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Key Points Optimized interstitial migration of leukocytes is necessary for their timely arrival at sites of tissue injury and microbial assault. This process is regulated by a multitude of cell-intrinsic and environmental factors. Intravital imaging studies have shed new light on the dynamics and regulation of interstitial leukocyte migration in non-lymphoid organs. These studies are discussed in this Review, with a focus on neutrophils and T cells. The actin cytoskeleton regulates the formation of a polarized cellular shape, which defines the 'amoeboid' migration mode of leukocytes in the interstitial space. Transendothelial migration of leukocytes and their entry into the interstitial space is regulated by the perivascular extravasation unit (PVEU), which is composed of endothelial cells, pericytes, perivascular macrophages, mast cells and the basement membrane. The PVEU provides physical and biochemical guidance for leukocytes during and after diapedesis. Neutrophil migration towards a focus of tissue injury is regulated by a multistep process defined by scouting, amplification and stabilization phases. Scouting is the initial process whereby scarce neutrophils accumulate at the focus. In a feedforward loop, these cells then attract waves of additional neutrophils, which form a cluster around the focus in order to contain tissue injury and pathogens. Directional decision making by migrating neutrophils is mediated by temporally and spatially coordinated gradients of chemoattractants and chemorepellents within tissues, and by physical guidance structures provided, for example, by pericytes. Multiple competing signals are integrated by intracellular signalling molecules in crawling neutrophils. Migrating effector T cell populations scan tissues for the presence of antigen. Signals delivered by the T cell receptor regulate both migratory stops — which are necessary for target cell interactions — and also the highly active migratory phenotype of T cells. Investigation of T cell population dynamics suggests that Lévy walk behaviour underlies the search strategies of T cells, and optimizes target screening behaviour. Functional impairment of T cells, such as a tolerized or exhausted state, is paralleled by impaired migration. Co-stimulatory and co-inhibitory pathways have been implicated in regulating the migration of functionally impaired T cells. A variety of innate immune cell subsets display active screening behaviour in non-lymphoid organs, which underlies the rapid detection of tissue debris or pathogens. This Review follows neutrophils and T cells as they journey from the blood into tissues in search of sites of infection or injury. It highlights the mediators, which form temporally and spatially coordinated gradients within the tissues, and the mechanisms, including physical structures, that guide this directional migration. Leukocyte migration through interstitial tissues is essential for mounting a successful immune response. Interstitial motility is governed by a vast array of cell-intrinsic and cell-extrinsic factors that together ensure the proper positioning of immune cells in the context of specific microenvironments. Recent advances in imaging modalities, in particular intravital confocal and multi-photon microscopy, have helped to expand our understanding of the cellular and molecular mechanisms that underlie leukocyte navigation in the extravascular space. In this Review, we discuss the key factors that regulate leukocyte motility within three-dimensional environments, with a focus on neutrophils and T cells in non-lymphoid organs.

The aim of this study was to evaluate the effects of leukocyte- and platelet-rich fibrin (L-PRF) on the pain and soft tissue healing after tooth extractions. Twenty-six patients (9 males and 17 females) were treated with multiple extractions (2 to 8), with a total of 108 extractions. This was an exploratory single blinded randomized clinical trial with a split-mouth design. The pain after the surgery was assessed in each patient by the VAS scale (1 to 10) at intervals of 24-48-72-96 hours. The soft tissue healing was clinically evaluated at 3, 7, 14, and 21 days after surgery by the same examiner surgeon, using the modified Healing Index (4 to 12). The mean value of postextraction pain was 3.2 ± 0.3 in the experimental sides and 4.1 ± 0.1 in the control sides. After 7 days from the extractions, the values of modified Healing Index in the experimental and control groups were, respectively, 4.8 ± 0.6 and 5.1 ± 0.9. The use of L-PRF in postextraction sockets filling can be proposed as a useful procedure in order to manage the postoperative pain and to promote the soft tissue healing process, reducing the early adverse effects of the inflammation.

Twenty years after the discovery of chemokines is an appropriate time to review leukocyte traffic and to assess the knowledge and opportunities that have arisen from countless studies of the large and tight-knit family of chemotactic proteins.

