Targeted Metabolomics on Rare Primary Cells.

Cellular function critically depends on metabolism, and the function of the underlying metabolic networks can be studied by measuring small molecule intermediates. However, obtaining accurate and reliable measurements of cellular metabolism, particularly in rare cell types like hematopoietic stem cells, has traditionally required pooling cells from multiple animals. A protocol now enables researchers to measure metabolites in rare cell types using only one mouse per sample while generating multiple replicates for more abundant cell types. This reduces the number of animals that are required for a given project. The protocol presented here involves several key differences over traditional metabolomics protocols, such as using 5 g/L NaCl as a sheath fluid, sorting directly into acetonitrile, and utilizing targeted quantification with rigorous use of internal standards, allowing for more accurate and comprehensive measurements of cellular metabolism. Despite the time required for the isolation of single cells, fluorescent staining, and sorting, the protocol can preserve differences among cell types and drug treatments to a large extent.

LC-MS-Based Targeted Metabolomics for FACS-Purified Rare Cells.

Metabolism plays a fundamental role in regulating cellular functions and fate decisions. Liquid chromatography-mass spectrometry (LC-MS)-based targeted metabolomic approaches provide high-resolution insights into the metabolic state of a cell. However, the typical sample size is in the order of 105-107 cells and thus not compatible with rare cell populations, especially in the case of a prior flow cytometry-based purification step. Here, we present a comprehensively optimized protocol for targeted metabolomics on rare cell types, such as hematopoietic stem cells and mast cells. Only 5000 cells per sample are required to detect up to 80 metabolites above background. The use of regular-flow liquid chromatography allows for robust data acquisition, and the omission of drying or chemical derivatization avoids potential sources of error. Cell-type-specific differences are preserved while the addition of internal standards, generation of relevant background control samples, and targeted metabolite with quantifiers and qualifiers ensure high data quality. This protocol could help numerous studies to gain thorough insights into cellular metabolic profiles and simultaneously reduce the number of laboratory animals and the time-consuming and costly experiments associated with rare cell-type purification.

Surprises from Intravital Imaging of the Innate Immune Response.

Successful immune responses depend on the spatiotemporal coordination of immune cell migration, interactions, and effector functions in lymphoid and parenchymal tissues. Real-time intravital microscopy has revolutionized our understanding of the dynamic behavior of many immune cell types in the living tissues of several species. Observing immune cells in their native environment has revealed many unanticipated facets of their biology, which were not expected from experiments outside a living organism. Here we highlight both classic and more recent examples of surprising discoveries that critically relied on the use of live in vivo imaging. In particular, we focus on five major cell types of the innate immune response (macrophages, microglia, neutrophils, dendritic cells, and mast cells), and how studying their dynamics in mouse tissues has helped us advance our current knowledge of immune cell-mediated tissue homeostasis, host defense, and inflammation.

Specification of CNS macrophage subsets occurs postnatally in defined niches.

All tissue-resident macrophages of the central nervous system (CNS)-including parenchymal microglia, as well as CNS-associated macrophages (CAMs1) such as meningeal and perivascular macrophages2-7-are part of the CNS endogenous innate immune system that acts as the first line of defence during infections or trauma2,8-10. It has been suggested that microglia and all subsets of CAMs are derived from prenatal cellular sources in the yolk sac that were defined as early erythromyeloid progenitors11-15. However, the precise ontogenetic relationships, the underlying transcriptional programs and the molecular signals that drive the development of distinct CAM subsets in situ are poorly understood. Here we show, using fate-mapping systems, single-cell profiling and cell-specific mutants, that only meningeal macrophages and microglia share a common prenatal progenitor. By contrast, perivascular macrophages originate from perinatal meningeal macrophages only after birth in an integrin-dependent manner. The establishment of perivascular macrophages critically requires the presence of arterial vascular smooth muscle cells. Together, our data reveal a precisely timed process in distinct anatomical niches for the establishment of macrophage subsets in the CNS.

Macrophage network dynamics depend on haptokinesis for optimal local surveillance.

Macrophages are key immune cells with important roles for tissue surveillance in almost all mammalian organs. Cellular networks made up of many individual macrophages allow for optimal removal of dead cell material and pathogens in tissues. However, the critical determinants that underlie these population responses have not been systematically studied. Here, we investigated how cell shape and the motility of individual cells influences macrophage network responses in 3D culture settings and in mouse tissues. We show that surveying macrophage populations can tolerate lowered actomyosin contractility, but cannot easily compensate for a lack of integrin-mediated adhesion. Although integrins were dispensable for macrophage chemotactic responses, they were crucial to control cell movement and protrusiveness for optimal surveillance by a macrophage population. Our study reveals that ß1 integrins are important for maintaining macrophage shape and network sampling efficiency in mammalian tissues, and sets macrophage motility strategies apart from the integrin-independent 3D migration modes of many other immune cell subsets.

