Impaired Adhesion Research Articles

Peptidylarginine Deiminase 4 Promotes the Renal Infiltration of Neutrophils and Exacerbates the TLR7 Agonist-Induced Lupus Mice

Peptidylarginine deiminase 4 (PAD4), encoded by PADI4, plays critical roles in the immune system; however, its contribution to the pathogenesis of lupus nephritis remains controversial. The pathological roles of PAD4 were investigated in lupus model mice. An imiquimod (IMQ)-induced lupus model was analyzed in wild-type (WT) and Padi4-knockout (KO) mice. Proteinuria, serum anti-double stranded DNA (anti-dsDNA) antibody, and renal infiltrated cells were evaluated. Neutrophil migration and adhesion were assessed using adoptive transfer and adhesion assay. PAD4-regulated pathways were identified by RNA-sequencing of Padi4 KO neutrophils. Padi4 KO mice exhibited significant improvements in proteinuria progression compared with WT mice, whereas, serum anti-dsDNA antibody and immune complex deposition in the glomeruli showed no difference between both mice strains. Padi4 KO mice showed decreased neutrophil infiltration in the kidneys. Adoptively transferred Padi4 KO neutrophils showed decreased migration to the kidneys of IMQ-treated WT mice, and adhesion to ICAM-1 was impaired in Padi4 KO neutrophils. Padi4 KO neutrophils exhibited reduced upregulation of p38 mitogen-activated protein kinase (MAPK) pathways. Toll-like receptor 7 (TLR7)-primed Padi4 KO neutrophils demonstrated reduced phosphorylation of p38 MAPK and lower expression of JNK-associated leucine zipper protein (JLP), a p38 MAPK scaffold protein. Neutrophils from heterozygous Jlp KO mice showed impaired adhesion to ICAM-1 and decreased migration to the kidneys of IMQ-treated WT mice. These results indicated a pivotal role of PAD4-p38 MAPK pathway in renal neutrophil infiltration in TLR7 agonist-induced lupus nephritis, and the importance of neutrophil-mediated kidney inflammation. Inhibition of the PAD4-p38 MAPK pathway may help in formulating a novel therapeutic strategy against lupus nephritis.

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy.

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features.

Enhanced Human Gingival Fibroblast Response and Reduced Porphyromonas gingivalis Adhesion with Titania Nanotubes.

Successful dental implants rely on stable osseointegration and soft-tissue integration. Titania nanotubes (TNTs) with a diameter of 100 nm could increase the mesenchymal stem cell response and simultaneously decrease Staphylococcus aureus adhesion. However, the interactions between the modified surface and surrounding soft tissues are still unknown. In the present study, we fully investigated the biological behavior of human gingival fibroblasts (HGFs) and the adhesion of Porphyromonas gingivalis (P. gingivalis). TNTs were synthesized on titanium (Ti) surfaces by electrochemical anodization at 10, 30, and 60 V, and the products were denoted as NT10, NT30, and NT60, respectively. NT10 (diameter: 30 nm) and NT30 (diameter: 100 nm) could enhance the HGF functions, such as cell attachment and proliferation and extracellular matrix- (ECM-) related gene expressions, with the latter showing higher enhancement. NT60 (diameter: 200 nm) clearly impaired cell adhesion and proliferation and ECM-related gene expressions. Bacterial adhesion on the TNTs decreased and reached the lowest value on NT30. Therefore, NT30 without pharmaceuticals can be used to substantially enhance the HGF response and reduce P. gingivalis adhesion to the utmost, thus demonstrating significant potential in the transgingival part of dental implants.

Ppp2r2a Knockout Mice Reveal That Protein Phosphatase 2A Regulatory Subunit, PP2A-B55α, Is an Essential Regulator of Neuronal and Epidermal Embryonic Development.