The role of Pannexin (PANX) channels during collective and single cell migration is increasingly recognized. Amongst many functions that are relevant to cell migration, here we focus on the role of PANX-mediated adenine nucleotide release and associated autocrine and paracrine signaling. We also summarize the contribution of PANXs with the cytoskeleton, which is also key regulator of cell migration. PANXs, as mechanosensitive ATP releasing channels, provide a unique link between cell migration and purinergic communication. The functional association with several purinergic receptors, together with a plethora of signals that modulate their opening, allows PANX channels to integrate physical and chemical cues during inflammation. Ubiquitously expressed in almost all immune cells, PANX1 opening has been reported in different immunological contexts. Immune activation is the epitome coordination between cell communication and migration, as leukocytes (i.e., T cells, dendritic cells) exchange information while migrating towards the injury site. In the current review, we summarized the contribution of PANX channels during immune cell migration and recruitment; although we also compile the available evidence for non-immune cells (including fibroblasts, keratinocytes, astrocytes, and cancer cells). Finally, we discuss the current evidence of PANX1 and PANX3 channels as a both positive and/or negative regulator in different inflammatory conditions, proposing a general mechanism of these channels contribution during cell migration.

To assess the role of lithium treatment in the relationship between bipolar disorder (BD) and leukocyte telomere length (LTL). We compared LTL between 131 patients with BD, with or without a history of lithium treatment, and 336 controls. We tested the association between genetically determined LTL and BD in two large genome-wide association datasets. Patients with BD with a history lithium treatment showed longer LTL compared with never-treated patients (p = 0.015), and similar LTL compared with controls. Patients never treated with lithium showed shorter LTL compared with controls (p = 0.029). Mendelian randomization analysis showed no association between BD and genetically determined LTL. Our data support previous findings showing that long-term lithium treatment might protect against telomere shortening.

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.

Leukocyte trafficking plays a critical role in determining the progress and resolution of inflammation. Although significant progress has been made in understanding the role of leukocyte activation in inflammation, dissecting the interactions between different leukocyte subpopulations during trafficking is hampered by the complexity of in vivo conditions and the lack of detail of current in vitro assays. To measure the effects of the interactions between neutrophils and monocytes migrating in response to various chemoattractants, at single-cell resolution, we developed a microfluidic platform that replicates critical features of focal inflammation sites. We integrated an elastase assay into the focal chemotactic chambers (FCCs) of our device that enabled us to distinguish between phlogistic and nonphlogistic cell recruitment. We found that lipoxin A ₄ and resolvin D1, in solution or incorporated into nano–proresolving medicines, reduced neutrophil and monocyte trafficking toward leukotriene B ₄. Lipoxin A ₄ also reduced the elastase release from homogenous and heterogenous mixtures of neutrophils and monocytes. Surprisingly, the effect of resolvin D1 on heterogenous mixtures was antisynergistic, resulting in a transient spike in elastase activity, which was quickly terminated, and the degraded elastin removed by the leukocytes inside the FCCs. Therefore, the microfluidic assay provides a robust platform for measuring the effect of leukocyte interactions during trafficking and for characterizing the effects of inflammation mediators.

Key Points Recognition of invading bacteria is a crucial task for the human innate immune system to initiate the first line of defence against infections. For this purpose various receptors have evolved, each with different specificities for conserved microbial molecules. One important function of these receptors is to promote the migration of immune cells to the centre of infection along a gradient of microbial- or host-derived chemoattractants. This mechanism is called chemotaxis and is a crucial prerequisite for the elimination of pathogens. Certain bacterial peptides can function as potent leukocyte attractants. Owing to the characteristics of bacterial translation, bacterial proteins are synthesized with a formylated methionine at the amino terminus. This feature is exploited by the human innate immune system because formylated peptides are identified as chemokines for leukocytes. The chemoattractant receptor repertoire is limited and comprises members of the family of G protein-coupled receptors (GPCRs). Among them, the human formyl peptide receptor 1 (FPR1) is specialized to detect formylated peptides. Among the two orthologues of FPR1, only FPR2 has been confirmed to detect microbial peptides; FPR2 also detects endogenous ligands. Recently, particular interest in FPR2 has arisen owing to the description of bacterial FPR2 ligands. These include the phenol-soluble modulin (PSM) peptides, which are cytotoxic at high concentrations and are secreted by Staphylococcus aureus and other staphylococci. FPR2 was further shown to respond differently to pathogenic and non-pathogenic staphylococcal strains according to the production of PSMs. In addition to staphylococci, other bacteria such as Helicobacter pylori or enterococci activate FPR2. However, only in some instances has the nature of the agonists been identified. Short-chain fatty acids are products of the energy metabolism of fermenting bacteria. They are recognized by the GPCRs GPR41 and GPR43 to mediate neutrophil chemotaxis and contribute to intestinal epithelial homeostasis. Bacteria have evolved strategies to evade the host response by synthesizing molecules that block chemokine receptors. The recognition of bacterial chemotactic molecules may be beneficial for the host or, in some instances, also for pathogens taking advantage of inflammation. Moreover, chemotactic molecules are not only crucial for infection, they are also secreted by commensal bacteria and regulate epithelial homeostasis in the gut. In conclusion, the field of research on microbial leukocyte chemoattractants offers interesting avenues for elucidating the complex interplay of pathogens and commensals with the immune system. Recognition of bacterial infection by the host innate immune system leads to chemotactic leukocyte influx. Recent studies have indicated that the recognition of microorganism-associated molecular patterns (MAMPs) by G protein-coupled receptors (GPCRs) involves a larger set of chemotactic MAMPs and corresponding GPCRs than was previously thought. Peschel and colleagues review bacterial leukocyte-attracting molecules, the corresponding human receptors, and their roles in antibacterial host defence. The innate immune system recognizes conserved microorganism-associated molecular patterns (MAMPs), some of which are sensed by G protein-coupled receptors (GPCRs), and this leads to chemotactic leukocyte influx. Recent studies have indicated that these processes are crucial for host defence and rely on a larger set of chemotactic MAMPs and corresponding GPCRs than was previously thought. Agonists, such as bacterial formyl peptides, enterococcal pheromone peptides, staphylococcal peptide toxins, bacterial fermentation products and the Helicobacter pylori peptide HP(2–20), stimulate specific GPCRs. The importance of leukocyte chemotaxis in host defence is highlighted by the fact that some bacterial pathogens produce chemotaxis inhibitors. How the various chemoattractants, receptors and antagonists shape antibacterial host defence represents an important topic for future research.

