Primary adhered neutrophils increase JNK1-MARCKSL1-mediated filopodia to promote secondary neutrophil transmigration.

During inflammation, leukocytes extravasate the vasculature to areas of inflammation in a process termed transendothelial migration. Previous research has shown that transendothelial migration hotspots exist, areas in the vasculature that are preferred by leukocytes to cross. Several factors that contribute to hotspot-mediated transmigration have been proposed already, but whether one leukocyte transmigration hotspot can be used subsequently by a second wave of leukocytes and thereby can increase the efficiency of leukocyte transmigration is not well understood. Here, we show that primary neutrophil adhesion to the endothelium triggers endothelial transmigration hotspots, allowing secondary neutrophils to cross the endothelium more efficiently. Mechanistically, we show that primary neutrophil adhesion increases the number of endothelial apical filopodia, resulting in an increase in the number of adherent secondary neutrophils. Using fluorescence resonance energy transfer (FRET)-based biosensors, we found that neutrophil adhesion did not trigger the activity of the small GTPase Cdc42. We used kinase translocation reporters to study the activity of mitogen-activated protein (MAP) kinases and Akt in endothelial cells on a single-cell level with a high temporal resolution during the process of leukocyte transmigration and found that c-Jun N-terminal kinase (JNK) is rapidly activated upon neutrophil adhesion, whereas extracellular regulated kinase (ERK), p38, and Akt are not. Additionally, we show that short-term chemical inhibition of endothelial JNK successfully prevents the adhesion of neutrophils to the endothelium. Furthermore, we show that neutrophil-induced endothelial JNK1 but not JNK2 increases the formation of filopodia and thereby the adhesion of secondary neutrophils. JNK1 needs its downstream substrate MARCKSL1 to trigger additional apical filopodia and consequently neutrophil adhesion. Overall, our data show that primary neutrophils can trigger the endothelial transmigration hotspot by activating JNK1 and MARCKSL1 to induce filopodia that trigger more neutrophils to transmigrate at the endothelial hotspot area.

Endothelial ICAM-1 Adhesome Recruits CD44 for Optimal Transcellular Migration of Human CTLs.

The endothelial lining of blood vessels is covered with a thin polysaccharide coat called the glycocalyx. This layer of polysaccharides contains hyaluronan that forms a protective coat on the endothelial surface. Upon inflammation, leukocytes leave the circulation and enter inflamed tissue by crossing inflamed endothelial cells, mediated by adhesion molecules such as ICAM-1/CD54. To what extent the glycocalyx participates in the regulation of leukocyte transmigration is not clear. During extravasation, leukocyte integrins cluster ICAM-1, resulting in the recruitment of a number of intracellular proteins and subsequent downstream effects in the endothelial cells. For our studies, we used primary human endothelial and immune cells. With an unbiased proteomics approach, we identified the full ICAM-1 adhesome and identified 93 (to our knowledge) new subunits of the ICAM-1 adhesome. Interestingly, we found the glycoprotein CD44 as part of the glycocalyx to be recruited to clustered ICAM-1 specifically. Our data demonstrate that CD44 binds hyaluronan to the endothelial surface, where it locally concentrates and presents chemokines that are essential for leukocytes to cross the endothelial lining. Taken together, we discover a link between ICAM-1 clustering and hyaluronan-mediated chemokine presentation by recruiting hyaluronan to sites of leukocyte adhesion via CD44.

Endothelial transmigration hotspots limit vascular leakage through heterogeneous expression of ICAM-1.

Upon inflammation, leukocytes leave the circulation by crossing the endothelial monolayer at specific transmigration "hotspot" regions. Although these regions support leukocyte transmigration, their functionality is not clear. We found that endothelial hotspots function to limit vascular leakage during transmigration events. Using the photoconvertible probe mEos4b, we traced back and identified original endothelial transmigration hotspots. Using this method, we show that the heterogeneous distribution of ICAM-1 determines the location of the transmigration hotspot. Interestingly, the loss of ICAM-1 heterogeneity either by CRISPR/Cas9-induced knockout of ICAM-1 or equalizing the distribution of ICAM-1 in all endothelial cells results in the loss of TEM hotspots but not necessarily in reduced TEM events. Functionally, the loss of endothelial hotspots results in increased vascular leakage during TEM. Mechanistically, we demonstrate that the 3 extracellular Ig-like domains of ICAM-1 are crucial for hotspot recognition. However, the intracellular tail of ICAM-1 and the 4th Ig-like dimerization domain are not involved, indicating that intracellular signaling or ICAM-1 dimerization is not required for hotspot recognition. Together, we discovered that hotspots function to limit vascular leakage during inflammation-induced extravasation.