The serine/threonine protein phosphatase 2A (PP2A) is a master regulator of the complex cellular signaling that occurs during all stages of mammalian development. PP2A is composed of a catalytic, a structural, and regulatory subunit, for which there are multiple isoforms. The association of specific regulatory subunits determines substrate specificity and localization of phosphatase activity, however, the precise role of each regulatory subunit in development is not known. Here we report the generation of the first knockout mouse for the Ppp2r2a gene, encoding the PP2A-B55α regulatory subunit, using CRISPR/Cas9. Heterozygous animals developed and grew as normal, however, homozygous knockout mice were not viable. Analysis of embryos at different developmental stages found a normal Mendelian ratio of Ppp2r2a–/– embryos at embryonic day (E) 10.5 (25%), but reduced Ppp2r2a–/– embryos at E14.5 (18%), and further reduced at E18.5 (10%). No live Ppp2r2a–/– pups were observed at birth. Ppp2r2a–/– embryos were significantly smaller than wild-type or heterozygous littermates and displayed a variety of neural defects such as exencephaly, spina bifida, and cranial vault collapse, as well as syndactyly and severe epidermal defects; all processes driven by growth and differentiation of the ectoderm. Ppp2r2a–/– embryos had incomplete epidermal barrier acquisition, associated with thin, poorly differentiated stratified epithelium with weak attachment to the underlying dermis. The basal keratinocytes in Ppp2r2a–/– embryos were highly disorganized, with reduced immunolabeling of integrins and basement membrane proteins, suggesting impaired focal adhesion and hemidesmosome assembly. The spinous and granular layers were thinner in the Ppp2r2a–/– embryos, with aberrant expression of adherens and tight junction associated proteins. The overlying stratum corneum was either absent or incomplete. Thus PP2A-B55α is an essential regulator of epidermal stratification, and is essential for ectodermal development during embryogenesis.

Proteome profile of neutrophils from a transgenic diabetic pig model shows distinct changes

INSC94Y transgenic pigs develop a stable diabetic phenotype early after birth and therefore allow studying the influence of hyperglycemia on primary immune cells in an early stage of diabetes mellitus in vivo. Since immune response is altered in diabetes mellitus, with deviant neutrophil function discussed as one of the possible causes in humans and mouse models, we investigated these immune cells in INSC94Y transgenic pigs and wild type controls at protein level. A total of 2371 proteins were quantified by label-free LC-MS/MS. Subsequent differential proteome analysis of transgenic animals and controls revealed clear differences in protein abundances, indicating a deviant behavior of granulocytes in the diabetic state. Interestingly, abundance of myosin regulatory light chain 9 (MLC-2C) was increased 5-fold in cells of diabetic pigs. MLC-2C directly affects cell contractility by regulating myosin ATPase activity, can act as transcription factor and was also associated with inflammation. It might contribute to impaired neutrophil cell adhesion, migration and phagocytosis.Our study provides novel insights into proteome changes in neutrophils from a large animal model for permanent neonatal diabetes mellitus and points to dysregulation of neutrophil function even in an early stage of this disease.Data are available via ProteomeXchange with identifier PXD017274. SignificanceOur studies provide novel basic information about the neutrophil proteome of pigs and contribute to a better understanding of molecular mechanisms involved in altered immune cell function in an early stage diabetes. We demonstrate proteins that are dysregulated in neutrophils from a transgenic diabetic pig and have not been described in this context so far. The data presented here are highly relevant for veterinary medicine and have translational quality for diabetes in humans.

Diabetic Serum Inhibits Osteoblast Adhesion to Titanium Surface Through Advanced Glycation End Products: An In Vitro Study.

Diabetes mellitus has been shown to delay osseointegration of titanium dental implants. This study tested the hypothesis that serum derived from diabetes negatively affects osteoblast adhesion to polystyrene and titanium surfaces, partly through the presence of advanced glycation end products (AGEs). Twenty-four Sprague-Dawley rats were divided into three groups: normoglycemic control, streptozotocin-induced diabetic group, and diabetic group treated with the AGE inhibitor aminoguanidine. Polystyrene or titanium disks were preincubated in serum derived from each group. Human osteoblasts transfected with green fluorescent protein (GFP) were cultured, and the number of adherent osteoblasts was quantified. High-pressure liquid chromatography (HPLC) was used to fractionate eluates, which were further characterized by western blot with AGE antibody and adhesion assays. In parallel, sera derived from healthy patients, patients with controlled diabetes, and patients with uncontrolled diabetes were utilized for osteoblast adhesion assay and western blot. Diabetic serum significantly reduced the number of adherent osteoblast and osteoblast aggregates on titanium disks, whereas aminoguanidine-treated serum rescued the effect of diabetes on the number of adherent osteoblast aggregates. Fractionated diabetic serum revealed distinct AGE bands at ~100 kDa and 44 kDa, whereas healthy serum did not express any. In human serum samples, both controlled and uncontrolled diabetes led to a significant reduction in the number of adherent osteoblasts on polystyrene and titanium surfaces compared with normoglycemic serum. This correlated with presence of AGEs in western blot in diabetic but not in healthy serum. Osteoblast adhesion on the titanium surface was greatly reduced by the exposure of serum derived from diabetic rats or humans. Recovery of osteoblast aggregates by aminoguanidine treatment suggests that AGEs played a role in this negative effect. The correlating presence of AGEs from the fractionated sera of diabetic rats or humans and impaired osteoblast adhesion on the titanium surface further supports this role.

