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Supplementary Table 1

Several solid malignancies trigger lymphangiogenesis, facilitating metastasis. Tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment (TME). Here, we have investigated the ability of tumoral lymphatic endothelial cells (LEC) to function as MHC class II–restricted antigen-presenting cells in the regulation of antitumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we have shown that tumoral LECs upregulate MHC class II and the MHC class II antigen-processing machinery, and that they promote regulatory T-cell (Treg) expansion ex vivo. In mice with LEC-restricted lack of MHC class II expression, tumor growth was severely impaired, whereas tumor-infiltrating effector T cells were increased. Reduction of tumor growth and reinvigoration of tumor-specific T-cell responses both resulted from alterations of the tumor-infiltrating Treg transcriptome and phenotype. Treg-suppressive functions were profoundly altered in tumors lacking MHC class II in LECs. No difference in effector T-cell responses or Treg phenotype and functions was observed in tumor-draining lymph nodes, indicating that MHC class II–restricted antigen presentation by LECs was required locally in the TME to confer potent suppressive functions to Tregs. Altogether, our study suggests that MHC class II–restricted antigen-presenting tumoral LECs function as a local brake, dampening T cell–mediated antitumor immunity and promoting intratumoral Treg-suppressive functions.

Supplementary Figures and Legends

The process of cancer immunosurveillance is a mechanism of tumour suppression that can protect the host from cancer development throughout its lifetime1,2. However, it is unknown whether the effectiveness of cancer immunosurveillance fluctuates over a single day. Here we demonstrate that the initial time of day of tumour engraftment dictates the ensuing tumour size across mouse cancer models. Using immunodeficient mice as well as mice lacking lineage-specific circadian functions, we show that dendritic cells (DCs) and CD8+ T cells exert circadian anti-tumour functions that control melanoma volume. Specifically, we find that rhythmic trafficking of DCs to the tumour draining lymph node governs a circadian response of tumour-antigen-specific CD8+ T cells that is dependent on the circadian expression of the co-stimulatory molecule CD80. As a consequence, cancer immunotherapy is more effective when synchronized with DC functions, shows circadian outcomes in mice and suggests similar effects in humans. These data demonstrate that the circadian rhythms of anti-tumour immune components are not only critical for controlling tumour size but can also be of therapeutic relevance.

The assessment of leukocyte activation in vivo is mainly based on surrogate parameters, such as cell shape changes and migration patterns. Consequently, additional parameters are required to dissect the complex spatiotemporal activation of leukocytes during inflammation. Here, we showed that intravital microscopy of myeloid leukocyte Ca2+signals with Ca2+reporter mouse strains combined with bioinformatic signal analysis provided a tool to assess their activation in vivo. We demonstrated by two-photon microscopy that tissue-resident macrophages reacted to sterile inflammation in the cremaster muscle with Ca2+transients in a distinct spatiotemporal pattern. Moreover, through high-resolution, intravital spinning disk confocal microscopy, we identified the intracellular Ca2+signaling patterns of neutrophils during the migration cascade in vivo. These patterns were modulated by the Ca2+channel Orai1 and Gαi-coupled GPCRs, whose effects were evident through analysis of the range of frequencies of the Ca2+signal (frequency spectra), which provided insights into the complex patterns of leukocyte Ca2+oscillations. Together, these findings establish Ca2+frequency spectra as an additional dimension to assess leukocyte activation and migration during inflammation in vivo.

The recruitment of neutrophils from the microvasculature to the site of injury or infection represents a key event in the inflammatory response. Vitronectin (VN) is a multifunctional macromolecule abundantly present in blood and extracellular matrix. The role of this glycoprotein in the extravasation process of circulating neutrophils remains elusive. Employing advanced in vivo/ex vivo imaging techniques in different mouse models as well as in vitro methods, we uncovered a previously unrecognized function of VN in the transition of dynamic to static intravascular interactions of neutrophils with microvascular endothelial cells. These distinct properties of VN require the heteromerization of this glycoprotein with plasminogen activator inhibitor-1 (PAI-1) on the activated venular endothelium and subsequent interactions of this protein complex with the scavenger receptor low-density lipoprotein receptor-related protein-1 (LRP-1) on intravascularly adhering neutrophils. This induces p38 mitogen-activated protein kinases (MAPK)-dependent intracellular signaling events which, in turn, regulates the proper clustering of the beta2 integrin lymphocyte function associated antigen-1 (LFA-1) on the surface of these immune cells. As a consequence of this molecular interplay, neutrophils become able to stabilize their adhesion to the microvascular endothelium and, subsequently, to extravasate to the perivascular tissue. Hence, endothelial- bound VN-PAI-1 heteromers stabilize intravascular adhesion of neutrophils by coordinating beta2 integrin clustering on the surface of these immune cells, thereby effectively controlling neutrophil trafficking to inflamed tissue. Targeting this protein complex might be beneficial for the prevention and treatment of inflammatory pathologies.