Code Red in the Supply Center: The Impact of Immune Activation on Hematopoiesis.

This Special Issue entitled "The Impact of Immune Activation on Hematopoiesis" aims to bring together review and primary articles focused on distinct types of immune activation that impact hematopoiesis [...].

Platelet number and function alterations in preclinical models of sterile inflammation and sepsis patients: implications in the pathophysiology and treatment of inflammation.

Platelets are the blood cells in charge of maintaining the body hemostasis, recognising the damaged vessel wall, and providing the appropriate cellular surface for the coagulation cascade to act locally. Additionally, platelets are active immunomodulators. At the crossroads of hemostasis and inflammation, platelets may exert either beneficial actions or participate in pathological manifestations, and have been associated with the prothrombotic nature of multi-organ failure in systemic inflammation. Platelet number alterations have been reported in septis, and platelet transfusions are given to thrombocytopenic patients. However, the risk to develop transfusion related acute lung injury (TRALI) is higher in sepsis patients. In this manuscript we show that platelets produced during inflammation in preclinical mouse models of sterile inflammation display lower aggregation capacity when stimulating certain receptors, while responses through other receptors remain intact, and we name them "inflammation-conditioned" platelets. In a cohort of sepsis patients, we observed, as previously reported, alterations in the number of platelets and platelet hyperreactivity. Furthermore, we identified a receptor-wise platelet aggregation response disbalance in these patients, although not similar to platelets from preclinical models of sterile inflammation. Interestingly, we generated evidence supporting the notion that platelet aggregation capacity disbalance was partially triggered by plasma components from sepsis patients. Our findings have implications in the indication of platelet transfusions in sepsis patients: Are fully functional platelets suitable for transfusion in sepsis patients? Current Clinical Trials (RESCUE) will answer whether platelet production stimulation with thrombopoietin receptor agonists (TPO-RAs) could be a substitute of platelet transfusions.

The endothelial diapedesis synapse regulates transcellular migration of human T lymphocytes in a CX3CL1- and SNAP23-dependent manner.

Understanding how cytotoxic T lymphocytes (CTLs) efficiently leave the circulation to target cancer cells or contribute to inflammation is of high medical interest. Here, we demonstrate that human central memory CTLs cross the endothelium in a predominantly paracellular fashion, whereas effector and effector memory CTLs cross the endothelium preferably in a transcellular fashion. We find that effector CTLs show a round morphology upon adhesion and induce a synapse-like interaction with the endothelium where ICAM-1 is distributed at the periphery. Moreover, the interaction of ICAM-1:ß2integrin and endothelial-derived CX3CL1:CX3CR1 enables transcellular migration. Mechanistically, we find that ICAM-1 clustering recruits the SNARE-family protein SNAP23, as well as syntaxin-3 and -4, for the local release of endothelial-derived chemokines like CXCL1/8/10. In line, silencing of endothelial SNAP23 drives CTLs across the endothelium in a paracellular fashion. In conclusion, our data suggest that CTLs trigger local chemokine release from the endothelium through ICAM-1-driven signals driving transcellular migration.

Age-Related Variation in Sympathetic Nerve Distribution in the Human Spleen.

