Periodic Reporting for period 1 - TakeupSLaCk (Role and regulation of dendritic cell functions by Solute Carrier Transporters)
Periodo di rendicontazione: 2019-01-01 al 2020-12-31
Phagocytes respond to billions of dying cells daily and a plethora of external pathogens. Dendritic cells (DCs) are a heterogenous group of phagocytes that express several phagocytic and pathogen recognition receptors (PRRs), and help maintain tissue homeostasis and prevent autoimmunity. Gene signatures initiated in dendritic cells during efferocytosis are not yet defined, and adaptation of dendritic cells to the metabolic challenge of ingesting efferocytic versus pathogenic cargo remain unclear. This project aimed to analyze the transcriptional programs and solute carrier transporter (SLC) signature in DC subsets upon engulfment in vitro and in vivo. My analysis revealed SLCs associated with amino acid metabolism were upregulated in efferocytic dendritic cells, while SLCs linked to intracellular pH regulation and nucleoside salvage/energy metabolism were upregulated upon ingesting pathogenic cargo. These studies addressing the uptake via dendritic cells, the role of SLCs, and altered DC responses could be useful for SLC-targeted drug development of both common and rare diseases, and to enhance the targeting of engulfment in healthy states.
To identify the pathways involved in phagocytosis by dendritic cells, I performed RNA sequencing of primary bone-marrow derived dendritic cells engulfing apoptotic human Jurkat cells (to undeniably distinguish phagocyte-derived RNA only) or after uptake of LPS-coated beads as a source of inflammatory trigger. My analysis revealed differential SLC programs that are initiated in dendritic cells during efferocytosis of apoptotic cells versus LPS-coated bead uptake. SLCs mediate transport of ions, aminoacids and small molecules across cellular membranes, are linked to >100 human diseases, and are also amenable for targeting by small molecules.
More particularly, SLCs associated with amino acid metabolism were upregulated and SLCs associated with oxidative phosphorylation (OXPHOS) were downregulated in efferocytic dendritic cells, while SLCs linked to intracellular pH regulation and nucleoside salvage/energy metabolism were upregulated upon ingesting pathogenic cargo. Using mouse models, I validated this SLC signature in DC subsets in vivo, in both tolerogenic and immunogenic conditions of phagocytosis.
Notably, efferocytic engulfment by dendritic cells indicated that an aminoacid transporter, which is highly expressed in skin-specific dendritic cell subsets, is upregulated in wounded skin in mice. Moreover, my in vitro studies show that switching off the gene function of the transporter specifically in dendritic cells, either by pharmacological inhibition or by gene deletion, promotes phagocytosis of dying cells. Therefore, I investigated whether this aminoacid transporter plays a role in the wound healing process, since removal of dead cells in the wounds by phagocytes, including dendritic cells, is essential for accelerating wound healing dynamics. My results demonstrated that topical administrator of a specific inhibitor of the transporter has both preventive and therapeutic effect in cutaneous wound healing in mouse models.
These results highlight dendritic cells and solute carrier transporters as critical players that regulate efferocytic clearance in the skin and open possibilities for more efficient treatment of chronic cutaneous wounds
More particularly, SLCs associated with amino acid metabolism were upregulated and SLCs associated with oxidative phosphorylation (OXPHOS) were downregulated in efferocytic dendritic cells, while SLCs linked to intracellular pH regulation and nucleoside salvage/energy metabolism were upregulated upon ingesting pathogenic cargo. Using mouse models, I validated this SLC signature in DC subsets in vivo, in both tolerogenic and immunogenic conditions of phagocytosis.
Notably, efferocytic engulfment by dendritic cells indicated that an aminoacid transporter, which is highly expressed in skin-specific dendritic cell subsets, is upregulated in wounded skin in mice. Moreover, my in vitro studies show that switching off the gene function of the transporter specifically in dendritic cells, either by pharmacological inhibition or by gene deletion, promotes phagocytosis of dying cells. Therefore, I investigated whether this aminoacid transporter plays a role in the wound healing process, since removal of dead cells in the wounds by phagocytes, including dendritic cells, is essential for accelerating wound healing dynamics. My results demonstrated that topical administrator of a specific inhibitor of the transporter has both preventive and therapeutic effect in cutaneous wound healing in mouse models.
These results highlight dendritic cells and solute carrier transporters as critical players that regulate efferocytic clearance in the skin and open possibilities for more efficient treatment of chronic cutaneous wounds
Dendritic cell-based interventions are traditionally used for therapies against cancer, inflammation, allergies, autoimmunity and vaccinations – therefore, there is a critical need for deeper understanding of DC biology and for the future development of efficient diagnostics and therapeutics. However, the role of dendritic cells in apoptotic cell clearance and skin homeostasis has not been addressed successfully.
The skin is the largest organ of the body and forms a barrier that protects the organism against infection, ultraviolet radiation or wounding. Deregulation of cell death mechanism or aberrant clearance of dead cells in the skin can lead to diseases like cancer or chronic skin inflammation. Immune cells in the skin, like macrophages and Langerhans cell, exert different roles in homeostatic conditions compared to wound repair, hair follicle regeneration or cancer progression. Despite the clear association between phagocytes and tissue damage repair, chronic inflammation and cancer, little is known about the role of dendritic cells in these processes. Dendritic cells (DCs) comprise an heterogenous group of phagocytes that are equipped with several phagocytic and pathogen recognition receptors (PRRs), and with the processing machinery to mediate efficient elimination of apoptotic or tumor cells. My studies validate dendritic cells as critical players that regulate wound healing in the skin and demonstrate aminoacid transporters as key modifiers of the phagocytes “appetite. My future studies will address the role of dendritic cells and of relevant solute carrier transporters, identified from this project, in models of skin tumors.
The skin is the largest organ of the body and forms a barrier that protects the organism against infection, ultraviolet radiation or wounding. Deregulation of cell death mechanism or aberrant clearance of dead cells in the skin can lead to diseases like cancer or chronic skin inflammation. Immune cells in the skin, like macrophages and Langerhans cell, exert different roles in homeostatic conditions compared to wound repair, hair follicle regeneration or cancer progression. Despite the clear association between phagocytes and tissue damage repair, chronic inflammation and cancer, little is known about the role of dendritic cells in these processes. Dendritic cells (DCs) comprise an heterogenous group of phagocytes that are equipped with several phagocytic and pathogen recognition receptors (PRRs), and with the processing machinery to mediate efficient elimination of apoptotic or tumor cells. My studies validate dendritic cells as critical players that regulate wound healing in the skin and demonstrate aminoacid transporters as key modifiers of the phagocytes “appetite. My future studies will address the role of dendritic cells and of relevant solute carrier transporters, identified from this project, in models of skin tumors.