Macrophages are immune cells in the body that remove dying cells and debris from tissues. They live in almost all the body's organs, surveilling for signs of infection and destroying microbes. They also migrate to wound sites, where they can eliminate foreign particles and stop microbes from entering the body. To perform their surveillance role, macrophages need to work together as a team. They form a network, coordinating their movements to optimise the removal of particles and dead cells. How this happens is something of a mystery. As individuals, cells travel through tissues using a balance of several activities: they change their shape, they contract and relax, and they grab hold of their surroundings using proteins called integrins. It is thought that the choice between these types of movement may affect the rest of the network. To investigate, Paterson and Lämmermann genetically engineered mouse macrophages grown in the laboratory so they would not produce working integrins. These macrophages were able to contract and relax, but they could not attach to the proteins in the structures they were exploring. Paterson and Lämmermann then placed these macrophages in gels studded with proteins that mimic a biological matrix to observe their behaviour. When these macrophages were exposed to the chemicals that indicate the presence of a wound, they moved normally, changing shape and contracting and relaxing. Paterson and Lämmermann confirmed this normal behaviour for macrophages moving to sites of injuries in the tissue of living mice. However, when it came to surveillance, the macrophages' abilities were seriously diminished, and they were unable to form an effective network to take up particles and dead cells. This work sheds light on how the movement of individual cells affects the entire immune surveillance network. A deeper understanding could lead to new insights into how to prevent inflammation. The next step is to map macrophage networks in healthy and diseased tissues to understand how cell movement affects surveillance under different conditions.

Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic defects in cytotoxicity.

BACKGROUND: Rubella virus-induced granulomas have been described in patients with various inborn errors of immunity. Most defects impair T-cell immunity, suggesting a critical role of T cells in rubella elimination. However, the molecular mechanism of virus control remains elusive. OBJECTIVE: This study sought to understand the defective effector mechanism allowing rubella vaccine virus persistence in granulomas. METHODS: Starting from an index case with Griscelli syndrome type 2 and rubella skin granulomas, this study combined an international survey with a literature search to identify patients with cytotoxicity defects and granuloma. The investigators performed rubella virus immunohistochemistry and PCR and T-cell migration assays. RESULTS: This study identified 21 patients with various genetically confirmed cytotoxicity defects, who presented with skin and visceral granulomas. Rubella virus was demonstrated in all 12 accessible biopsies. Granuloma onset was typically before 2 years of age and lesions persisted from months to years. Granulomas were particularly frequent in MUNC13-4 and RAB27A deficiency, where 50% of patients at risk were affected. Although these proteins have also been implicated in lymphocyte migration, 3-dimensional migration assays revealed no evidence of impaired migration of patient T cells. Notably, patients showed no evidence of reduced control of concomitantly given measles, mumps, or varicella live-attenuated vaccine or severe infections with other viruses. CONCLUSIONS: This study identified lymphocyte cytotoxicity as a key effector mechanism for control of rubella vaccine virus, without evidence for its need in control of live measles, mumps, or varicella vaccines. Rubella vaccine-induced granulomas are a novel phenotype with incomplete penetrance of genetic disorders of cytotoxicity.

Age-related changes in the local milieu of inflamed tissues cause aberrant neutrophil trafficking and subsequent remote organ damage.

Aging is associated with dysregulated immune functions. Here, we investigated the impact of age on neutrophil diapedesis. Using confocal intravital microscopy, we found that in aged mice, neutrophils adhered to vascular endothelium in inflamed tissues but exhibited a high frequency of reverse transendothelial migration (rTEM). This retrograde breaching of the endothelium by neutrophils was governed by enhanced production of the chemokine CXCL1 from mast cells that localized at endothelial cell (EC) junctions. Increased EC expression of the atypical chemokine receptor 1 (ACKR1) supported this pro-inflammatory milieu in aged venules. Accumulation of CXCL1 caused desensitization of the chemokine receptor CXCR2 on neutrophils and loss of neutrophil directional motility within EC junctions. Fluorescent tracking revealed that in aged mice, neutrophils undergoing rTEM re-entered the circulation and disseminated to the lungs where they caused vascular leakage. Thus, neutrophils stemming from a local inflammatory site contribute to remote organ damage, with implication to the dysregulated systemic inflammation associated with aging.

A Subset of Skin Macrophages Contributes to the Surveillance and Regeneration of Local Nerves.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.

Resident Macrophages Cloak Tissue Microlesions to Prevent Neutrophil-Driven Inflammatory Damage.

