PDGFRα-lineage origin directs monocytes to trafficking proficiency to support peripheral immunity.

Multiple embryonic precursors give rise to leukocytes in adults while the lineage-based functional impacts are underappreciated. Mesodermal precursors expressing PDGFRα appear transiently during E7.5-8.5 descend to a subset of Lin- Sca1+ Kit+ hematopoietic progenitors found in adult BM. By analyzing a PDGFRα-lineage tracing mouse line, we here report that PDGFRα-lineage BM F4/80+ SSClo monocytes/macrophages are solely Ly6C+ LFA-1hi Mac-1hi monocytes enriched on the abluminal sinusoidal endothelium while Ly6C- LFA-1lo Mac-1lo macrophages are mostly from non-PDGFRα-lineage in vivo. Monocytes with stronger integrin profiles outcompete macrophages for adhesion on an endothelial monolayer or surfaces coated with ICAM-1-Fc or VCAM-1-Fc. Egress of PDGFRα-lineage-rich monocytes and subsequent differentiation to peripheral macrophages spatially segregates them from non-PDGFRα-lineage BM-resident macrophages and allows functional specialization since macrophages derived from these egressing monocytes differ in morphology, phenotype, and functionality from BM-resident macrophages in culture. Extravasation preference for blood PDGFRα-lineage monocytes varies by tissues and governs the local lineage composition of macrophages. More PDGFRα-lineage classical monocytes infiltrated into skin and colon but not into peritoneum. Accordingly, transcriptomic analytics indicated augmented inflammatory cascades in dermatitis skin of BM-chimeric mice harbouring only PDGFRα-lineage leukocytes. Thus, the PDGFRα-lineage origin biasedly generates monocytes predestined for BM exit to support peripheral immunity following extravasation and macrophage differentiation.

Synthesized HMGB1 peptide attenuates liver inflammation and suppresses fibrosis in mice.

The liver has a high regenerative ability and can induce spontaneous regression of fibrosis when early liver damage occurs; however, these abilities are lost when chronic liver damage results in decompensated cirrhosis. Cell therapies, such as mesenchymal stem cell (MSC) and macrophage therapies, have attracted attention as potential strategies for mitigating liver fibrosis. Here, we evaluated the therapeutic effects of HMGB1 peptide synthesized from box A of high mobility group box 1 protein. Liver damage and fibrosis were evaluated using a carbon tetrachloride (CCl4)-induced cirrhosis mouse model. The effects of HMGB1 peptide against immune cells were evaluated by single-cell RNA-seq using liver tissues, and those against monocytes/macrophages were further evaluated by in vitro analyses. Administration of HMGB1 peptide did not elicit a rapid response within 36 h, but attenuated liver damage after 1 week and suppressed fibrosis after 2 weeks. Fibrosis regression developed over time, despite continuous liver damage, suggesting that administration of this peptide could induce fibrolysis. In vitro analyses could not confirm a direct effect of HMGB1 peptide against monocyte/macrophages. However, macrophages were the most affected immune cells in the liver, and the number of scar-associated macrophages (Trem2+Cd9+ cells) with anti-inflammatory markers increased in the liver following HMGB1 treatment, suggesting that indirect effects of monocytes/macrophages were important for therapeutic efficacy. Overall, we established a new concept for cell-free therapy using HMGB1 peptide for cirrhosis through the induction of anti-inflammatory macrophages.

PECAM-1 supports leukocyte diapedesis by tension-dependent dephosphorylation of VE-cadherin.