Background High tumor levels of urokinase-type plasminogen activator (uPA)-plasminogen activator inhibitor-1 (PAI-1) heteromers independently predict poor survival in early breast cancer. The pathogenetic role of this protein complex, however, remains largely obscure. Material and Methods Neutrophil trafficking was analyzed in orthotopic (multi-channel flow cytometry) and heterotopic (ear; multi-channel in vivo microscopy) mouse models of 4T1 breast cancer, in a mouse peritonitis assay (multi-channel flow cytometry), as well as in the mouse cremaster muscle (multi-channel in vivo microscopy). Cytokine expression in tumors was determined by multiplex ELISA. Phenotypic and functional properties of primary mouse neutrophils, microvascular endothelial cells (cell line bEnd.3), macrophages (cell line RAW 264.7), and breast cancer cells (cell line 4T1) were characterized in different in vitro assays. uPA/PAI-1 expression and neutrophil infiltration in human breast cancer samples were assessed by RNA sequencing, immunhistochemistry, and ELISA. Results and Discussion Here, we demonstrate that uPA-PAI-1 heteromerization multiplies the potential of the single proteins to attract pro-tumorigenic neutrophils. To this end, tumor-released uPA-PAI-1 activates peritumoral macrophages (VLDL receptor- and ERK/MAPK-pathway). This promotes neutrophil trafficking to cancerous lesions (enhanced β2 integrin activation and clustering) and primes these immune cells towards a pro-tumorigenic phenotype (elevated neutrophil elastase expression), thus supporting tumor growth and metastasis. Blockade of uPA-PAI-1 heteromerization by a novel inhibitor effectively interfered with these events and prevented tumor progression. Conclusions Here, we identified an already therapeutically targetable interplay between hemostasis and innate immunity that drives advanced stages of breast cancer as well as characterized the underlying mechanisms of this process. As a personalized immunotherapeutic strategy, blockade of uPA-PAI-1 heteromerization might be particularly beneficial for patients with highly aggressive uPA-PAI-1high tumors. This study was supported by Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich (SFB) 914 Disclosure Information B. Uhl: None. L. Mittmann: None. J. Dominik: None. R. Hennel: None. B. Smiljanov: None. F. Haring: None. J. Schaubacher: None. C. Braun: None. L. Padovan: None. R. Pick: None. M. Canis: None. C. Schulz: None. M. Mack: None. E. Gutjahr: None. P. Sinn: None. J. Heil: None. K. Steiger: None. S.M. Kanse: None. W. Weichert: None. M. Sperandio: None. K. Lauber: None. F. Krombach: None. C. Reichel: None.

The authors show that autophagy in lymphatic endothelial cells (LECs) dampens collagen-induced arthritis by regulating two distinct pathways: the promotion of T reg cells in LNs and the inhibition of S1P-dependent migration of pathogenic Th17 cells in inflamed organs., Lymphatic endothelial cells (LECs) present peripheral tissue antigens to induce T cell tolerance. In addition, LECs are the main source of sphingosine-1-phosphate (S1P), promoting naive T cell survival and effector T cell exit from lymph nodes (LNs). Autophagy is a physiological process essential for cellular homeostasis. We investigated whether autophagy in LECs modulates T cell activation in experimental arthritis. Whereas genetic abrogation of autophagy in LECs does not alter immune homeostasis, it induces alterations of the regulatory T cell (T reg cell) population in LNs from arthritic mice, which might be linked to MHCII-mediated antigen presentation by LECs. Furthermore, inflammation-induced autophagy in LECs promotes the degradation of Sphingosine kinase 1 (SphK1), resulting in decreased S1P production. Consequently, in arthritic mice lacking autophagy in LECs, pathogenic Th17 cell migration toward LEC-derived S1P gradients and egress from LNs are enhanced, as well as infiltration of inflamed joints, resulting in exacerbated arthritis. Our results highlight the autophagy pathway as an important regulator of LEC immunomodulatory functions in inflammatory conditions.