Introduction: The cholinergic anti-inflammatory pathway (CAIP) has been proposed as an efferent neural pathway dampening the systemic inflammatory response via the spleen. The CAIP activates the splenic neural plexus and a subsequent series of intrasplenic events, which at least require a close association between sympathetic nerves and T cells. Knowledge on this pathway has mostly been derived from rodent studies and only scarce information is available on the innervation of the human spleen. This study aimed to investigate the sympathetic innervation of different structures of the human spleen, the topographical association of nerves with T cells and age-related variations in nerve distribution. Materials and Methods: Spleen samples were retrieved from a diagnostic archive and were allocated to three age groups; neonates, 10-25 and 25-70 years of age. Sympathetic nerves and T cells were identified by immunohistochemistry for tyrosine hydroxylase (TH) and the membrane marker CD3, respectively. The overall presence of sympathetic nerves and T cells was semi-automatically quantified and expressed as total area percentage. A predefined scoring system was used to analyze the distribution of nerves within different splenic structures. Results: Sympathetic nerves were observed in all spleens and their number appeared to slightly increase from birth to adulthood and to decrease afterward. Irrespective to age, more than halve of the periarteriolar lymphatic sheaths (PALSs) contained sympathetic nerves in close association with T cells. Furthermore, discrete sympathetic nerves were observed in the capsule, trabeculae and red pulp and comparable to the total amount of sympathetic nerves, showed a tendency to decrease with age. No correlation was found between the number of T cells and sympathetic nerves. Conclusion: The presence of discrete sympathetic nerves in the splenic parenchyma, capsule and trabecular of human spleens could suggest a role in functions other than vasoregulation. In the PALS, sympathetic nerves were observed to be in proximity to T cells and is suggestive for the existence of the CAIP in humans. Since sympathetic nerve distribution shows interspecies and age-related variation, and our general understanding of the relative and spatial contribution of splenic innervation in immune regulation is incomplete, it remains difficult to estimate the anti-inflammatory potential of targeting splenic nerves in patients.

Transendothelial migration induces differential migration dynamics of leukocytes in tissue matrix.

Leukocyte extravasation into inflamed tissue is a complex process that is difficult to capture as a whole in vitro. We employed a blood-vessel-on-a-chip model in which human endothelial cells were cultured in a tube-like lumen in a collagen-1 matrix. The vessels are leak tight, creating a barrier for molecules and leukocytes. Addition of inflammatory cytokine TNF-α (also known as TNF) caused vasoconstriction, actin remodelling and upregulation of ICAM-1. Introducing leukocytes into the vessels allowed real-time visualization of all different steps of the leukocyte transmigration cascade, including migration into the extracellular matrix. Individual cell tracking over time distinguished striking differences in migratory behaviour between T-cells and neutrophils. Neutrophils cross the endothelial layer more efficiently than T-cells, but, upon entering the matrix, neutrophils display high speed but low persistence, whereas T-cells migrate with low speed and rather linear migration. In conclusion, 3D imaging in real time of leukocyte extravasation in a vessel-on-a-chip enables detailed qualitative and quantitative analysis of different stages of the full leukocyte extravasation process in a single assay. This article has an associated First Person interview with the first authors of the paper.

Overexpression of Transmembrane TNF Drives Development of Ectopic Lymphoid Structures in the Bone Marrow and B Cell Lineage Alterations in Experimental Spondyloarthritis.

TNF is important in immune-mediated inflammatory diseases, including spondyloarthritis (SpA). Transgenic (tg) mice overexpressing transmembrane TNF (tmTNF) develop features resembling human SpA. Furthermore, both tmTNF tg mice and SpA patients develop ectopic lymphoid aggregates, but it is unclear whether these contribute to pathology. Therefore, we characterized the lymphoid aggregates in detail and studied potential alterations in the B and T cell lineage in tmTNF tg mice. Lymphoid aggregates developed in bone marrow (BM) of vertebrae and near the ankle joints prior to the first SpA features and displayed characteristics of ectopic lymphoid structures (ELS) including presence of B cells, T cells, germinal centers, and high endothelial venules. Detailed flow cytometric analyses demonstrated more germinal center B cells with increased CD80 and CD86 expression, along with significantly more T follicular helper, T follicular regulatory, and T regulatory cells in tmTNF tg BM compared with non-tg controls. Furthermore, tmTNF tg mice exhibited increased IgA serum levels and significantly more IgA+ plasma cells in the BM, whereas IgA+ plasma cells in the gut were not significantly increased. In tmTNF tg × TNF-RI-/- mice, ELS were absent, consistent with reduced disease symptoms, whereas in tmTNF tg × TNF-RII-/- mice, ELS and clinical symptoms were still present. Collectively, these data show that tmTNF overexpression in mice results in osteitis and ELS formation in BM, which may account for the increased serum IgA levels that are also observed in human SpA. These effects are mainly dependent on TNF-RI signaling and may underlie important aspects of SpA pathology.