TAK1 is a pivotal therapeutic target for tumor progression and bone destruction in myeloma.

Along with tumor progression, the bone marrow microenvironment is skewed in multiple myeloma (MM), which underlies the unique pathophysiology of MM and confers aggressiveness and drug resistance in MM cells. TGF-b-activated kinase-1 (TAK1) mediates a wide range of intracellular signaling pathways. We demonstrate here that TAK1 is constitutively overexpressed and phosphorylated in MM cells, and that TAK1 inhibition suppresses the activation of NF-κB, p38MAPK, ERK and STAT3 in order to decrease the expression of critical mediators for MM growth and survival, including PIM2, MYC, Mcl- 1, IRF4, and Sp1, along with a substantial reduction in the angiogenic factor VEGF in MM cells. Intriguingly, TAK1 phosphorylation was also induced along with upregulation of vascular cell adhesion molecule-1 (VCAM-1) in bone marrow stromal cells (BMSC) in cocultures with MM cells, which facilitated MM cell-BMSC adhesion while inducing IL-6 production and receptor activator of nuclear factor κ-B ligand (RANKL) expression by BMSC. TAK1 inhibition effectively impaired MM cell adhesion to BMSC to disrupt the support of MM cell growth and survival by BMSC. Furthermore, TAK1 inhibition suppressed osteoclastogenesis enhanced by RANKL in cocultures of bone marrow cells with MM cells, and restored osteoblastic differentiation suppressed by MM cells or inhibitory factors for osteoblastogenesis overproduced in MM. Finally, treatment with the TAK1 inhibitor LLZ1640-2 markedly suppressed MM tumor growth and prevented bone destruction and loss in mouse MM models. Therefore, TAK1 inhibition may be a promising therapeutic option targeting not only MM cells but also the skewed bone marrow microenvironment in MM.

Magnetic nanoparticles of Ga‐substituted ε‐Fe2O3 for biomedical applications: Magnetic properties, transverse relaxivity, and effects of silica‐coated particles on cytoskeletal networks

Magnetic nanoparticles of ε-Fe1.76 Ga0.24 O3 with the volume-weighted mean size of 17 nm were prepared by thermal treatment of a mesoporous silica template impregnated with metal nitrates and were coated with silica shell of four different thicknesses in the range 6-24 nm. The bare particles exhibited higher magnetization than the undoped compound, 22.4 Am2 kg-1 at 300 K, and were characterized by blocked state with the coercivity of 1.2 T at 300 K, being thus the very opposite of superparamagnetic iron oxides. The relaxometric study of the silica-coated samples at 0.47 T revealed promising properties for MRI, specifically, transverse relaxivity of 89-168 s-1 mmol(f.u.)-1 L depending on the shell thickness was observed. We investigated the effects of the silica-coated nanoparticles on human A549 and MCF-7 cells. Cell viability, proliferation, cell cycle distribution, and the arrangement of actin cytoskeleton were assessed, as well as formation and maturation of focal adhesions. Our study revealed that high concentrations of silica-coated particles with larger shell thicknesses of 16-24 nm interfere with the actin cytoskeletal networks, inducing thus morphological changes. Consequently, the focal adhesion areas were significantly decreased, resulting in impaired cell adhesion.

Impaired integrin α5 /β1 -mediated hepatocyte growth factor release by stellate cells of the aged liver.