Several solid malignancies trigger lymphangiogenesis, facilitating metastasis. Tumor-associated lymphatic vessels significantly contribute to the generation of an immunosuppressive tumor microenvironment (TME). Here, we have investigated the ability of tumoral lymphatic endothelial cells (LEC) to function as MHC class II-restricted antigen-presenting cells in the regulation of antitumor immunity. Using murine models of lymphangiogenic tumors engrafted under the skin, we have shown that tumoral LECs upregulate MHC class II and the MHC class II antigen-processing machinery, and that they promote regulatory T-cell (Treg) expansion ex vivo In mice with LEC-restricted lack of MHC class II expression, tumor growth was severely impaired, whereas tumor-infiltrating effector T cells were increased. Reduction of tumor growth and reinvigoration of tumor-specific T-cell responses both resulted from alterations of the tumor-infiltrating Treg transcriptome and phenotype. Treg-suppressive functions were profoundly altered in tumors lacking MHC class II in LECs. No difference in effector T-cell responses or Treg phenotype and functions was observed in tumor-draining lymph nodes, indicating that MHC class II-restricted antigen presentation by LECs was required locally in the TME to confer potent suppressive functions to Tregs. Altogether, our study suggests that MHC class II-restricted antigen-presenting tumoral LECs function as a local brake, dampening T cell-mediated antitumor immunity and promoting intratumoral Treg-suppressive functions.

Migration of leukocytes from the skin to lymph nodes (LNs) via afferent lymphatic vessels (LVs) is pivotal for adaptive immune responses1,2. Circadian rhythms have emerged as important regulators of leukocyte trafficking to LNs via the blood3,4. Here, we demonstrate that dendritic cells (DCs) have a circadian migration pattern into LVs, which peaks during the rest phase in mice. This migration pattern is determined by rhythmic gradients in the expression of the chemokine CCL21 and of adhesion molecules in both mice and humans. Chronopharmacological targeting of the involved factors abrogates circadian migration of DCs. We identify cell-intrinsic circadian oscillations in skin lymphatic endothelial cells (LECs) and DCs that cogovern these rhythms, as their genetic disruption in either cell type ablates circadian trafficking. These observations indicate that circadian clocks control the infiltration of DCs into skin lymphatics, a process that is essential for many adaptive immune responses and relevant for vaccination and immunotherapies., Scheiermann and colleagues show that circadian clocks control the infiltration of dendritic cells into skin lymphatics in mice and humans, with a peak migration to the lymph nodes during the rest phase.

Background High tumor levels of urokinase-type plasminogen activator (uPA)-plasminogen activator inhibitor-1 (PAI-1) heteromers independently predict poor survival in early breast cancer. The pathogenetic role of this protein complex, however, remains largely obscure. Materials and Methods Neutrophil trafficking was analyzed in orthotopic (multi-channel flow cytometry) and heterotopic (ear; multi-channel in vivo microscopy) mouse models of 4T1 breast cancer, in a mouse peritonitis assay (multi-channel flow cytometry), as well as in the mouse cremaster muscle (multi-channel in vivo microscopy). Cytokine expression in tumors was determined by multiplex ELISA. Phenotypic and functional properties of primary mouse neutrophils, microvascular endothelial cells (cell line bEnd.3), macrophages (cell line RAW 264.7), and breast cancer cells (cell line 4T1) were characterized in different in vitro assays. uPA/PAI-1 expression and neutrophil infiltration in human breast cancer samples were assessed by RNA sequencing, immunhistochemistry, and ELISA. Results Here, we demonstrate that uPA-PAI-1 heteromerization multiplies the potential of the single proteins to attract pro-tumorigenic neutrophils. To this end, tumor-released uPA-PAI-1 utilizes very low density lipoprotein receptor and ERK mitogen-activated protein kinases to initiate a pro-inflammatory program in peritumoral macrophages. This promotes neutrophil trafficking to cancerous lesions and primes these immune cells towards a pro-tumorigenic phenotype, thus supporting tumor growth and metastasis. Blockade of uPA-PAI-1 heteromerization by a novel inhibitor effectively interfered with these events and prevented tumor progression. Conclusions Our findings identify an already therapeutically targetable interplay between hemostasis and innate immunity that drives advanced stages of breast cancer. As a personalized immunotherapeutic strategy, blockade of uPA-PAI-1 heteromerization might be particularly beneficial for patients with highly aggressive uPA-PAI-1hightumors. This study was supported by Deutsche Forschungsgemeinschaft (DFG), Sonderforschungsbereich (SFB) 914. Disclosure Information B. Uhl: None. L. Mittmann: None. J. Dominik: None. J. Schaubacher: None. C. Braun: None. R. Pick: None. M. Canis: None. S. Kanse: None. W. Weichert: None. M. Sperandio: None. K. Lauber: None. F. Krombach: None. C.A. Reichel: None.