The RhoGEF Trio: A Protein with a Wide Range of Functions in the Vascular Endothelium.

Many cellular processes are controlled by small GTPases, which can be activated by guanine nucleotide exchange factors (GEFs). The RhoGEF Trio contains two GEF domains that differentially activate the small GTPases such as Rac1/RhoG and RhoA. These small RhoGTPases are mainly involved in the remodeling of the actin cytoskeleton. In the endothelium, they regulate junctional stabilization and play a crucial role in angiogenesis and endothelial barrier integrity. Multiple extracellular signals originating from different vascular processes can influence the activity of Trio and thereby the regulation of the forementioned small GTPases and actin cytoskeleton. This review elucidates how various signals regulate Trio in a distinct manner, resulting in different functional outcomes that are crucial for endothelial cell function in response to inflammation.

Endothelial junctional membrane protrusions serve as hotspots for neutrophil transmigration.

Upon inflammation, leukocytes rapidly transmigrate across the endothelium to enter the inflamed tissue. Evidence accumulates that leukocytes use preferred exit sites, alhough it is not yet clear how these hotspots in the endothelium are defined and how they are recognized by the leukocyte. Using lattice light sheet microscopy, we discovered that leukocytes prefer endothelial membrane protrusions at cell junctions for transmigration. Phenotypically, these junctional membrane protrusions are present in an asymmetric manner, meaning that one endothelial cell shows the protrusion and the adjacent one does not. Consequently, leukocytes cross the junction by migrating underneath the protruding endothelial cell. These protrusions depend on Rac1 activity and by using a photo-activatable Rac1 probe, we could artificially generate local exit-sites for leukocytes. Overall, we have discovered a new mechanism that uses local induced junctional membrane protrusions to facilitate/steer the leukocyte escape/exit from inflamed vessel walls.

The Bone Marrow as Sanctuary for Plasma Cells and Memory T-Cells: Implications for Adaptive Immunity and Vaccinology.

The bone marrow (BM) is key to protective immunological memory because it harbors a major fraction of the body's plasma cells, memory CD4+ and memory CD8+ T-cells. Despite its paramount significance for the human immune system, many aspects of how the BM enables decade-long immunity against pathogens are still poorly understood. In this review, we discuss the relationship between BM survival niches and long-lasting humoral immunity, how intrinsic and extrinsic factors define memory cell longevity and show that the BM is also capable of adopting many responsibilities of a secondary lymphoid organ. Additionally, with more and more data on the differentiation and maintenance of memory T-cells and plasma cells upon vaccination in humans being reported, we discuss what factors determine the establishment of long-lasting immunological memory in the BM and what we can learn for vaccination technologies and antigen design. Finally, using these insights, we touch on how this holistic understanding of the BM is necessary for the development of modern and efficient vaccines against the pandemic SARS-CoV-2.

Diversity, localization, and (patho)physiology of mature lymphocyte populations in the bone marrow.

The bone marrow (BM) is responsible for generating and maintaining lifelong output of blood and immune cells. In addition to its key hematopoietic function, the BM acts as an important lymphoid organ, hosting a large variety of mature lymphocyte populations, including B cells, T cells, natural killer T cells, and innate lymphoid cells. Many of these cell types are thought to visit the BM only transiently, but for others, like plasma cells and memory T cells, the BM provides supportive niches that promote their long-term survival. Interestingly, accumulating evidence points toward an important role for mature lymphocytes in the regulation of hematopoietic stem cells (HSCs) and hematopoiesis in health and disease. In this review, we describe the diversity, migration, localization, and function of mature lymphocyte populations in murine and human BM, focusing on their role in immunity and hematopoiesis. We also address how various BM lymphocyte subsets contribute to the development of aplastic anemia and immune thrombocytopenia, illustrating the complexity of these BM disorders and the underlying similarities and differences in their disease pathophysiology. Finally, we summarize the interactions between mature lymphocytes and BM resident cells in HSC transplantation and graft-versus-host disease. A better understanding of the mechanisms by which mature lymphocyte populations regulate BM function will likely improve future therapies for patients with benign and malignant hematologic disorders.

