Periodic Reporting for period 3 - IM-ID (Defining the intrinsic transcriptional programs and the microenvironmental signals tailoring lung Interstitial Macrophage IDentity)
Okres sprawozdawczy: 2022-01-01 do 2023-06-30
Resident tissue macrophages (ResMacs) are present in most tissues and are recognized as an integral part of the tissues, where they play key roles in development, homeostasis, metabolism and repair. The diversity and identity (ID) of ResMacs is thought to rely on signals they receive from their microenvironment (i.e. the local niche), which trigger activation of specific transcription factors and differentiation programs, thus tailoring a particular identity that fulfills tissue-specific functions. We have previously identified lung interstitial macrophages (IM) as crucial regulators of lung tissue homeostasis at steady-state. However, they have been mainly investigated as a bulk population in functional studies. The ERC grant “IM-ID” aims to define the identity of IM at the highest resolution, model the differentiation of IM from monocytes using an innovative transgenic mouse model allowing IM niche depletion and refilling in vivo, and thereby investigate the intrinsic transcriptional programs and the niche-derived signals tailoring lung IM ID and sustaining lung homeostasis. This project will increase our understanding of the basic mechanisms underlying the fine-tuning of tolerogenic IM and will thus provide robust foundations for novel IM-targeted approaches promoting health and preventing lung diseases in which IM (dys)functions have been implicated.
Thanks to high-dimensional single-cell RNA-sequencing technologies, we discovered that lung IM were heterogenous and encompassed to functionally distinct subsets of long-lived monocyte-derived IM residing in different niches. CD206high IM are peribronchial self-maintaining ResMacs that constitutively produce high levels of chemokines and immunosuppressive cytokines, while CD206low IM preferentially populate the alveolar interstitium and exhibit features of antigen-presenting cells. To investigate the intrinsic transcriptional programs and extrinsic niche-derived cues regulating IM subset differentiation from monocytic precursors and IM identity, we generated and characterized a transgenic mouse model that allows the complete depletion of the IM niche. In this model, we can specifically trigger a bolus depletion of both IM subsets by diphteria toxin to create an empty niche, which is then rapidly repopulated by monocytes differentiating into newly differentiated IM that are similar to the native IM. We performed single cell RNA-sequencing on monocytes, differentiating IM and niche cells at different time points after DT injection, which revealed for the first time a comprehensive high-resolution picture of the of IM differentiation process and the niche of IM. Computational analyses and validation experiments revealed important IM-intrinsic transcription factors and niche ligand-IM receptor interactions involved in IM differentiation and identity. The functional relevance of these transcription factors and intercellular communications are currently investigated in-depth in vivo.
Having identified promising intrinsic and extrinsic molecules and molecular circuits that could be crucial for IM ID and lung homeostasis, we will formally investigate their roles in vivo. Conventional models of conditional gene deletion in IM or niche cells will be used on the one hand, to the extent of the resources available. On the other hand, we are currently developing and implementing a CRISP-seq-based strategy in order to formally and simultaneously test the function of many gene products, alone or in combination. In brief, the CRISP-seq strategy combines CRISPR-based genome editing with a single-cell RNA-sequencing read-out, thus allowing the profiling of the perturbation (i.e. in our case, the IM-specific knock-out of genes coding for transcription factors or receptors binding niche-derived signals) and of the transcriptome in single IM. We have already achieved several milestones in the implementation of CRISP-seq and want to succeed in this innovative method and model that is beyond the state-of-the-art. By the end of the project, we expect to identify the main niche signals activating specific transcription factors in IM subsets, thus endowing them with a particular identity and sustaining lung homeostasis.