Neutrophils are the first leukocytes to arrive at sites of injury during the acute inflammatory response. To maintain the polarized morphology during migration, nonmuscle myosins class II are essential, but studies using genetic models to investigate the role of Myh9 for neutrophil migration were missing. In this study, we analyzed the functional role of Myh9 on neutrophil trafficking using genetic downregulation of Myh9 in Vav-iCre+/Myh9wt/fl mice because the complete knockout of Myh9 in the hematopoietic system was lethal. Migration velocity and Euclidean distance were significantly diminished during mechanotactic migration of Vav-iCre+/Myh9wt/fl neutrophils compared with Vav-iCre−/Myh9wt/fl control neutrophils. Similar results were obtained for transmigration and migration in confined three-dimensional environments. Stimulated emission depletion nanoscopy revealed that a certain threshold of Myh9 was required to maintain proper F-actin dynamics in the front of the migrating cell. In laser-induced skin injury and in acute peritonitis, reduced Myh9 expression in the hematopoietic system resulted in significantly diminished neutrophil extravasation. Investigation of bone marrow chimeric mice in the peritonitis model revealed that the migration defect was cell intrinsic. Expression of Myh9-EGFP rescued the Myh9-related defects in two-dimensional and three-dimensional migration of Hoxb8-SCF cell–derived neutrophils generated from fetal liver cells with a Myh9 knockdown. Live cell imaging provided evidence that Myh9 was localized in branching lamellipodia and in the uropod where it may enable fast neutrophil migration. In summary, the severe migration defects indicate an essential and fundamental role of Myh9 for neutrophil trafficking in innate immunity.

Neutrophil extravasation and interstitial migration are important steps during the recruitment of neutrophils to sites of inflammation. In the present study, we addressed the functional importance of the unconventional class I myosin 1f (Myo1f) for neutrophil trafficking during acute inflammation. In contrast to leukocyte rolling and adhesion, the genetic absence of Myo1f severely compromised neutrophil extravasation into the inflamed mouse cremaster tissue when compared with Myo1f+/+ mice as studied by intravital microscopy. Similar results were obtained in experimental models of acute peritonitis and acute lung injury. In contrast to 2-dimensional migration, which occurred independently of Myo1f, Myo1f was indispensable for neutrophil migration in 3-dimensional (3D) environments, that is, transmigration and migration in collagen networks as it regulated squeezing and dynamic deformation of the neutrophil nucleus during migration through physical barriers. Thus, we provide evidence for an important role of Myo1f in neutrophil trafficking during inflammation by specifically regulating neutrophil extravasation and migration in 3D environments.

This article contains errors in Figs. 5 and 6, for which we apologize. In Fig. 5f, the image ‘E12.5 tail’ was inadvertently replaced with a duplicate of the image ‘E12.5 trunk’ from the same panel. In Figure 6d, the image ‘E9.5/OH-TAM E8.5, embryo’ was inadvertently replaced with a duplicate of the image ‘E10.5/ OH-TAM E8.5, embryo’ from Fig. 6b. The corrected versions of these figures appear in the Author Correction associated with this Article.

Leukocyte recruitment into inflamed tissue is highly dependent on the activation and binding of integrins to their respective ligands, followed by the induction of various signaling events within the cell referred to as outside-in signaling. Src family kinases (SFK) are the central players in the outside-in signaling process, assigning them a critical role for proper immune cell function. Our study investigated the role of SFK on neutrophil recruitment in vivo using Hck-/- Fgr-/- Lyn-/- mice, which lack SFK expressed in neutrophils. We show that loss of SFK strongly reduces neutrophil adhesion and post-arrest modifications in a shear force dependent manner. Additionally, we found that in the absence of SFK, neutrophils display impaired Rab27a-dependent surface mobilization of neutrophil elastase, VLA3 and VLA6 containing vesicles. This results in a defect in neutrophil vascular basement membrane penetration and thus strongly impaired extravasation. Taken together, we demonstrate that SFK play a role in neutrophil post-arrest modifications and extravasation during acute inflammation. These findings may support the current efforts to use SFK-inhibitors in inflammatory diseases with unwanted neutrophil recruitment.

The number of leukocytes circulating in blood in mammals is under circadian control (i.e., ∼24h). We summarize here latest findings on the mechanisms governing leukocyte migration from the blood into various organs, focusing on the distinct leukocyte subtype- and tissue-specific molecules involved. We highlight the oscillatory expression patterns of adhesion molecules, chemokines, and their receptors that are expressed on endothelial cells and leukocytes, and which are crucial regulators of rhythmic leukocyte recruitment. We also discuss the relevance of clock genes for leukocyte function and migration. Finally, we compare immune cell rhythms under steady-state conditions as well as during inflammation and disease, and we postulate how these findings provide potential new avenues for therapeutic intervention.