Leukocyte extravasation is an essential step during the immune response and requires the destabilization of endothelial junctions. We have shown previously that this process depends in vivo on the dephosphorylation of VE-cadherin-Y731. Here, we reveal the underlying mechanism. Leukocyte-induced stimulation of PECAM-1 triggers dissociation of the phosphatase SHP2 which then directly targets VE-cadherin-Y731. The binding site of PECAM-1 for SHP2 is needed for VE-cadherin dephosphorylation and subsequent endocytosis. Importantly, the contribution of PECAM-1 to leukocyte diapedesis in vitro and in vivo was strictly dependent on the presence of Y731 of VE-cadherin. In addition to SHP2, dephosphorylation of Y731 required Ca2+ -signaling, non-muscle myosin II activation, and endothelial cell tension. Since we found that ß-catenin/plakoglobin mask VE-cadherin-Y731 and leukocyte docking to endothelial cells exert force on the VE-cadherin-catenin complex, we propose that leukocytes destabilize junctions by PECAM-1-SHP2-triggered dephosphorylation of VE-cadherin-Y731 which becomes accessible by actomyosin-mediated mechanical force exerted on the VE-cadherin-catenin complex.

Incidental diagnosis of leukocyte adhesion deficiency type II following ABO typing.

Individuals with the Bombay phenotype (Oh) in the ABO blood group system do not express the H, A, and B antigens but have no clinical symptoms. Bombay phenotype with clinical symptoms has been described in leukocyte adhesion deficiency type II (LAD II), a fucosylation disorder caused by mutations in SLC35C1. Only few LAD II patients have been described so far. Here we describe an additional patient, a 22-year old male, born to unrelated parents, presenting with inflammatory skin disease, periodontitis, growth, and mental retardation, admitted to the department of dentistry for treatment under general anesthesia. Pre-operative routine investigations revealed the presence of the Bombay phenotype (Oh). Genomic sequencing identified two novel triplet deletions of the SLC35C1 gene. Functional investigations confirmed the diagnosis of LAD II. Therapy with oral fucose led to the disappearance of the chronic skin infections and improvements in behavior and attention span.

Actin-Binding Protein Cortactin Promotes Pathogenesis of Experimental Autoimmune Encephalomyelitis by Supporting Leukocyte Infiltration into the Central Nervous System.

Leukocyte entry into the central nervous system (CNS) is essential for immune surveillance but is also the basis for the development of pathologic inflammatory conditions within the CNS, such as multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). The actin-binding protein, cortactin, in endothelial cells is an important player in regulating the interaction of immune cells with the vascular endothelium. Cortactin has been shown to control the integrity of the endothelial barrier and to support neutrophil transendothelial migration in vitro and in vivo in the skin. Here we use cortactin gene-inactivated male and female mice to study the role of this protein in EAE. Inducing EAE by immunization with a myelin oligodendrocyte glycoprotein peptide (MOG35-55) revealed an ameliorated disease course in cortactin gene-deficient female mice compared with WT mice. However, proliferation capacity and expression of IL-17A and IFNγ by cortactin-deficient and WT splenocytes did not differ, suggesting that the lack of cortactin does not affect induction of the immune response. Rather, cortactin deficiency caused decreased vascular permeability and reduced leukocyte infiltration into the brains and spinal cords of EAE mice. Accordingly, cortactin gene-deficient mice had smaller numbers of proinflammatory cuffs, less extensive demyelination, and reduced expression levels of proinflammatory cytokines within the neural tissue compared with WT littermates. Thus, cortactin contributes to the development of neural inflammation by supporting leukocyte transmigration through the blood-brain barrier and, therefore, represents a potential candidate for targeting CNS autoimmunity.SIGNIFICANCE STATEMENT Multiple sclerosis is an autoimmune neuroinflammatory disorder, based on the entry of inflammatory leukocytes into the CNS where these cells cause demyelination and neurodegeneration. Here, we use a mouse model for multiple sclerosis, experimental autoimmune encephalomyelitis, and show that gene inactivation of cortactin, an actin binding protein that modulates actin dynamics and branching, protects against neuroinflammation in experimental autoimmune encephalomyelitis. Leukocyte infiltration into the CNS was inhibited in cortactin-deficient mice, and lack of cortactin in cultured primary brain endothelial cells inhibited leukocyte transmigration. Expression levels of proinflammatory cytokines in the CNS and induction of vascular permeability were reduced. We conclude that cortactin represents a novel potential target for the treatment of multiple sclerosis.