Extracellular Vesicles Derived From Adult and Fetal Bone Marrow Mesenchymal Stromal Cells Differentially Promote ex vivo Expansion of Hematopoietic Stem and Progenitor Cells.

Recently, we and others have illustrated that extracellular vesicles (EVs) have the potential to support hematopoietic stem and progenitor cell (HSPC) expansion; however, the mechanism and processes responsible for the intercellular communication by EVs are still unknown. In the current study, we investigate whether primary human bone marrow derived mesenchymal stromal cells (BMSC) EVs isolated from two different origins, fetal (fEV) and adult (aEV) tissue, can increase the relative low number of HSPCs found in umbilical cord blood (UCB) and which EV-derived components are responsible for ex vivo HSPC expansion. Interestingly, aEVs and to a lesser extent fEVs, showed supportive ex vivo expansion capacity of UCB-HSPCs. Taking advantage of the two BMSC sources with different supportive effects, we analyzed the EV cargo and investigated how gene expression is modulated in HSPCs after incubation with aEVs and fEVs. Proteomics analyses of the protein cargo composition of the supportive aEV vs. the less-supportive fEV identified 90% of the Top100 exosome proteins present in the ExoCarta database. Gene Ontology (GO) analyses illustrated that the proteins overrepresented in aEVs were annotated to oxidation-reduction process, mitochondrial ATP synthesis coupled proton transport, or protein folding. In contrast, the proteins overrepresented in fEVs were annotated to extracellular matrix organization positive regulation of cell migration or transforming growth factor beta receptor (TGFBR) signaling pathway. Small RNA sequencing identified different molecular signatures between aEVs and fEVs. Interestingly, the microRNA cluster miR-99b/let-7e/miR-125a, previously identified to increase the number of HSPCs by targeting multiple pro-apoptotic genes, was highly and significantly enriched in aEVs. Although we identified significant differences in the supportive effects of aEVs and fEVs, RNAseq analyses of the 24 h treated HSPCs indicated that a limited set of genes was differentially regulated when compared to cells that were treated with cytokines only. Together, our study provides novel insights into the complex biological role of EVs and illustrates that aEVs and fEVs differentially support ex vivo expansion capacity of UCB-HSPCs. Together opening new means for the application of EVs in the discovery of therapeutics for more efficient ex vivo HSPC expansion.

Bone Marrow Harbors a Unique Population of Dendritic Cells with the Potential to Boost Neutrophil Formation upon Exposure to Fungal Antigen.

Apart from controlling hematopoiesis, the bone marrow (BM) also serves as a secondary lymphoid organ, as it can induce naïve T cell priming by resident dendritic cells (DC). When analyzing DCs in murine BM, we uncovered that they are localized around sinusoids, can (cross)-present antigens, become activated upon intravenous LPS-injection, and for the most part belong to the cDC2 subtype which is associated with Th2/Th17 immunity. Gene-expression profiling revealed that BM-resident DCs are enriched for several c-type lectins, including Dectin-1, which can bind beta-glucans expressed on fungi and yeast. Indeed, DCs in BM were much more efficient in phagocytosis of both yeast-derived zymosan-particles and Aspergillus conidiae than their splenic counterparts, which was highly dependent on Dectin-1. DCs in human BM could also phagocytose zymosan, which was dependent on ß1-integrins. Moreover, zymosan-stimulated BM-resident DCs enhanced the differentiation of hematopoietic stem and progenitor cells towards neutrophils, while also boosting the maintenance of these progenitors. Our findings signify an important role for BM DCs as translators between infection and hematopoiesis, particularly in anti-fungal immunity. The ability of BM-resident DCs to boost neutrophil formation is relevant from a clinical perspective and contributes to our understanding of the increased susceptibility for fungal infections following BM damage.

Integrins Control Vesicular Trafficking; New Tricks for Old Dogs.