The role of integrin-mediated adhesion during T cell progenitor homing to and differentiation within the thymus is ill-defined, mainly due to functional overlap. To circumvent compensation, we disrupted the hematopoietic integrin regulator kindlin-3 in mice and found a progressive thymus atrophy that is primarily caused by an impaired homing capacity of T cell progenitors to the vascularized thymus. Notably, the low shear flow conditions in the vascular system at midgestation allow kindlin-3-deficient fetal liver-derived T cell progenitors to extravasate via pharyngeal vessels and colonize the avascular thymus primordium. Once in the thymus, kindlin-3 promotes intrathymic T cell proliferation by facilitating the integrin-dependent crosstalk with thymic antigen presenting cells, while intrathymic T cell migration, maturation into single positive CD4 and CD8 T cells and release into the circulation proceed without kindlin-3. Thus, kindlin-3 is dispensable for integrin-mediated T cell progenitor adhesion and signalling at low and indispensable at high shear forces.

Neutrophil recruitment during acute inflammation critically depends on the spatial and temporal regulation of βThis review provides a comprehensive overview of our current knowledge on proteins interacting with the cytoplasmic tail of CD18, the conserved β-subunit of βA total of 22 proteins including Talin, Kindlin 3 and Coronin 1A have been reported to interact with the CD18 cytoplasmic tail. Here, proteins binding to the cytoplasmic domain of CD18 in experiments using purified, recombinant proteins or peptides in, for example, pull-down assays, are defined as direct interactors. Proteins that have been shown to interact with the cytoplasmic domain of CD18 using whole cell lysates in, for example, pull-down experiments are claimed as interacting proteins without evidence for direct interaction. In summary, βThe knowledge of proteins interacting with β

The ability to escape apoptosis is a hallmark of cancer-initiating cells and a key factor of resistance to oncolytic therapy. Here, we identify FAM96A as a ubiquitous, evolutionarily conserved apoptosome-activating protein and investigate its potential pro-apoptotic tumor suppressor function in gastrointestinal stromal tumors (GISTs). Interaction between FAM96A and apoptotic peptidase activating factor 1 (APAF1) was identified in yeast two-hybrid screen and further studied by deletion mutants, glutathione-S-transferase pull-down, co-immunoprecipitation and immunofluorescence. Effects of FAM96A overexpression and knock-down on apoptosis sensitivity were examined in cancer cells and zebrafish embryos. Expression of FAM96A in GISTs and histogenetically related cells including interstitial cells of Cajal (ICCs), "fibroblast-like cells" (FLCs) and ICC stem cells (ICC-SCs) was investigated by Northern blotting, reverse transcription-polymerase chain reaction, immunohistochemistry and Western immunoblotting. Tumorigenicity of GIST cells and transformed murine ICC-SCs stably transduced to re-express FAM96A was studied by xeno- and allografting into immunocompromised mice. FAM96A was found to bind APAF1 and to enhance the induction of mitochondrial apoptosis. FAM96A protein or mRNA was dramatically reduced or lost in 106 of 108 GIST samples representing three independent patient cohorts. Whereas ICCs, ICC-SCs and FLCs, the presumed normal counterparts of GIST, were found to robustly express FAM96A protein and mRNA, FAM96A expression was much reduced in tumorigenic ICC-SCs. Re-expression of FAM96A in GIST cells and transformed ICC-SCs increased apoptosis sensitivity and diminished tumorigenicity. Our data suggest FAM96A is a novel pro-apoptotic tumor suppressor that is lost during GIST tumorigenesis.

The relationship between physical distance to program, different types of family contact, and youth behavioral outcomes, discharge assessment, and 6-month follow-up was examined. Data came from clinical record information for 350 youth who had been admitted to a group home program between July 2009 and October 2011. Path analysis was used to examine the hypothesized pattern of interrelationships among the variables. Study results found that home visits of youth with their families were related to better outcomes, while program visits by family were unrelated to outcomes. Phone contacts during the week were also unrelated to youth outcomes, whereas weekend phone contacts were significantly related to less successful outcomes. Findings suggest that family contact is important for the health and wellbeing of youth in residential care, but that not all family/friend contacts are equally beneficial and that some can even undermine a youth’s progress. Specific recommendations are made for future research to inform practice on how to make the most of these opportunities. Ultimately, all family and friend contact should be a positive factor in the youth’s treatment and outcomes.

