Periodic Reporting for period 1 - TLRstorm (Spatial and temporal control of self/non-self discrimination in innate immunity)
Período documentado: 2019-07-01 hasta 2021-06-30
This project aimed to understand how the regulation of nucleic acid-sensing Toll-like receptors (TLRs) and its various interactions are integrated into the cellular architecture. It has long been appreciated that the endosomal localization of this class of immune receptors is critical for proper self/non-self discrimination of nucleic acids, and that the breakdown of this compartmentalization can lead to autoimmune or autoinflammatory diseases. Here, I proposed to elucidate the spatial and functional segregation of endosomal TLRs to systematically dissect how their subcellular distribution impacts signaling and maintains immune tolerance.
The dysregulation of nucleic acid-sensing TLRs, including TLR7, TLR8, and TLR9, is known to play a major role in numerous human autoimmune diseases. Interestingly, the cellular mechanisms that promote loss of self-tolerance by individual TLRs seem to differ. For example, while overexpression of TLR7 is sufficient to break the ‘tolerance’ mechanisms that normally prevent reactivity to self, merely increasing the number of TLR9 molecules in the cell does not lead to aberrant activation of TLR9. These findings point to fine-tuned regulation of each TLR family member, the details of which are only recently coming to light. Thus, the differential regulation of nucleic acid-sensing TLRs is of medical relevance for understanding the pathomechanisms of autoimmunity and autoinflammatory disease. This work will provide a conceptual framework for how TLR signaling is controlled in space and time and define the molecular principles that maintain self-tolerance to nucleic acids.
The formal objectives of this MSCA have been to 1) define the precise subcellular localization (single-molecule resolution) of nucleic acid-sensing TLRs, to 2) identify and characterize the intracellular TLR signaling compartments, and to 3) investigate TLR signaling dynamics under normal and dysregulated (autoimmune-prone) conditions.
The fellowship has been prematurely suspended as I accepted a Research Group Leader position at the Max Planck Institute for Infection Biology (MPIIB) in Berlin. I will continue to explore the same research questions in my own laboratory.
The dysregulation of nucleic acid-sensing TLRs, including TLR7, TLR8, and TLR9, is known to play a major role in numerous human autoimmune diseases. Interestingly, the cellular mechanisms that promote loss of self-tolerance by individual TLRs seem to differ. For example, while overexpression of TLR7 is sufficient to break the ‘tolerance’ mechanisms that normally prevent reactivity to self, merely increasing the number of TLR9 molecules in the cell does not lead to aberrant activation of TLR9. These findings point to fine-tuned regulation of each TLR family member, the details of which are only recently coming to light. Thus, the differential regulation of nucleic acid-sensing TLRs is of medical relevance for understanding the pathomechanisms of autoimmunity and autoinflammatory disease. This work will provide a conceptual framework for how TLR signaling is controlled in space and time and define the molecular principles that maintain self-tolerance to nucleic acids.
The formal objectives of this MSCA have been to 1) define the precise subcellular localization (single-molecule resolution) of nucleic acid-sensing TLRs, to 2) identify and characterize the intracellular TLR signaling compartments, and to 3) investigate TLR signaling dynamics under normal and dysregulated (autoimmune-prone) conditions.
The fellowship has been prematurely suspended as I accepted a Research Group Leader position at the Max Planck Institute for Infection Biology (MPIIB) in Berlin. I will continue to explore the same research questions in my own laboratory.
The transition for my Research Group Leader position at the MPIIB, as well as the Covid-19 pandemic lead to some deviations and delays from my original workplan. In some instances, I had to re-prioritize my action plan in order to quickly produce preliminary data for my job interview and conceptually expand my research plan into a 5-10 years research program. However, the main core objectives of my fellowship proposal remained the same. In the following section I will explain the goals and objectives of the MSCA IF proposal that have been met, as well as the actions that deviated from the original objectives due to the aforementioned reasons.
1) Define the precise subcellular localization of nucleic acid-sensing TLRs
Within the first three months of my funding period, I successfully implemented the primary Hoxb8 macrophage culture system in the host lab and received the necessary approval for S2 work by the ethics authorities. For timely reasons to quickly obtain preliminary superresolution microcopy data for my upcoming Max Planck selection symposium I decided to generate stable cell lines expressing various tagged-versions of TLRs using retroviral delivery, instead of generating endogenous CRISPR knock-in lines.
I obtained multiple cell lines within a few weeks and was able to compare them across multiple superresolution microscopy platforms, including dSTORM, STED, and SIM. In each case, I aimed to visualize TLRs in conjunction with one or two additional endosome markers to study their colocalization. The Lattice SIM Elyra 7 Microscope (Zeiss) offered the best platform for my research for its outstanding combination of 3D superresolution and acquisition speed. I purchased this microscope for my research group at the MPIIB.
2) Identify and characterize the intracellular TLR signaling compartments
For this aim, I generated stable Hoxb8 macrophages that would allow me to visualize TLR signaling in real-time. By fluorescently tagging Myd88, one of the proximal signaling adaptor of TLRs, TLR activation and signaling can be visualized through the assembly of supramolecular structures called the Myddosome. Myd88 KO Hoxb8 macrophages were reconstituted with Myd88-RFP using retroviral delivery and imaged for Myddosome formation after TLR7 stimulation. Due to the lock-down these experiments were performed during a remote demo workshop with Zeiss, in which we imaged live cells on the Lattice SIM Elyra 7. TLR7 stimulation of the Myd88-RFP line resulted in Myddosome assemblies that appeared in close proximity to endosomes, suggesting that this labeling strategy can successfully be used to identify TLR signaling endosomes. In the future, I want to utilize this labeling strategy to further characterize the nature of different TLR signaling endosomes.