This study reports on what we believe are novel mechanism(s) of the vascular protective action of adiponectin. We used intravital microscopy to measure leukocyte-endothelium interactions in adiponectin-deficient (Ad(-/-)) mice and found that adiponectin deficiency was associated with a 2-fold increase in leukocyte rolling and a 5-fold increase in leukocyte adhesion in the microcirculation. Measurement of endothelial NO (eNO) revealed that adiponectin deficiency drastically reduced levels of eNO in the vascular wall. Immunohistochemistry demonstrated increased expression of E-selectin and VCAM-1 in the vascular endothelium of Ad(-/-) mice. Systemic administration of the recombinant globular adiponectin domain (gAd) to Ad(-/-) mice significantly attenuated leukocyte-endothelium interactions and adhesion molecule expression in addition to restoring physiologic levels of eNO. Importantly, prior administration of gAd also protected WT mice against TNF-alpha-induced leukocyte-endothelium interactions, indicating a pharmacologic action of gAd. Mechanistically, blockade of eNOS with N(omega)-nitro-L-arginine methyl ester ( L-NAME) abolished the inhibitory effect of gAd on leukocyte adhesion, demonstrating the obligatory role of eNOS signaling in the antiinflammatory action of gAd. We believe this is the first demonstration that gAd protects the vasculature in vivo via increased NO bioavailability with suppression of leukocyte-endothelium interactions. Overall, we provide evidence that loss of adiponectin induces a primary state of endothelial dysfunction with increased leukocyte-endothelium adhesiveness.

Key Points Immune responses depend on the ability of leukocytes to move from the circulation into tissues. Leukocyte extravasation is guided and controlled by endothelial cells that capture circulating leukocytes and open a path for diapedesis. Regulation of integrin-mediated adhesion controls the slowing down of leukocyte rolling, leukocyte arrest, crawling and migration through the blood vessel wall. Each of these cellular functions is tightly regulated and depends on different extracellular factors and binding partners with varying signalling requirements. A central step in the diapedesis process involves a mechanism that stimulates the opening of endothelial cell junctions, which depends on regulating the function of VE-cadherin. An alternative, although less common, pathway is the transcellular route through endothelial cells. The diapedesis process involves many functions of leukocytes and endothelial cells, from stopping intraluminal crawling at suitable exit sites to loosening of endothelial cell contacts, preventing plasma leakage, extending the membrane surface area at endothelial cell junctions, active leukocyte migration through the junctional cleft, preventing reverse transmigration and sealing of the junction after diapedesis. After crossing the endothelial barrier, leukocytes crawl along pericytes to reach preferential sites of exit through the basement membrane that are characterized by low levels of expression of certain protein components. Understanding the process of leukocyte diapedesis in more detail will help to identify molecular targets, to interfere with various inflammatory processes. Reciprocal interactions between leukocytes and endothelial cells enable leukocytes to recognize the blood vessel endothelium in areas of inflammation and transmigrate across the endothelial barrier to mount an immune response in infected and/or damaged tissues. Immune responses depend on the ability of leukocytes to move from the circulation into tissue. This is enabled by mechanisms that guide leukocytes to the right exit sites and allow them to cross the barrier of the blood vessel wall. This process is regulated by a concerted action between endothelial cells and leukocytes, whereby endothelial cells activate leukocytes and direct them to extravasation sites, and leukocytes in turn instruct endothelial cells to open a path for transmigration. This Review focuses on recently described mechanisms that control and open exit routes for leukocytes through the endothelial barrier.