The primary defense machinery to combat inflammation involves neutrophil granulocytes which in order to execute their functions rely on the efficiency of different cellular mechanisms including adhesion, spreading, migration in different environments, and phagocytosis. These functions require an accurately regulated actin network as well as the activation and adjustment of various signaling pathways. Mammalian filamins (FLNs) comprise three highly homologous large actin-binding proteins that are obvious candidates to control these processes as FLNs have been described to play a role in migration, spreading and adhesion in a variety of different cell types. The present study analyzed the role of filamin A (FLNa) in human neutrophil-like HL-60 cells. We found a strong enrichment of FLNa at the uropod of migrating neutrophils, and show that deficiency of FLNa caused a decrease in speed of migration both in 2D and 3D that is accompanied by a reduced activation of myosin-II. In addition, we show that FLNa plays a role in neutrophil phagocytosis. We also identified a hitherto unknown interaction of FLNa with coronin 1A that is mediated by FLNa repeats 9–18. FLNa deficiency had no or only minor effects on cell adhesion and spreading. In summary, deficiency of FLNa in human neutrophil-like HL-60 cells resulted in a surprisingly subtle phenotype. Our data indicate that FLNa is not essential for the regulation of mechanical properties during migration, but contributes to motility in a modulatory manner probably through its action at the uropod.

Trafficking of polymorphonuclear neutrophils (PMNs) during inflammation critically depends on the β2 integrins lymphocyte function-associated antigen 1 (LFA-1) (CD11a/CD18) and macrophage-1 antigen (CD11b/CD18). Here, we identify coronin 1A (Coro1A) as a novel regulator of β2 integrins that interacts with the cytoplasmic tail of CD18 and is crucial for induction of PMN adhesion and postadhesion events, including adhesion strengthening, spreading, and migration under flow conditions. Transition of PMN rolling to firm adhesion critically depends on Coro1A by regulating the accumulation of high-affinity LFA-1 in focal zones of adherent cells. Defective integrin affinity regulation in the genetic absence of Coro1A impairs leukocyte adhesion and extravasation in inflamed cremaster muscle venules in comparison with control animals. In a Helicobacter pylori mouse infection model, PMN infiltration into the gastric mucosa is dramatically reduced in Coro1A-/- mice, resulting in an attenuated gastric inflammation. Thus, Coro1A represents an important novel player in integrin biology, with key functions in PMN trafficking during innate immunity.

Summary The number of leukocytes present in circulation varies throughout the day, reflecting bone marrow output and emigration from blood into tissues. Using an organism-wide circadian screening approach, we detected oscillations in pro-migratory factors that were distinct for specific vascular beds and individual leukocyte subsets. This rhythmic molecular signature governed time-of-day-dependent homing behavior of leukocyte subsets to specific organs. Ablation of BMAL1, a transcription factor central to circadian clock function, in endothelial cells or leukocyte subsets demonstrated that rhythmic recruitment is dependent on both microenvironmental and cell-autonomous oscillations. These oscillatory patterns defined leukocyte trafficking in both homeostasis and inflammation and determined detectable tumor burden in blood cancer models. Rhythms in the expression of pro-migratory factors and migration capacities were preserved in human primary leukocytes. The definition of spatial and temporal expression profiles of pro-migratory factors guiding leukocyte migration patterns to organs provides a resource for the further study of the impact of circadian rhythms in immunity., Graphical Abstract, Highlights • Leukocyte subsets show time-of-day-dependent migration patterns to organs • This relies on lineage- and tissue-specific oscillations in pro-migratory factors • Loss of circadian clocks in the endothelium or leukocytes ablates rhythmicity • The efficacy of blocking leukocyte migration is time-of-day dependent, Leukocytes continuously circulate throughout the body. He et al. demonstrate that trafficking patterns of major leukocyte subsets occur in a rhythmic manner dependent on the time-of-day-dependent expression of lineage- and tissue-specific factors. This influences the inflammatory response and leukemic tumor burden and translates to the migration behavior of human primary lymphocytes.

Site-directed trafficking of polymorphonuclear neutrophils (PMN) to their target regions within the tissue is an important prerequisite for efficient host defense during the acute inflammatory response. This process requires intraluminal crawling of PMN on the activated endothelial cells to their extravasation sites. Upon transendothelial diapedesis, PMN migrate in the interstitial tissue to sites of inflammation. These crucial steps within the recruitment cascade are defined as intraluminal crawling and interstitial migration. In this review, we will focus on the molecular mechanisms that control and fine-tune these migratory processes and discuss the role of adhesion molecules of the β2 integrin (CD11/CD18) family for these cellular functions.