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α5/β1 is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α5 and β1 decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α5 and β1 knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α5 and laminin gene expression whereas integrin β1 remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α5 knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α5/β1 in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.

Genetic deletion of microRNA biogenesis in muscle cells reveals a hierarchical non-clustered network that controls focal adhesion signaling during muscle regeneration

ObjectiveDecreased muscle mass is a major contributor to age-related morbidity, and strategies to improve muscle regeneration during ageing are urgently needed. Our aim was to identify the subset of relevant microRNAs (miRNAs) that partake in critical aspects of muscle cell differentiation, irrespective of computational predictions, genomic clustering or differential expression of the miRNAs. MethodsmiRNA biogenesis was deleted in primary myoblasts using a tamoxifen-inducible CreLox system and combined with an add-back miRNA library screen. RNA-seq experiments, cellular signalling events, and glycogen synthesis, along with miRNA inhibitors, were performed in human primary myoblasts. Muscle regeneration in young and aged mice was assessed using the cardiotoxin (CTX) model. ResultsWe identified a hierarchical non-clustered miRNA network consisting of highly (miR-29a), moderately (let-7) and mildly active (miR-125b, miR-199a, miR-221) miRNAs that cooperate by directly targeting members of the focal adhesion complex. Through RNA-seq experiments comparing single versus combinatorial inhibition of the miRNAs, we uncovered a fundamental feature of this network, that miRNA activity inversely correlates to miRNA cooperativity. During myoblast differentiation, combinatorial inhibition of the five miRNAs increased activation of focal adhesion kinase (FAK), AKT, and p38 mitogen-activated protein kinase (MAPK), and improved myotube formation and insulin-dependent glycogen synthesis. Moreover, antagonizing the miRNA network in vivo following CTX-induced muscle regeneration enhanced muscle mass and myofiber formation in young and aged mice. ConclusionOur results provide novel insights into the dynamics of miRNA cooperativity and identify a miRNA network as therapeutic target for impaired focal adhesion signalling and muscle regeneration during ageing.

Cofilin1-driven actin dynamics controls migration of thymocytes and is essential for positive selection in the thymus.

Actin dynamics is essential for T-cell development. We show here that cofilin1 is the key molecule for controlling actin filament turnover in this process. Mice with specific depletion of cofilin1 in thymocytes showed increased steady-state levels of actin filaments, and associated alterations in the pattern of thymocyte migration and adhesion. Our data suggest that cofilin1 is controlling oscillatory F-actin changes, a parameter that influences the migration pattern in a 3-D environment. In a collagen matrix, cofilin1 controls the speed and resting intervals of migrating thymocytes. Cofilin1 was not involved in thymocyte proliferation, cell survival, apoptosis or surface receptor trafficking. However, in cofilin1 mutant mice, impaired adhesion and migration resulted in a specific block of thymocyte differentiation from CD4/CD8 double-positive thymocytes towards CD4 and CD8 single-positive cells. Our data suggest that tuning of the dwelling time of thymocytes in the thymic niches is tightly controlled by cofilin1 and essential for positive selection during T-cell differentiation. We describe a novel role of cofilin1 in the physiological context of migration-dependent cell differentiation.

EBF1-deficient bone marrow stroma elicits persistent changes in HSC potential.

Crosstalk between mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) is essential for hematopoietic homeostasis and lineage output. Here, we investigate how transcriptional changes in bone marrow (BM) MSCs result in long-lasting effects on HSCs. Single-cell analysis of Cxcl12-abundant reticular (CAR) cells and PDGFRα+Sca1+ (PαS) cells revealed an extensive cellular heterogeneity but uniform expression of the transcription factor gene Ebf1. Conditional deletion of Ebf1 in these MSCs altered their cellular composition, chromatin structure and gene expression profiles, including the reduced expression of adhesion-related genes. Functionally, the stromal-specific Ebf1 inactivation results in impaired adhesion of HSCs, leading to reduced quiescence and diminished myeloid output. Most notably, HSCs residing in the Ebf1-deficient niche underwent changes in their cellular composition and chromatin structure that persist in serial transplantations. Thus, genetic alterations in the BM niche lead to long-term functional changes of HSCs.