Integrins are transmembrane receptors that transduce biochemical and mechanical signals across the plasma membrane and promote cell adhesion and migration. In addition, integrin adhesion complexes are functionally and structurally linked to components of the intracellular trafficking machinery and accumulating data now reveal that they are key regulators of endocytosis and exocytosis in a variety of cell types. Here, we highlight recent insights into integrin control of intracellular trafficking in processes such as degranulation, mechanotransduction, cell-cell communication, antibody production, virus entry, Toll-like receptor signaling, autophagy, and phagocytosis, as well as the release and uptake of extracellular vesicles. We discuss the underlying molecular mechanisms and the implications for a range of pathophysiological contexts, including hemostasis, immunity, tissue repair, cancer, and viral infection.

Hematopoietic stem and progenitor cells use podosomes to transcellularly cross the bone marrow endothelium.

Bone marrow endothelium plays an important role in the homing of hematopoietic stem and progenitor cells upon transplantation, but surprisingly little is known on how the bone marrow endothelial cells regulate local permeability and hematopoietic stem and progenitor cells transmigration. We show that temporal loss of vascular endothelial-cadherin function promotes vascular permeability in BM, even upon low-dose irradiation. Loss of vascular endothelial-cadherin function also enhances homing of transplanted hematopoietic stem and progenitor cells to the bone marrow of irradiated mice although engraftment is not increased. Intriguingly, stabilizing junctional vascular endothelial-cadherin in vivo reduced bone marrow permeability, but did not prevent hematopoietic stem and progenitor cells migration into the bone marrow, suggesting that hematopoietic stem and progenitor cells use the transcellular migration route to enter the bone marrow. Indeed, using an in vitro migration assay, we show that human hematopoietic stem and progenitor cells predominantly cross bone marrow endothelium in a transcellular manner in homeostasis by inducing podosome-like structures. Taken together, vascular endothelial-cadherin is crucial for BM vascular homeostasis but dispensable for the homing of hematopoietic stem and progenitor cells. These findings are important in the development of potential therapeutic targets to improve hematopoietic stem and progenitor cell homing strategies.

GITR shapes humoral immunity by controlling the balance between follicular T helper cells and regulatory T follicular cells.

Follicular helper CD4+ T-cells (Tfh) control humoral immunity by driving affinity maturation and isotype-switching of activated B-cells. Tfh localize within B-cell follicles and, upon encounter with cognate antigen, drive B-cell selection in germinal centers (GCs) as GC-Tfh. Tfh functionality is controlled by Foxp3-expressing Tfh, which are known as regulatory T follicular cells (Tfr). Thus far, it remains unclear which factors determine the balance between these functionally opposing follicular T-cell subsets. Here, we demonstrate in human and mouse that Tfh and GC-Tfh, as well as their regulatory counterparts, express glucocorticoid-induced TNF receptor related protein (GITR) on their surface. This costimulatory molecule not only helps to identify follicular T-cell subsets, but also increases the ratio of Tfh vs. Tfr, both within and outside the GC. Correspondingly, GITR triggering increases the number of IL-21 producing CD4+ T-cells, which also produce more IFN-γ and IL-10. The latter are known switch factors for IgG2c and IgG1, respectively, which corresponds to a concomitant increase in IgG2c and IgG1 production upon GITR-mediated costimulation. These results demonstrate that GITR can skew the functional balance between Tfh and Tfr, which offers new therapeutic possibilities in steering humoral immunity.

Activation and suppression of hematopoietic integrins in hemostasis and immunity.

Integrins are a large family of heterodimeric cell surface receptors that bind prototypic ligands on neighboring cells or in the extracellular matrix. Numerous studies have revealed key roles for platelet and leukocyte integrins in adhesion and migration and, thereby, their significance for hemostasis and immunity. The clinical importance of these integrins has also become clear, because aberrant integrin expression and/or behavior are associated with bleeding disorders, immunodeficiency, or autoimmune diseases. Importantly, overwhelming evidence gathered over recent years shows that regulation of integrin function is far more complex than previously assumed; a picture has emerged of multiple cytoplasmic, cell surface, and extracellular regulators working together to ensure cell type-specific and integrin-specific control of integrin functions. Here, we discuss recent insights into the dynamic activation and suppression of hematopoietic integrins, as well as their implications for platelet and leukocyte function in health and disease.