Protocol for standardized intrathymic injection in mice.

Currently available intrathymic injection techniques cause postoperative complications or difficulties in equipment acquisition. Here, we describe a standardized intrathymic injection protocol that requires only basic equipment with a minimally invasive procedure. We detail steps to identify injection sites for intrathymic delivery. We then describe how to visualize a successful intrathymic injection by including Indian ink in the injected solution. For complete details on the use and execution of this protocol, please refer to Tsai et al. (2022).1.

IFN-stimulated metabolite transporter ENT3 facilitates viral genome release.

An increasing amount of evidence emphasizes the role of metabolic reprogramming in immune cells to fight infections. However, little is known about the regulation of metabolite transporters that facilitate and support metabolic demands. In this study, we found that the expression of equilibrative nucleoside transporter 3 (ENT3, encoded by solute carrier family 29 member 3, Slc29a3) is part of the innate immune response, which is rapidly upregulated upon pathogen invasion. The transcription of Slc29a3 is directly regulated by type I interferon-induced signaling, demonstrating that this metabolite transporter is an interferon-stimulated gene (ISG). Suprisingly, we unveil that several viruses, including SARS-CoV-2, require ENT3 to facilitate their entry into the cytoplasm. The removal or suppression of Slc29a3 expression is sufficient to significantly decrease viral replication in vitro and in vivo. Our study reveals that ENT3 is a pro-viral ISG co-opted by some viruses to gain a survival advantage.

Thymic macrophages consist of two populations with distinct localization and origin.

Tissue-resident macrophages are essential to protect from pathogen invasion and maintain organ homeostasis. The ability of thymic macrophages to engulf apoptotic thymocytes is well appreciated, but little is known about their ontogeny, maintenance, and diversity. Here, we characterized the surface phenotype and transcriptional profile of these cells and defined their expression signature. Thymic macrophages were most closely related to spleen red pulp macrophages and Kupffer cells and shared the expression of the transcription factor (TF) SpiC with these cells. Single-cell RNA sequencing (scRNA-Seq) showed that the macrophages in the adult thymus are composed of two populations distinguished by the expression of Timd4 and Cx3cr1. Remarkably, Timd4+ cells were located in the cortex, while Cx3cr1+ macrophages were restricted to the medulla and the cortico-medullary junction. Using shield chimeras, transplantation of embryonic thymuses, and genetic fate mapping, we found that the two populations have distinct origins. Timd4+ thymic macrophages are of embryonic origin, while Cx3cr1+ macrophages are derived from adult hematopoietic stem cells. Aging has a profound effect on the macrophages in the thymus. Timd4+ cells underwent gradual attrition, while Cx3cr1+ cells slowly accumulated with age and, in older mice, were the dominant macrophage population in the thymus. Altogether, our work defines the phenotype, origin, and diversity of thymic macrophages.

Heparin is required for the formation of granules in connective tissue mast cells.

Mast cells are innate immune cells strategically positioned around blood vessels near body surfaces. Their primary weapons are bioactive amines, mast cell-specific proteases, and cytokines stored in preformed granules. Mast cells granules constituents are packaged efficiently with the help of the highly negatively charged Heparan sulfate-derivative, Heparin. Heparin is one of the most widely used drugs to treat coagulation disorders, yet, it is not found in the circulation at a steady state, casting doubt that the prevention of blood clotting is its physiological function. Early studies using Ndst2 -/- mice have shown that Heparin is essential for mast cells granules formation. However, these mice could still produce less sulfated Heparan sulfate that could potentially replace Heparin. Here, we have created and validated a novel genetic model for Heparin deficiency, specifically in connective tissue mast cells, to address the physiological role of this molecule. Using this model, we have demonstrated that Heparin is required for mast cell granules formation; without it, mast cells are reduced in the peritoneal cavity and the skin. The absence of Heparin impaired the response to passive cutaneous anaphylaxis but, surprisingly, enhanced ear swelling in an irritant dermatitis model and reduced the lesion size and bacterial burden in a Staphylococcus aureus necrotizing dermatitis model. The altered function of Heparin-deficient mast cells in the latter two models was not mediated through enhanced Histamine or TNFα release. However, the Mrgprb2 receptor was up-regulated in knock-out mast cells, potentially explaining the enhanced response of mutant mice to irritant and necrotizing dermatitis. Altogether our results expand our current understanding of the physiological role of Heparin and provide unique tools to further dissect its importance.

Multiomics reveal the central role of pentose phosphate pathway in resident thymic macrophages to cope with efferocytosis-associated stress.

Tissue-resident macrophages (TRMs) are heterogeneous cell populations found throughout the body. Depending on their location, they perform diverse functions maintaining tissue homeostasis and providing immune surveillance. To survive and function within, TRMs adapt metabolically to the distinct microenvironments. However, little is known about the metabolic signatures of TRMs. The thymus provides a nurturing milieu for developing thymocytes yet efficiently removes those that fail the selection, relying on the resident thymic macrophages (TMφs). This study harnesses multiomics analyses to characterize TMφs and unveils their metabolic features. We find that the pentose phosphate pathway (PPP) is preferentially activated in TMφs, responding to the reduction-oxidation demands associated with the efferocytosis of dying thymocytes. The blockade of PPP in Mφs leads to decreased efferocytosis, which can be rescued by reactive oxygen species (ROS) scavengers. Our study reveals the key role of the PPP in TMφs and underscores the importance of metabolic adaptation in supporting Mφ efferocytosis.

Evaluation of Mitochondria Content and Function in Live Cells by Multicolor Flow Cytometric Analysis.