The Heparin-Binding Hemagglutinin of Nocardia cyriacigeorgica GUH-2 Stimulates Inflammatory Cytokine Secretion Through Activation of Nuclear Factor κB and Mitogen-Activated Protein Kinase Pathways via TLR4.

Heparin-binding hemagglutinin (HBHA) from mycobacteria is involved in the dissemination of infection and the activation of the host immune response. However, the interaction of Nocardia cyriacigeorgica HBHA with the host cells remains unknown. In the present study, we describe N. cyriacigeorgica HBHA interactions with epithelial cells and organ colonization. We then investigate the mechanisms by which HBHA induces the production of inflammatory cytokines in macrophages. Immunofluorescent microscopy showed that HBHA adhered to A549 cells and HeLa cells and that the C-terminal fragment, which contains a Pro-Ala-Lys–rich domain, was responsible for adhesion. The deletion of the hbha gene in N. cyriacigeorgica mutant strains impaired adhesion to A549 cells and HeLa cells. In addition, the HBHA protein activated the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) signaling pathways and promoted the production of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-10 in macrophages. HBHA-mediated TNF-α production was dependent on the activation of the c-Jun N-terminal kinase (JNK) signal pathways, and the IL-6 and IL-10 production was dependent on the activation of extracellular regulated kinase (ERK) 1/2, MAPK p38 (p38), JNK, and nuclear NF-κB signaling pathways. Additionally, the HBHA-mediated activation of innate immunity was dependent on Toll-like receptor 4 (TLR4). Taken together, these results indicate that N. cyriacigeorgica HBHA not only adheres to epithelial cells and may be involved in organ colonization, but also plays a critical role in the modulation of innate immunity through the MAPK and NF-κB signaling pathways via TLR4.

Receptor recognition by meningococcal type IV pili relies on a specific complex N-glycan

Bacterial infections are frequently based on the binding of lectin-like adhesins to specific glycan determinants exposed on host cell receptors. These interactions confer species-specific recognition and tropism for particular host tissues and represent attractive antibacterial targets. However, the wide structural diversity of carbohydrates hampers the characterization of specific glycan determinants. Here, we characterized the receptor recognition of type IV pili (Tfp), a key adhesive factor present in numerous bacterial pathogens, using Neisseria meningitidis as a model organism. We found that meningococcal Tfp specifically recognize a triantennary sialylated poly-N-acetyllactosamine-containing N-glycan exposed on the human receptor CD147/Basigin, while fucosylated derivatives of this N-glycan impaired bacterial adhesion. Corroborating the inhibitory role of fucosylation on receptor recognition, adhesion of the meningococcus on nonhuman cells expressing human CD147 required prior defucosylation. These findings reveal the molecular basis of the selective receptor recognition by meningococcal Tfp and thereby, identify a potential antibacterial target.

Uncombable hair syndrome with loose anagen syndrome: A rare association

Uncombable hair syndrome presents with dry, coarse, silvery hairs that stand straight from the scalp surface. Loose anagen syndrome is characterized by easy pluckability of the hair due to impaired adhesion between the cuticle of the inner root sheath and the cuticle of the hair shaft. We report the case of a 7-year-old female who presented with features of both these conditions. She had difficulty in combing hair for 4 years. Her hairs did not grow and were dry and frizzy. Hairs were easily pluckable, and microscopy showed predominantly dystrophic anagen hair, with the shaft having undulations and longitudinal groove.