Recruitment of polymorphonuclear neutrophils (PMNs) to sites of acute inflammation critically depends on β2 integrins (CD11/CD18). Recently, the mammalian actin-binding protein 1 (mAbp1) was identified as an important adaptor protein regulating PMN trafficking downstream of β2 integrins. Here, we show that mAbp1 constitutively co-immunoprecipitated with hematopoietic progenitor kinase 1 (HPK1) in neutrophil-like differentiated HL-60 (dHL-60) cells. HPK1 was enriched at the lamellipodium of polarized dHL-60 cells, where it colocalized with mAbp1 and actin. Functional analysis of PMNs from HPK1-deficient mice showed that HPK1 was critical for CXCL1-induced lymphocyte function-associated antigen 1 (LFA-1)-mediated PMN adhesion to ICAM-1 under flow conditions. Accordingly, CXCL1-mediated induction of high-affinity LFA-1 required HPK1, but macrophage antigen 1 (Mac-1) affinity regulation was independent of HPK1. Intravital microscopy of the mouse cremaster muscle confirmed the defect of CXCL1-induced leukocyte adhesion in HPK1-deficient mice. Furthermore, β2 integrin-mediated post-adhesion processes-adhesion strengthening, spreading, and directed mechanotactic crawling of PMNs under flow conditions-involved HPK1 in vitro and in vivo. Upon intrascrotal administration of tumor necrosis factor α (TNF-α), PMN adhesion and extravasation were severely compromised in HPK1-deficient mice. In summary, our results indicate that HPK1 is critically involved in LFA-1-mediated PMN trafficking during acute inflammation.

The Pediatric Quality of Life assessment (PedsQL™) is the most widely used measure for assessing adolescent health-related quality of life (HRQoL). While youth in residential treatment facilities face many physical and mental health, behavioral, education, and familial challenges that could impact their HRQoL, no research has sought to assess the factor structure of the PedsQL™ among youth receiving residential care. High school–aged youth (N = 229) attending a large residential treatment center in Omaha, NE were recruited to complete a data collection packet comprised of various health assessments including the PedsQL. Four competing confirmatory factor analysis models were used to test the hypothesized internal structure of the PedsQL™ 4.0 Teen Report. Models A, B, and C had acceptable CFI (≥.90), TLI (≥.90), and RMSEA (≤.08) fit indicators. However, factor loadings for items 5 and 6 were problematic. After removing the two problematic items, Model D was fit to the data and proved to be the superior of the four models. This model included two first-order factors (physical health problems; school attendance problems) and one second-order factor (psychological health problems). The findings suggest that researchers and practitioners studying youth in residential settings can reliably use the PedsQL™ to assess their HRQoL.

Background: Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the context of arterial myeloid cell recruitment. Methods: Intravital microscopy of the carotid artery, the jugular vein, and cremasteric arterioles and venules in Apoe –/– and CatG-deficient mice ( Apoe –/– Ctsg –/– ) was used to study site-specific myeloid cell behavior after high-fat diet feeding or tumor necrosis factor stimulation. Atherosclerosis development was assessed in aortic root sections after 4 weeks of high-fat diet, whereas lung inflammation was assessed after inhalation of lipopolysaccharide. Endothelial deposition of CatG and CCL5 was quantified in whole-mount preparations using 2-photon and confocal microscopy. Results: Our observations elucidated a crucial role for CatG during arterial leukocyte adhesion, an effect not found during venular adhesion. Consequently, CatG deficiency attenuates atherosclerosis but not acute lung inflammation. Mechanistically, CatG is immobilized on arterial endothelium where it activates leukocytes to firmly adhere engaging integrin clustering, a process of crucial importance to achieve effective adherence under high-shear flow. Therapeutic neutralization of CatG specifically abrogated arterial leukocyte adhesion without affecting myeloid cell adhesion in the microcirculation. Repetitive application of CatG-neutralizing antibodies permitted inhibition of atherogenesis in mice. Conclusions: Taken together, these findings present evidence of an arterial-specific recruitment pattern centered on CatG-instructed adhesion strengthening. The inhibition of this process could provide a novel strategy for treatment of arterial inflammation with limited side effects.

This study examined the 12-month post-departure outcomes for youth who exited a residential treatment program at differing levels of restrictiveness. Study participants were 120 youth who entered an integrated residential continuum of care at its most restrictive level and then either departed the program at the same level or stepped down and departed at a lower level of restrictiveness. Results indicate that youth who stepped down and exited at the lowest level of restrictiveness were the most likely to be living at home or in a homelike setting and experienced fewer formal post-departure out-of-home placements. However, there were no differences in post-departure rates of substance use, arrests, or being in school or having graduated. These results suggest that youth who were served in the integrated continuum and departed at the lowest level of restrictiveness had more positive outcomes at 12-month post-discharge.

