In mammals, megakaryocytes (MKs) in the bone marrow (BM) produce blood platelets, required for hemostasis and thrombosis. MKs originate from hematopoietic stem cells and are thought to migrate from an endosteal niche towards the vascular sinusoids during their maturation. Through imaging of MKs in the intact BM, here we show that MKs can be found within the entire BM, without a bias towards bone-distant regions. By combining in vivo two-photon microscopy and in situ light-sheet fluorescence microscopy with computational simulations, we reveal surprisingly slow MK migration, limited intervascular space, and a vessel-biased MK pool. These data challenge the current thrombopoiesis model of MK migration and support a modified model, where MKs at sinusoids are replenished by sinusoidal precursors rather than cells from a distant periostic niche. As MKs do not need to migrate to reach the vessel, therapies to increase MK numbers might be sufficient to raise platelet counts.
In recent years, the myocardium has been rediscovered under the lenses of immunology, and lymphocytes have been implicated in the pathogenesis of cardiomyopathies with different etiologies. Aging is an important risk factor for heart diseases, and it also has impact on the immune system. Thus, we sought to determine whether immunological activity would influence myocardial structure and function in elderly mice. Morphological, functional, and molecular analyses revealed that the age-related myocardial impairment occurs in parallel with shifts in the composition of tissueresident leukocytes and with an accumulation of activated CD4 + Foxp3 − (forkhead box P3) IFN-γ + T cells in the heart-draining lymph nodes. A comprehensive characterization of different aged immune-deficient mouse strains revealed that T cells significantly contribute to age-related myocardial inflammation and functional decline. Upon adoptive cell transfer, the T cells isolated from the mediastinal lymph node (med-LN) of aged animals exhibited increased cardiotropism, compared with cells purified from young donors or from other irrelevant sites. Nevertheless, these cells caused rather mild effects on cardiac functionality, indicating that myocardial aging might stem from a combination of intrinsic and extrinsic (immunological) factors. Taken together, the data herein presented indicate that heart-directed immune responses may spontaneously arise in the elderly, even in the absence of a clear tissue damage or concomitant infection. These observations might shed new light on the emerging role of T cells in myocardial diseases, which primarily affect the elderly population.T he myocardial cellular composition has been revisited in recent years, and leukocyte subsets residing in the healthy heart have been described (1-10). Cardiac-resident macrophages exhibiting an M2-like gene expression profile were found to be distributed in close association with the coronary vascular bed (3), and niches for dendritic cells (CD11c + MHC-II high CD80/86 low ) were found near the cardiac valves of the intact heart (1). It was also demonstrated that cardiac-resident MHCII + cells process and present myosin heavy chain-alpha-derived peptides under steady-state conditions (11, 12) and prime T cells ex vivo (1). However, whether lymphocytes can seed the intact myocardium and whether T-cell priming with myocardial antigens can occur in the absence of an infection or autoimmune myocarditis remain elusive.More recently, accumulating evidence indicated that noninfectious myocardial diseases are modulated by T cells. During the last couple of years, our group demonstrated that ischemic, sterile myocardial injuries can elicit lymphocyte activation directed against cardiac antigens (13-16). Our previous data, showing for the first time that CD4 + T cells reactive to cardiac components can foster the healing process that takes place after myocardial infarction, were corroborated by several other reports (13,15,(17)(18)(19)(20). However, these autoreactive T cells can also b...
We demonstrate the first, to our knowledge, integration of stimulated emission depletion (STED) with selective plane illumination microscopy (SPIM). Using this method, we were able to obtain up to 60% improvements in axial resolution with lateral resolution enhancements in control samples and zebrafish embryos. The integrated STED-SPIM method combines the advantages of SPIM with the resolution enhancement of STED, and thus provides a method for fast, high-resolution imaging with >100 μm deep penetration into biological tissue.
While the extravasation cascade of lymphocytes is well characterized, data on their intraepithelial positioning and morphology are scant. However, the latter process is presumably crucial for many immune functions. Integrin ␣ E (CD103) 7 has previously been implicated in epithelial retention of some T cells through binding to E-cadherin. Our current data suggest that ␣ E (CD103) 7 also determines shape and motility of some lymphocytes. Time-lapse microscopy showed that wild-type ␣ E (CD103) 7 conferred the ability to form cell protrusions/filopodia and to move in an amoeboid fashion on E-cadherin, an activity that was abrogated by ␣ E (CD103) 7 -directed antibodies or cytochalasin D. The ␣ E -dependent motility was further increased (P < .001) when point-mutated ␣ E (CD103) locked in a constitutively active conformation was expressed. Moreover, different yellow fluorescent protein-coupled ␣ E (CD103) species demonstrated that the number and length of filopodia extended toward purified E-cadherin, cocultured keratinocytes, cryostat-cut skin sections, or epidermal sheets depended on functional ␣ E (CD103). The in vivo relevance of these findings was demonstrated by wild-type dendritic epidermal T cells (DETCs), which showed significantly more dendrites and spanned larger epidermal areas as compared with DETCs of ␣ E (CD103)-deficient mice (P < .001). Thus, integrin ␣ E (CD103) 7 is not only involved in epithelial retention, but also in shaping and proper intraepithelial morphogenesis of some leukocytes. IntroductionPositioning and locomotion of leukocytes within tissues provide the basis for the molecular crosstalk with other cells and are prerequisites for a functional immune system. To date, the recruitment cascade of initial endothelial adhesion, activation, firm adhesion, transmigration, and subsequent localization into the connective tissue is one of the best established concepts in leukocyte biology. 1,2 However, many effector functions of immunocytes are exerted within parenchymatous organs, mostly epithelia. It is therefore somewhat surprising that we know relatively little about locomotion and function-determining morphogenesis of lymphocytes within epithelial tissues, such as the epidermis of the skin. 3 An adhesion receptor that is thought to mediate retention of lymphocytes within epithelial tissues is integrin ␣ E (CD103) 7 . 4 First described 2 decades ago as a selective marker for intestinal intraepithelial lymphocytes, 5 ␣ E (CD103) 7 has been implicated in epithelial T-cell retention through binding to E-cadherin. 6,7 Indeed, ␣ E (CD103)-deficient mice exhibited a reduced number of mucosal intraepithelial T cells. 8 However, ␣ E (CD103) 7 has later been found to be also expressed by some lymphocytes within other epithelia, such as the epidermis of the skin, 9 where it presumably contributes to recruitment of T cells in inflamed human skin 10 as well as dendritic epidermal T cells (DETCs) in murine skin. 11 Thymic DETC precursor cells express integrin ␣ E (CD103) 7 before their migration ...
Understanding the spatiotemporal changes of cellular and molecular events within an organism is crucial to elucidate the complex immune processes involved in infections, autoimmune disorders, transplantation, and neoplastic transformation and metastasis. Here we introduce a novel multicolor light sheet fluorescence microscopy (LSFM) approach for deciphering immune processes in large tissue specimens on a single-cell level in 3 dimensions. We combined and optimized antibody penetration, tissue clearing, and triple-color illumination to create a method for analyzing intact mouse and human tissues. This approach allowed us to successfully quantify changes in expression patterns of mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and T cell responses in Peyer's patches following stimulation of the immune system. In addition, we employed LSFM to map individual T cell subsets after hematopoietic cell transplantation and detected rare cellular events. Thus, we present a versatile imaging technology that should be highly beneficial in biomedical research.
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