Recurrent corneal erosion: modern view of the problem

Recurrent corneal erosion syndrome (RCES) is a common chronic disease associated with repeated episodes of epithelial disruption and is characterized by sudden onset of sharp pain in the eye, usually at night or upon waking, as well as redness, photophobia and lacrimation. The cause of the disease is considered to be impaired adhesion of the epithelial layer to the stroma, which is a consequence of abnormal regeneration of the basement membrane of corneal epithelium. A number of biological factors, including inflammatory mediators and proteinases in high concentrations, contribute to the destruction of the formed adhesion complexes. The basis of RCES diagnosis is characteristic medical history and biomicroscopy. An area of epithelium absence can be detected under direct illumination - more often in paracentral view - mainly in the lower third of the cornea, where an area of loose and irregular epithelium with microcysts or grayish inclusions can be seen. There may also be a card-like or «fingerprint» pattern. First of all, it is necessary to differentiate RCES from infectious lesions, which require a fundamentally different treatment regimen. It is believed that the first stage of treatment should include lubricants, drugs that promote epithelialization, inhibitors of matrix metalloproteinases and lipases. The «second line» of conservative treatment can include blood derivatives. If conservative therapy is ineffective, it is advised to use phototherapeutic keratectomy (PTK) or perform diamond burr polishing of Bowman's layer. The choice of a method for treating recurrent erosion should be based on the effectiveness of drug therapy, frequency of recurrence, duration of the disease, severity of clinical symptoms, availability of specific ophthalmic equipment and experience with it.

Autoantibody-mediated desialylation impairs human thrombopoiesis and platelet lifespan.

Immune thrombocytopenia is a common bleeding disease caused by autoantibody-mediated accelerated platelet clearance and impaired thrombopoiesis. Accumulating evidence suggests that desialylation affects platelet lifespan in immune thrombocytopenia. Herein, we report on novel effector functions of autoantibodies from patients with immune thrombocytopenia, which might interfere with the clinical picture of the disease. Data from our study show that a subgroup of autoantibodies is able to induce cleavage of sialic acid residues from the surface of human platelets and megakaryocytes. Moreover, autoantibody-mediated desialylation interferes with the interaction between cells and extracellular matrix proteins leading to impaired platelet adhesion and megakaryocyte differentiation. Using a combination of an ex vivo model of thrombopoiesis, a humanized animal model, and a clinical cohort study, we demonstrate that cleavage of sialic acid induces significant impairment of the production, survival as well as function of human platelets. These data may indicate that prevention of desialylation should be investigated in the future in clinical studies as a potential therapeutic approach to treat bleeding in immune thrombocytopenia.

Biallelic loss-of-function mutations in JAM2 cause primary familial brain calcification.

Primary familial brain calcification is a monogenic disease characterized by bilateral calcifications in the basal ganglia and other brain regions, and commonly presents motor, psychiatric, and cognitive symptoms. Currently, four autosomal dominant (SLC20A2, PDGFRB, PDGFB, XPR1) and one autosomal recessive (MYORG) causative genes have been identified. Compared with patients with autosomal dominant primary familial brain calcification, patients with the recessive form of the disease present with more severe clinical and imaging phenotypes, and deserve more clinical and research attention. Biallelic mutations in MYORG cannot explain all autosomal recessive primary familial brain calcification cases, indicating the existence of novel autosomal recessive genes. Using homozygosity mapping and whole genome sequencing, we detected a homozygous frameshift mutation (c.140delT, p.L48*) in the JAM2 gene in a consanguineous family with two affected siblings diagnosed with primary familial brain calcification. Further genetic screening in a cohort of 398 probands detected a homozygous start codon mutation (c.1A>G, p.M1?) and compound heterozygous mutations [c.504G>C, p.W168C and c.(67+1_68-1)_(394+1_395-1), p.Y23_V131delinsL], respectively, in two unrelated families. The clinical phenotypes of the four patients included parkinsonism (3/4), dysarthria (3/4), seizures (1/4), and probable asymptomatic (1/4), with diverse onset ages. All patients presented with severe calcifications in the cortex in addition to extensive calcifications in multiple brain areas (lenticular nuclei, caudate nuclei, thalamus, cerebellar hemispheres, ± brainstem; total calcification scores: 43-77). JAM2 encodes junctional adhesion molecule 2, which is highly expressed in neurovascular unit-related cell types (endothelial cells and astrocytes) and is predominantly localized on the plasma membrane. It may be important in cell-cell adhesion and maintaining homeostasis in the CNS. In Chinese hamster ovary cells, truncated His-tagged JAM2 proteins were detected by western blot following transfection of p.Y23_V131delinsL mutant plasmid, while no protein was detected following transfection of p.L48* or p.1M? mutant plasmids. In immunofluorescence experiments, the p.W168C mutant JAM2 protein failed to translocate to the plasma membrane. We speculated that mutant JAM2 protein resulted in impaired cell-cell adhesion functions and reduced integrity of the neurovascular unit. This is similar to the mechanisms of other causative genes for primary familial brain calcification or brain calcification syndromes (e.g. PDGFRB, PDGFB, MYORG, JAM3, and OCLN), all of which are highly expressed and functionally important in the neurovascular unit. Our study identifies a novel causative gene for primary familial brain calcification, whose vital function and high expression in the neurovascular unit further supports impairment of the neurovascular unit as the root of primary familial brain calcification pathogenesis.