To evaluate how a cell responds to the external stimuli, treatment, or alteration of the microenvironment, the quantity and quality of mitochondria are commonly used as readouts. However, it is challenging to apply mitochondrial analysis to the samples that are composed of mixed cell populations originating from tissues or when multiple cell populations are of interest, using methods such as Western blot, electron microscopy, or extracellular flux analysis.Flow cytometry is a technique allowing the detection of individual cell status and its identity simultaneously when used in combination with surface markers. Here we describe how to combine mitochondria-specific dyes or the dyes targeting the superoxide produced by mitochondria with surface marker staining to measure the mitochondrial content and activity in live cells by flow cytometry. This method can be applied to all types of cells in suspension and is particularly useful for analysis of samples composed of heterogeneous cell populations.

Heparan sulfate is essential for thymus growth.

Thymus organogenesis and T cell development are coordinated by various soluble and cell-bound molecules. Heparan sulfate (HS) proteoglycans can interact with and immobilize many soluble mediators, creating fields or gradients of secreted ligands. While the role of HS in the development of many organs has been studied extensively, little is known about its function in the thymus. Here, we examined the distribution of HS in the thymus and the effect of its absence on thymus organogenesis and T cell development. We found that HS was expressed most abundantly on the thymic fibroblasts and at lower levels on endothelial, epithelial, and hematopoietic cells. To study the function of HS in the thymus, we eliminated most of HS in this organ by genetically disrupting the glycosyltransferase Ext1 that is essential for its synthesis. The absence of HS greatly reduced the size of the thymus in fetal thymic organ cultures and in vivo, in mice, and decreased the production of T cells. However, no specific blocks in T cell development were observed. Wild-type thymic fibroblasts were able to physically bind the homeostatic chemokines CCL19, CCL21, and CXCL12 ex vivo. However, this binding was abolished upon HS degradation, disrupting the CCL19/CCL21 chemokine gradients and causing impaired migration of dendritic cells in thymic slices. Thus, our results show that HS plays an essential role in the development and growth of the thymus and in regulating interstitial cell migration.

Examination of Fas-Induced Apoptosis of Murine Thymocytes in Thymic Tissue Slices Reveals That Fas Is Dispensable for Negative Selection.

The death receptor Fas can induce cell death through the extrinsic pathway of apoptosis in a variety of cells, including developing thymocytes. Although Fas-induced cell death has been researched and modeled extensively, most of the studies have been done in vitro because of the lethality of Fas triggering in vivo. Thus, little is known about the time line of this type of cell death in vivo, specifically, how does the presence of macrophages and pro-survival cytokines affect apoptosis progression. In addition, although the sequence and timing of events during intrinsic pathway activation in thymocytes in situ have been described, no corresponding data for the extrinsic pathway are available. To address this gap in our knowledge, we established a novel system to study Fas-induced thymocyte cell death using tissue explants. We found that within 1 h of Fas ligation, caspase 3 was activated, within 2 h phosphatidylserine was externalized to serve as an "eat-me" signal, and at the same time, we observed signs of cell loss, likely due to efferocytosis. Both caspase 3 activation and phosphatidylserine exposure were critical for cell loss. Although Fas ligand (FasL) was delivered simultaneously to all cells, we observed significant variation in the entry into the cell death pathway. This model also allowed us to revisit the role of Fas in negative selection, and we ruled out an essential part for it in the deletion of autoreactive thymocytes. Our work provides a timeline for the apoptosis-associated events following Fas triggering in situ and confirms the lack of involvement of Fas in the negative selection of thymocytes.

A role for phagocytosis in inducing cell death during thymocyte negative selection.

Autoreactive thymocytes are eliminated during negative selection in the thymus, a process important for establishing self-tolerance. Thymic phagocytes serve to remove dead thymocytes, but whether they play additional roles during negative selection remains unclear. Here, using a murine thymic slice model in which thymocytes undergo negative selection in situ, we demonstrate that phagocytosis promotes negative selection, and provide evidence for the escape of autoreactive CD8 T cells to the periphery when phagocytosis in the thymus is impaired. We also show that negative selection is more efficient when the phagocyte also presents the negative selecting peptide. Our findings support a model for negative selection in which the death process initiated following strong TCR signaling is facilitated by phagocytosis. Thus, the phagocytic capability of cells that present self-peptides is a key determinant of thymocyte fate.

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

Equilibrative Nucleoside Transporter 3 Regulates T Cell Homeostasis by Coordinating Lysosomal Function with Nucleoside Availability.

T cells are a versatile immune cell population responding to challenges by differentiation and proliferation followed by contraction and memory formation. Dynamic metabolic reprogramming is essential for T cells to meet the biosynthetic needs and the reutilization of biomolecules, processes that require active participation of metabolite transporters. Here, we show that equilibrative nucleoside transporter 3 (ENT3) is highly expressed in peripheral T cells and has a key role in maintaining T cell homeostasis by supporting the proliferation and survival of T cells. ENT3 deficiency leads to an enlarged and disturbed lysosomal compartment, resulting in accumulation of surplus mitochondria, elevation of intracellular reactive oxygen species, and DNA damage in T cells. Our results identify ENT3 as a vital metabolite transporter that supports T cell homeostasis and activation by regulating lysosomal integrity and the availability of nucleosides. Moreover, we uncovered that T cell lysosomes are an important source of salvaged metabolites for survival and proliferation.

Two-photon imaging of the immune system.

Two-photon microscopy is a powerful method for visualizing biological processes as they occur in their native environment in real time. The immune system uniquely benefits from this technology as most of its constituent cells are highly motile and interact extensively with each other and with the environment. Two-photon microscopy has provided many novel insights into the dynamics of the development and function of the immune system that could not have been deduced by other methods and has become an indispensible tool in the arsenal of immunologists. In this unit, we provide several protocols for preparation of various organs for imaging by two-photon microscopy that are intended to introduce the new user to some basic aspects of this method.