3) Investigate TLR signaling dynamics under normal and dysregulated (autoimmune-prone) conditions
In preparation for my Research Group Leader position I scientifically deviated from Aim 3 and instead focused my attention on systems that would allow me to actively manipulate cellular organization to examine its effect on TLR signaling and self-tolerance. I reasoned that after having mapped the localization of endosomal TLRs and identified their specific signaling endosomes, I would now need a system to functionally explore these cellular coordinates. Specifically, I started to investigate how endosome positioning would affect TLR responses.
The correct localization of late endosomes is relevant in several cellular processes, including antigen presentation, metabolic sensing, or microbial killing. Perturbation of endosome positioning contributes to the pathogenesis of various diseases, such as cancer, neurodegeneration and autoimmunity. Interestingly, late endosomes exhibit functional heterogeneity based on their positioning in the cell, with peripheral endosomes being less acidic and proteolytic compared to perinuclear ones. As TLR function depends on proteolytic processing of the receptor by endosomal proteases, I hypothesized that endosome positioning may influence the signaling function of TLRs and impact the recognition of self-molecules. I manipulated endosome positioning in macrophages by depleting components of motor protein complexes that regulate endosome movement in the cell. My preliminary results indicate that altering endosome location has profound impacts on TLR signaling. I am currently investigating the underlying mechanisms of this regulation
1) Define the precise subcellular localization of nucleic acid-sensing TLRs
Within the first three months of my funding period, I successfully implemented the primary Hoxb8 macrophage culture system in the host lab and received the necessary approval for S2 work by the ethics authorities. For timely reasons to quickly obtain preliminary superresolution microcopy data for my upcoming Max Planck selection symposium I decided to generate stable cell lines expressing various tagged-versions of TLRs using retroviral delivery, instead of generating endogenous CRISPR knock-in lines.
I obtained multiple cell lines within a few weeks and was able to compare them across multiple superresolution microscopy platforms, including dSTORM, STED, and SIM. In each case, I aimed to visualize TLRs in conjunction with one or two additional endosome markers to study their colocalization. The Lattice SIM Elyra 7 Microscope (Zeiss) offered the best platform for my research for its outstanding combination of 3D superresolution and acquisition speed. I purchased this microscope for my research group at the MPIIB.
2) Identify and characterize the intracellular TLR signaling compartments
For this aim, I generated stable Hoxb8 macrophages that would allow me to visualize TLR signaling in real-time. By fluorescently tagging Myd88, one of the proximal signaling adaptor of TLRs, TLR activation and signaling can be visualized through the assembly of supramolecular structures called the Myddosome. Myd88 KO Hoxb8 macrophages were reconstituted with Myd88-RFP using retroviral delivery and imaged for Myddosome formation after TLR7 stimulation. Due to the lock-down these experiments were performed during a remote demo workshop with Zeiss, in which we imaged live cells on the Lattice SIM Elyra 7. TLR7 stimulation of the Myd88-RFP line resulted in Myddosome assemblies that appeared in close proximity to endosomes, suggesting that this labeling strategy can successfully be used to identify TLR signaling endosomes. In the future, I want to utilize this labeling strategy to further characterize the nature of different TLR signaling endosomes.
3) Investigate TLR signaling dynamics under normal and dysregulated (autoimmune-prone) conditions
In preparation for my Research Group Leader position I scientifically deviated from Aim 3 and instead focused my attention on systems that would allow me to actively manipulate cellular organization to examine its effect on TLR signaling and self-tolerance. I reasoned that after having mapped the localization of endosomal TLRs and identified their specific signaling endosomes, I would now need a system to functionally explore these cellular coordinates. Specifically, I started to investigate how endosome positioning would affect TLR responses.
The correct localization of late endosomes is relevant in several cellular processes, including antigen presentation, metabolic sensing, or microbial killing. Perturbation of endosome positioning contributes to the pathogenesis of various diseases, such as cancer, neurodegeneration and autoimmunity. Interestingly, late endosomes exhibit functional heterogeneity based on their positioning in the cell, with peripheral endosomes being less acidic and proteolytic compared to perinuclear ones. As TLR function depends on proteolytic processing of the receptor by endosomal proteases, I hypothesized that endosome positioning may influence the signaling function of TLRs and impact the recognition of self-molecules. I manipulated endosome positioning in macrophages by depleting components of motor protein complexes that regulate endosome movement in the cell. My preliminary results indicate that altering endosome location has profound impacts on TLR signaling. I am currently investigating the underlying mechanisms of this regulation
I have been able to image intracellular TLRs at unprecedented optical resolution and prepared the groundwork to now systematically explore their subcellular localization. I've been the first one to visualize TLR signaling events emanating from endosomes in real-time, which will now allow me to identify and characterize the specific signaling endosomes of nucleic acid-sensing TLRs. I've discovered a new regulatory pathway of nucleic acid-sensing TLRs, which involves endosome positioning. Dissecting the underlying molecular mechanism will improve the understanding of the basic principles of nucleic acid immunity.
As the fellowship has been prematurely suspended, no additional results will be expected from this funding period.
As the fellowship has been prematurely suspended, no additional results will be expected from this funding period.