Autoantibody Mediated Desialylation Impairs Human Thrombopoiesis and Platelet Life Span

Background: The low platelet count in autoimmune thrombocytopenia (ITP) is caused by enhanced destruction of opsonised platelets in the spleen upon binding of the anti-platelet autoantibodies (AAbs) to the glycoproteins (GPs) express on PLT's surface. Data from animal model suggested that desialylation may contribute to PLT destruction in ITP. However, accumulating evidence suggests that reduction of PLT generation from megakaryocytes (MKs) in bone morrow is also responsible thrombocytopenia in ITP. Based on these considerations, we hypothesized that AAb-mediated desialylation of the GPs expressed on PLT and MKs may interfere with PLT formation and life span. Methods: Sera from 100 ITP patients were investigated in this study. AAb-induced desialylation was detected using a lectin binding assay (LBA) by flow cytometry (FC). To investigate the impact of desialylation on the life-span of human PLTs, the NSG mouse model was used. PLTs and MKs functions were assessed after AAb treatment using proplatelet formation test and adhesion assays on different surfaces. Results: Sera from 35/100 (35%) ITP patients induced cleavage of sialic acid from PLT surface. Injection of desialylating AAbs in vivo resulted in accelerated clearance of human PLTs which was significantly reduced by a specific sialidase inhibitor that prevents desialylation on the PLT surface (survival after 5h: 29%, range 22-40% vs. 48%, range 41-53%, p=0.014, respectively). Desialylating AAbs caused a significant reduction in PLT adhesion to fibrinogen and von Willebrand factor (mean of % adherent PLTs compared to control IgG: 34±6%, p=0.004 and 26±2%, p=0.001, respectively). Interestingly, PLT adhesion was recovered in the presence of a sialidase inhibitor (mean of % adherent PLTs: 86±6%, p=0.001 and 67±10, p=0.020, respectively). IgG fractions from 7/10 (70%) ITP-sera were able to cleave sialic acid and induce exposure of ß-galactose residues on CD34+-derived MKs. Desialylating AAbs induced lower ability to form proplatelet extensions compared to control IgG, which was significantly increased in the presence of the sialidase inhibitor (mean of % proplatelet forming MKs: 42±11% vs. 90±9%, p=0.032, respectively). Conclusion: Our findings show that AAbs from a subgroup of ITP patients are not only able to cleave sialic acid on surface of human PLTs, but also on MKs leading to accelerate PLT destruction and impaired thrombopoiesis, respectively. In addition, we observed that AAb-mediated receptor desialyation interferes with cell interaction with extracellular matrix proteins leading to impaired PLT adhesion, MK differentiation and thrombopoiesis. These novel findings highlight the multiple effects of AAbs in ITP and add to the existing evidence that ITP is rather a group of disorders sharing common characteristics, namely loss of immune tolerance toward PLT and MK antigens and increased bleeding tendency. Disclosures No relevant conflicts of interest to declare.

Neddylation Pathway Regulates Treg Differentiation and T Cell Function in Chronic Lymphocytic Leukemia (CLL) Ex Vivo and Murine In Vivo Studies