Blood flow guides sequential support of neutrophil arrest and diapedesis by PILR-ß1 and PILR-α.

Arrest of rapidly flowing neutrophils in venules relies on capturing through selectins and chemokine-induced integrin activation. Despite a long-established concept, we show here that gene inactivation of activating paired immunoglobulin-like receptor (PILR)-ß1 nearly halved the efficiency of neutrophil arrest in venules of the mouse cremaster muscle. We found that this receptor binds to CD99, an interaction which relies on flow-induced shear forces and boosts chemokine-induced ß2-integrin-activation, leading to neutrophil attachment to endothelium. Upon arrest, binding of PILR-ß1 to CD99 ceases, shifting the signaling balance towards inhibitory PILR-α. This enables integrin deactivation and supports cell migration. Thus, flow-driven shear forces guide sequential signaling of first activating PILR-ß1 followed by inhibitory PILR-α to prompt neutrophil arrest and then transmigration. This doubles the efficiency of selectin-chemokine driven neutrophil arrest by PILR-ß1 and then supports transition to migration by PILR-α.

Endothelial CD99 supports arrest of mouse neutrophils in venules and binds to neutrophil PILRs.

CD99 is a crucial regulator of the transmigration (diapedesis) of leukocytes through the blood vessel wall. Here, we report that CD99 acts at 2 different steps in the extravasation process. In agreement with previous antibody-blocking experiments, we found that CD99 gene inactivation caused neutrophil accumulation between venular endothelial cells and the basement membrane in the inflamed cremaster. Unexpectedly, we additionally found that leukocyte attachment to the luminal surface of the venular endothelium was impaired in the absence of CD99. Intravital video microscopy revealed that CD99 supported rapid chemokine-induced leukocyte arrest. Inhibition of leukocyte attachment and extravasation were both solely due to the absence of CD99 on endothelial cells, whereas CD99 on leukocytes was irrelevant. Therefore, we searched for heterophilic ligands of endothelial CD99 on neutrophils. We found that endothelial cells bind to the paired immunoglobulinlike receptors (PILRs) in a strictly CD99-dependent way. In addition, endothelial CD99 was coprecipitated with PILRs from neutrophils that adhered to endothelial cells. Furthermore, soluble CD99 carrying a transferable biotin tag could transfer this tag covalently to PILR when incubated with intact neutrophils. Binding of neutrophils under flow to a surface coated with P-selectin fragment crystallizable (Fc) and intercellular adhesion molecule 1 (ICAM-1) Fc became more shear resistant if CD99 Fc was coimmobilized. This increased shear resistance was lost if neutrophils were preincubated with anti-PILR antibodies. We concluded that endothelial CD99 promotes leukocyte attachment to endothelium in inflamed vessels by a heterophilic ligand. In addition, CD99 binds to PILRs on neutrophils, an interaction that leads to increased shear resistance of the neutrophil attachment to ICAM-1.

Platelet-cytokine Complex Suppresses Tumour Growth by Exploiting Intratumoural Thrombin-dependent Platelet Aggregation.

Tumours constitute unique microenvironments where various blood cells and factors are exposed as a result of leaky vasculature. In the present study, we report that thrombin enrichment in B16F10 melanoma led to platelet aggregation, and this property was exploited to administer an anticancer cytokine, interferon-gamma induced protein 10 (IP10), through the formation of a platelet-IP10 complex. When intravenously infused, the complex reached platelet microaggregates in the tumour. The responses induced by the complex were solely immune-mediated, and tumour cytotoxicity was not observed. The complex suppressed the growth of mouse melanoma in vivo, while both platelets and the complex suppressed the accumulation of FoxP3(+) regulatory T cells in the tumour. These results demonstrated that thrombin-dependent platelet aggregation in B16F10 tumours defines platelets as a vector to deliver anticancer cytokines and provide specific treatment benefits.