Background Neutrophil recruitment during acute inflammation critically depends on the spatial and temporal regulation of beta(2) integrins (CD11/CD18). This regulation occurs by inside-out and outside-in signalling via interaction of cytoplasmic proteins with the intracellular domains of the integrin alpha- and beta-subunits. The underlying molecular mechanisms regulating beta(2) integrins in neutrophils are still incompletely understood. Aim Results This review provides a comprehensive overview of our current knowledge on proteins interacting with the cytoplasmic tail of CD18, the conserved beta-subunit of beta(2) integrins, their regulation and their functional importance for neutrophil trafficking during acute inflammation. A total of 22 proteins including Talin, Kindlin 3 and Coronin 1A have been reported to interact with the CD18 cytoplasmic tail. Here, proteins binding to the cytoplasmic domain of CD18 in experiments using purified, recombinant proteins or peptides in, for example, pull-down assays, are defined as direct interactors. Proteins that have been shown to interact with the cytoplasmic domain of CD18 using whole cell lysates in, for example, pull-down experiments are claimed as interacting proteins without evidence for direct interaction. In summary, beta(2) integrin activation and signalling depend on a specific subset of proteins interacting with CD18 and their precise regulation. If disturbed, profound defects of neutrophil recruitment and activation become evident compromising the innate immune response. Conclusions The knowledge of proteins interacting with beta(2) integrins and their regulation during neutrophil trafficking does not only improve our basic understanding of innate immunity but may pave the way to novel therapeutic strategies in the treatment of inflammatory diseases.

At the time of entry into residential treatment, many youth have prescriptions for one or more psychotropic medications. It is not well understood why some youth are prescribed only one psychotropic medication while others may be prescribed more. We sought to determine if differences existed between youth entering residential treatment with monopharmacy (n = 77) versus youth entering with polypharmacy (n = 103). Youth were compared across demographic, family, behavioral, mental health, and psychotropic medication variables. The only significant differences identified were related to the numbers of youth with prescriptions across the different psychotropic classes. As would be expected, youth experiencing polypharmacy were more likely to have prescriptions within each class. Implications, limitations, and recommendations for future research are discussed.

Youth departing from out-of-home care settings face numerous challenges as they adapt to new settings or return to placements that have been unsuccessful in the past. Although several thousand youth face this transition annually, little is known about their specific needs and risks at departure. To better identify needs and risks, we evaluated the discharge data of 640 youth served in a residential group care setting by addressing the following questions: (a) to what settings do youth depart following a stay in residential group care, (b) what are the demographic, family, educational, behavioral, and departure characteristics of youth at departure, and (c) do these characteristics differ for youth departing to different levels of restrictiveness? Results indicate significant differences on youth characteristics based on levels of restrictiveness at departure placement. As one might expect, youth departing to more restrictive placements presented a broad host of challenges across domains, while those departing to less restrictive settings demonstrated fewer needs and departed with greater educational and behavioral gains. Results provide support for the development and planning of targeted aftercare programs designed to promote the short and long term functioning of youth served in out-of-home care.

The RNA alphavirus Semliki Forest (SFV) triggers apoptosis in various mammalian cells, but it has remained controversial at what infection stage and by which signalling pathways host cells are killed. Both RNA synthesis-dependent and -independent initiation processes and mitochondrial as well as death receptor signalling pathways have been implicated. Here, we show that SFV-induced apoptosis is initiated at the level of RNA replication or thereafter. Moreover, by expressing antiapoptotic genes from recombinant SFV (replicons) and by using neutralizing reagents and gene-knockout cells, we provide clear evidence that SFV does not require CD95L-, TRAIL (tumor necrosis factor-related apoptosis-inducing ligand)- or tumor necrosis factor-mediated signalling but mitochondrial Bak to trigger cytochrome c release, the fall in the mitochondrial membrane potential, apoptotic protease-activating factor-1/caspase-9 apoptosome formation and caspase-3/-7 activation. Of seven BH3-only proteins tested, only Bid contributed to effective SFV-induced apoptosis. However, caspase-8 activation and Bid cleavage occurred downstream of Bax/Bak, indicating that truncated Bid formation serves to amplify rather than trigger SFV-induced apoptosis. Our data show that SFV sequentially activates a mitochondrial, Bak-mediated, caspase-8-dependent and Bid-mediated death signalling pathway that can be accurately dissected with gene-knockout cells and SFV replicons carrying antiapoptotic genes.