Neutrophils are attracted to and generate dense swarms at sites of cell damage in diverse tissues, often extending the local disruption of organ architecture produced by the initial insult. Whether the inflammatory damage resulting from such neutrophil accumulation is an inescapable consequence of parenchymal cell death has not been explored. Using a combination of dynamic intravital imaging and confocal multiplex microscopy, we report here that tissue-resident macrophages rapidly sense the death of individual cells and extend membrane processes that sequester the damage, a process that prevents initiation of the feedforward chemoattractant signaling cascade that results in neutrophil swarms. Through this "cloaking" mechanism, the resident macrophages prevent neutrophil-mediated inflammatory damage, maintaining tissue homeostasis in the face of local cell injury that occurs on a regular basis in many organs because of mechanical and other stresses. VIDEO ABSTRACT.

Cochaperone Mzb1 is a key effector of Blimp1 in plasma cell differentiation and ß1-integrin function.

Plasma cell differentiation involves coordinated changes in gene expression and functional properties of B cells. Here, we study the role of Mzb1, a Grp94 cochaperone that is expressed in marginal zone (MZ) B cells and during the terminal differentiation of B cells to antibody-secreting cells. By analyzing Mzb1-/-Prdm1+/gfp mice, we find that Mzb1 is specifically required for the differentiation and function of antibody-secreting cells in a T cell-independent immune response. We find that Mzb1-deficiency mimics, in part, the phenotype of Blimp1 deficiency, including the impaired secretion of IgM and the deregulation of Blimp1 target genes. In addition, we find that Mzb1-/- plasmablasts show a reduced activation of ß1-integrin, which contributes to the impaired plasmablast differentiation and migration of antibody-secreting cells to the bone marrow. Thus, Mzb1 function is required for multiple aspects of plasma cell differentiation.

Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival.

We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6ß1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.

Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo.

Neutrophil recruitment from blood to extravascular sites of sterile or infectious tissue damage is a hallmark of early innate immune responses, and the molecular events leading to cell exit from the bloodstream have been well defined. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects. The molecular players that direct this response at the single-cell and population levels within the complexity of an inflamed tissue are unknown. Using two-photon intravital microscopy in mouse models of sterile injury and infection, we show a critical role for intercellular signal relay among neutrophils mediated by the lipid leukotriene B4, which acutely amplifies local cell death signals to enhance the radius of highly directed interstitial neutrophil recruitment. Integrin receptors are dispensable for long-distance migration, but have a previously unappreciated role in maintaining dense cellular clusters when congregating neutrophils rearrange the collagenous fibre network of the dermis to form a collagen-free zone at the wound centre. In this newly formed environment, integrins, in concert with neutrophil-derived leukotriene B4 and other chemoattractants, promote local neutrophil interaction while forming a tight wound seal. This wound seal has borders that cease to grow in kinetic concert with late recruitment of monocytes and macrophages at the edge of the displaced collagen fibres. Together, these data provide an initial molecular map of the factors that contribute to neutrophil swarming in the extravascular space of a damaged tissue. They reveal how local events are propagated over large-range distances, and how auto-signalling produces coordinated, self-organized neutrophil-swarming behaviour that isolates the wound or infectious site from surrounding viable tissue.

A spatially-organized multicellular innate immune response in lymph nodes limits systemic pathogen spread.

The lymphatic network that transports interstitial fluid and antigens to lymph nodes constitutes a conduit system that can be hijacked by invading pathogens to achieve systemic spread unless dissemination is blocked in the lymph node itself. Here, we show that a network of diverse lymphoid cells (natural killer cells, γδ T cells, natural killer T cells, and innate-like CD8+ T cells) are spatially prepositioned close to lymphatic sinus-lining sentinel macrophages where they can rapidly and efficiently receive inflammasome-generated IL-18 and additional cytokine signals from the pathogen-sensing phagocytes. This leads to rapid IFNγ secretion by the strategically positioned innate lymphocytes, fostering antimicrobial resistance in the macrophage population. Interference with this innate immune response loop allows systemic spread of lymph-borne bacteria. These findings extend our understanding of the functional significance of cellular positioning and local intercellular communication within lymph nodes while emphasizing the role of these organs as highly active locations of innate host defense.

Cancer-associated epithelial cell adhesion molecule (EpCAM; CD326) enables epidermal Langerhans cell motility and migration in vivo.

