cells, and how this interaction in turn affects the pathogen’s physiology, could provide critical information on how infections can be controlled, and how microbial persistence in the body could be counteracted. This knowledge can on the one hand help to improve the prevention and treatment of infectious diseases. On the other hand, by revealing how the immune system’s plasticity and dynamics is affected by the interaction with the pathogen, the findings obtained in the project will open new possibilities to modulate immune responses in the context of a variety of diseases ranging from cancer through autoimmunity.
In order to elucidate the interplay between pathogen proliferation and the dynamics and plasticity of the immune system, used a biosensor that permits measuring the growth rate of the microbe Leishmania major in real-time and in the ongoing infection. This enabled us to determine (1) whether fast versus slow growing pathogens occupy different cellular niches in the infected individual, (2) whether immune cells show a differential behavior when confronted with microbes of different growth rates, and (3) how molecular activation signals and the metabolic state of immune cells are connected to the growth activity of the pathogen.
We have characterized the cell types in which efficient growth of Leishmania major takes place in the infected skin. Using intravital 2-photon imaging of the tissue infected with Leishmania major that expresses a biosensor for growth, we could determine that the growth rate of the pathogen is strongly linked to specific cell types. Using a series of experiments to synchronize the arrival of cells, mainly monocytic phagocytes (monocytes) at the site of infection, and by in-depth single cell analysis, we could show that the monocytes infected by Leishmania major at the site of infection are heterogenic, ranging from pro- through anti-inflammatory phenotypes which coexist at the same tissue site. These findings are not only important for the elucidation of the cell tropism of the pathogen Leishmania major, but also expand our knowledge on how recruited monocytes are activated and exert their function upon entry of the site of infection.
The pro-inflammatory, newly recruited monocytes were revealed to be the cell type which harbours mainly fast-growing pathogens. This suggested that high pathogen proliferation is linked with the transfer of Leishmania major to new host cell, which we could show is the case and, since it was outside of the focus of the funded research, lead to the application of a follow-up project dealing exclusively with the question of pathogen exit from the infected host cell. As efficient Leishmania proliferation required newly skin-recruited monocytes, and since the antimicrobial effector nitric oxide (NO) can inhibit entry of these cells into infected tissues, we investigated, whether NO dampens Leishmania proliferation indirectly, limiting the pathogen’s cellular niche. Indeed, we could show that restriction of proliferation-permissive host cell recruitment by NO represents a mechanism that controls Leishmania major infection.
The anti-inflammatory cells on the other hand, infected with mainly slow-proliferating Leishmania major were found to be the cell type most different from any other monocyte subset investigated. We found a number of genes, including IL7R and Cathepsin B specifically up-regulated in this cell population. Ablation of these genes, both globally and limited to myeloid cells (of which monocytes constitute the largest part at the site of infection), resulted in an enhanced control of Leishmania major. For IL7R it was shown that a main effect in the cell population infected by slow proliferating Leishmania major (termed LowP) might be the reorganization of the extracellular matrix at the site of infection, while Cathepsin B seems to inhibit efficient T cell activation. Therefore, the LowP population might serve as a “control centre” that enables pathogen persistence at the site of infection, possibly via IL7R and Cathepsin B mediated mechanisms.
The results obtained in the project were included in nine publications, seven of which included first and/or corresponding authorships by members of the workforce (Heyde et al., 2018; Brandt et al., 2020; Seiß et al., 2019; Baars et al., 2021; Regli et al., 2020; Handschuh et al., 2020; Kleinholz et al., 2021; Formaglio*, Alabdullah* et al., 2021; Krone*, Fu* et al., 2022).
The results were also presented in grant applications for competitive funding by the German research foundation DFG, resulting in two successful DFG research grants to the PI (DFG MU 3744/5-1. DFG MU 3744/6-1).
The pro-inflammatory, newly recruited monocytes were revealed to be the cell type which harbours mainly fast-growing pathogens. This suggested that high pathogen proliferation is linked with the transfer of Leishmania major to new host cell, which we could show is the case and, since it was outside of the focus of the funded research, lead to the application of a follow-up project dealing exclusively with the question of pathogen exit from the infected host cell. As efficient Leishmania proliferation required newly skin-recruited monocytes, and since the antimicrobial effector nitric oxide (NO) can inhibit entry of these cells into infected tissues, we investigated, whether NO dampens Leishmania proliferation indirectly, limiting the pathogen’s cellular niche. Indeed, we could show that restriction of proliferation-permissive host cell recruitment by NO represents a mechanism that controls Leishmania major infection.
The anti-inflammatory cells on the other hand, infected with mainly slow-proliferating Leishmania major were found to be the cell type most different from any other monocyte subset investigated. We found a number of genes, including IL7R and Cathepsin B specifically up-regulated in this cell population. Ablation of these genes, both globally and limited to myeloid cells (of which monocytes constitute the largest part at the site of infection), resulted in an enhanced control of Leishmania major. For IL7R it was shown that a main effect in the cell population infected by slow proliferating Leishmania major (termed LowP) might be the reorganization of the extracellular matrix at the site of infection, while Cathepsin B seems to inhibit efficient T cell activation. Therefore, the LowP population might serve as a “control centre” that enables pathogen persistence at the site of infection, possibly via IL7R and Cathepsin B mediated mechanisms.
The results obtained in the project were included in nine publications, seven of which included first and/or corresponding authorships by members of the workforce (Heyde et al., 2018; Brandt et al., 2020; Seiß et al., 2019; Baars et al., 2021; Regli et al., 2020; Handschuh et al., 2020; Kleinholz et al., 2021; Formaglio*, Alabdullah* et al., 2021; Krone*, Fu* et al., 2022).
The results were also presented in grant applications for competitive funding by the German research foundation DFG, resulting in two successful DFG research grants to the PI (DFG MU 3744/5-1. DFG MU 3744/6-1).
The project has deciphered the link between the in vivo growth rate of Leishmania major and the behaviour of infected host cells. First, we have defined the cellular environment of pathogen proliferation in detail, yielding important information about the architecture and dynamic processes of inflammatory tissues. Second, we have shown in the ongoing infection the spread of the microbe from one cell to the next, and identified critical cell types in the process, which contributes to the better understanding of how infectious organisms can persist and disseminate at the site of infection. Finally, as we could show the importance of newly recruited cells for the establishment of an infection by Leishmania major, we found that an immunomodulatory mechanism has an important additional role in controlling the pathogen’s proliferation. The methods that we have established to link the in vivo behavior of a pathogen and the recruitment kinetics of monocytes in the inflammatory tissue with single cell RNA sequencing constitute an important tool to study host-pathogen interactions on a cellular level in the living tissue.
Taken together, the project expands our understanding of how the monocyte populations recruited to an infection site mature and are activated over time, and in the context of pathogen proliferation. The exploration of the interactions of these cells on a molecular and cellular level with the pathogen, and with other cells of the immune system, will continue to increase our general understanding in how the immune system can be manipulated in order to better treat infections, but also immunodeficiency, cancer or autoimmunity.
Taken together, the project expands our understanding of how the monocyte populations recruited to an infection site mature and are activated over time, and in the context of pathogen proliferation. The exploration of the interactions of these cells on a molecular and cellular level with the pathogen, and with other cells of the immune system, will continue to increase our general understanding in how the immune system can be manipulated in order to better treat infections, but also immunodeficiency, cancer or autoimmunity.