After activation, Langerhans cells (LC), a distinct subpopulation of epidermis-resident dendritic cells, migrate from skin to lymph nodes where they regulate the magnitude and quality of immune responses initiated by epicutaneously applied antigens. Modulation of LC-keratinocyte adhesion is likely to be central to regulation of LC migration. LC express high levels of epithelial cell adhesion molecule (EpCAM; CD326), a cell-surface protein that is characteristic of some epithelia and many carcinomas and that has been implicated in intercellular adhesion and metastasis. To gain insight into EpCAM function in a physiologic context in vivo, we generated conditional knockout mice with EpCAM-deficient LC and characterized them. Epidermis from these mice contained increased numbers of LC with normal levels of MHC and costimulatory molecules and T-cell-stimulatory activity in vitro. Migration of EpCAM-deficient LC from skin explants was inhibited, but chemotaxis of dissociated LC was not. Correspondingly, the ability of contact allergen-stimulated, EpCAM-deficient LC to exit epidermis in vivo was delayed, and strikingly fewer hapten-bearing LC subsequently accumulated in lymph nodes. Attenuated migration of EpCAM-deficient LC resulted in enhanced contact hypersensitivity responses as previously described in LC-deficient mice. Intravital microscopy revealed reduced translocation and dendrite motility in EpCAM-deficient LC in vivo in contact allergen-treated mice. These results conclusively link EpCAM expression to LC motility/migration and LC migration to immune regulation. EpCAM appears to promote LC migration from epidermis by decreasing LC-keratinocyte adhesion and may modulate intercellular adhesion and cell movement within in epithelia during development and carcinogenesis in an analogous fashion.

The extracellular matrix of the spleen as a potential organizer of immune cell compartments.

Until recently little information was available on the molecular details of the extracellular matrix (ECM) of secondary lymphoid tissues. There is now growing evidence that these ECMs are unique structures, combining characteristics of basement membranes and interstitial or fibrillar matrices, resulting in scaffolds that are strong and highly flexible and, in certain secondary lymphoid compartments, also forming conduit networks for rapid fluid transport. This review will address the structural characteristics of the ECM of the murine spleen and its potential role as an organizer of immune cell compartments, with reference to the lymph node where relevant.

The microanatomy of T-cell responses.

The priming of a T cell results from its physical interaction with a dendritic cell (DC) that presents the cognate antigenic peptide. The success rate of such interactions is extremely low, because the precursor frequency of a naive T cell recognizing a specific antigen is in the range of 1:10(5)-10(6). To make this principle practicable, encounter frequencies between DCs and T cells are maximized within lymph nodes (LNs) that are compact immunological projections of the peripheral tissue they drain. But LNs are more than passive meeting places for DCs that immigrated from the tissue and lymphocytes that recirculated via the blood. The microanatomy of the LN stroma actively organizes the cellular encounters by providing preformed migration tracks that create dynamic but highly ordered movement patterns. LN architecture further acts as a sophisticated filtration system that sieves the incoming interstitial fluid at different levels and guarantees that immunologically relevant antigens are loaded on DCs or B cells while inert substances are channeled back into the blood circulation. This review focuses on the non-hematopoietic infrastructure of the lymph node. We describe the association between fibroblastic reticular cell, conduit, DC, and T cell as the essential functional unit of the T-cell cortex.

Beta1 integrins: zip codes and signaling relay for blood cells.

At least eight of the twelve known members of the beta1 integrin family are expressed on hematopoietic cells. Among these, the VCAM-1 receptor alpha4beta1 has received most attention as a main factor mediating firm adhesion to the endothelium during blood cell extravasation. Therapeutic trials are ongoing into the use of antibodies and small molecule inhibitors to target this interaction and hence obtain anti-inflammatory effects. However, extravasation is only one possible process that is mediated by beta1 integrins and there is evidence that they also mediate leukocyte retention and positioning in the tissue, lymphocyte activation and possibly migration within the interstitium. Genetic mouse models where integrins are selectively deleted on blood cells have been used to investigate these functions and further studies will be invaluable to critically evaluate therapeutic trials.

gamma-Parvin is dispensable for hematopoiesis, leukocyte trafficking, and T-cell-dependent antibody response.

Integrins regulate cell behavior through the assembly of multiprotein complexes at the site of cell adhesion. Parvins are components of such a multiprotein complex. They consist of three members (alpha-, beta-, and gamma-parvin), form a functional complex with integrin-linked kinase (ILK) and PINCH, and link integrins to the actin cytoskeleton. Whereas alpha- and beta-parvins are widely expressed, gamma-parvin has been reported to be expressed in hematopoietic organs. In the present study, we report the expression pattern of the parvins in hematopoietic cells and the phenotypic analysis of gamma-parvin-deficient mice. Whereas alpha-parvin is not expressed in hematopoietic cells, beta-parvin is only found in myeloid cells and gamma-parvin is present in both cells of the myeloid and lymphoid lineages, where it binds ILK. Surprisingly, loss of gamma-parvin expression had no effect on blood cell differentiation, proliferation, and survival and no consequence for the T-cell-dependent antibody response and lymphocyte and dendritic cell migration. These data indicate that despite the high expression of gamma-parvin in hematopoietic cells it must play a more subtle role for blood cell homeostasis.