Introduction: Inhibitors of B-cell receptor associated kinases (e.g., Bruton tyrosine kinase and phoshpoinotiside-3 kinase) have led to significant improvement in outcomes of patients with CLL. Furthermore, such therapies have immunomodulatory effects. This is particularly relevant given that T cells from patients with CLL are functionally compromised and demonstrate impaired immune synapse formation, adhesion, migration and cytotoxicity. It is critical to improve understanding of the immunomodulatory properties of the novel agents as this will help understand their effect on the immune system in CLL, mitigate toxicities as well as inform future drug development. Pevonedistat, (MLN4924) forms an irreversible covalent adduct with NEDD8, a ubiquitin-like modifier, thereby inhibiting the NEDD8-activating enzyme (NAE). This leads to decreased neddylation and reduced activity of cullin-RING E3 ubiquitin ligases (CRLs). Ultimately, a decrease in CRL activity leads to reduced ubiquitination and proteasomal degradation of CRL substrates, extending the half-life of these proteins, including inhibitor of NFκB (IκB). We have shown that pevonedistat abrogates NFκB activation in CLL cells. Importantly, NFκB activation is indispensable in T-cell activation. However, there is paucity of data regarding the immune effects of targeting neddylation. Here we address this knowledge gap and demonstrate that NAE inhibition may have favorable immunomodulatory effects in CLL. Methods: Peripheral blood mononuclear cells were isolated from patients with CLL and T cells were purified using Dynabeads. Pevonedistat was provided by Millennium Pharmaceuticals, Inc. (Cambridge, MA). For gene expression analysis, FACS-sorted naïve CD4+ T cells were pre-treated with pevonedistat for 1 hour prior to T-cell receptor (TCR; αCD3/CD28) stimulation; RNA was harvested 3 or 24 hours after stimulation and analyzed on a Clariom S microarray chip. For polarization assays, FACS-sorted naïve CD4+ T cells were TCR-activated for 7 days under Th1/2/17/Treg-differentiation conditions. BALB/c mice were administered 60 mg/kg pevonedistat SC twice weekly for 3 weeks and T cell populations were analyzed by flow cytometry. Results: In vitro treatment of T cells with pevonedistat led to rapid reduction in neddylated cullins and stabilization of pIκBα. NAE inhibition did not impede proximal TCR signaling following TCR stimulation (pZAP70, pERK). GSEA demonstrated downmodulation of NFκB and IL-2 signaling pathways in pevonedistat-treated cells by 3 h. Despite this, CD4/CD8+ T cells exhibited normal induction of early activation markers (CD40L, CD69). By contrast, we observed reduced expression of CD38, HLA-DR and PD-1 and diminished CD25 following continuous treatment with pevonedistat for 72 h. This was accompanied by dose-dependent decrease in IL-2 secretion and reduced proliferation of the CD4/CD8+ T cell subsets (CFSE), but no apoptosis. Sorted naive T cells treated with pevonedistat in Th/Treg polarizing conditions exhibited an increase in IFNγ secretion and a decrease in IL-4, suggesting a shift toward Th1 phenotype. Furthermore, we observed a robust decrease of the inducible Treg (iTreg) FoxP3+ population. Loss of iTregs was accompanied by ablated IL-2/STAT5 signaling. Concurrently, we observed a modest increase in Th17 subpopulation following NAE inhibition. We found increased expression of HIF-1α, a CRL target, in pevonedistat-treated T cells, which may have contributed to this phenomenon. To mimic the clinical pharmacokinetics of pevonedistat, we performed 2 h pulse treatment with pevonedistat prior to TCR stimulation. Under these conditions NFκB activity fully recovered by 24 h. Importantly, allogeneic (OCI-LY19 cells) and autologous (CD40L-stimulated CLL cells) T-cell cytotoxicity, perforin and granzyme B production were not disrupted by NAE inhibition. In vivo administration of pevonedistat in immunocompetent BALB/c mice resulted in a decrease of Treg population, confirming in vitro data. Conclusions: Our data suggest that targeting neddylation may help rebalance T cells towards healthy immune subsets in CLL via the reduction of the Treg/Th2 phenotypes. Combined with our earlier reports that targeting NAE kills CLL cells under lymph node-mimicking conditions, these data provide a strong rationale for continued investigation of pevonedistat in CLL and lymphoid malignancies. Disclosures Berger: Millennium Pharmaceuticals, Inc., Cambridge, MA, a wholly owned subsidiary of Takeda Pharmaceutical Company Limited: Employment. Danilov:TG Therapeutics: Consultancy; Takeda Oncology: Research Funding; MEI: Research Funding; Verastem Oncology: Consultancy, Other: Travel Reimbursement , Research Funding; AstraZeneca: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Seattle Genetics: Consultancy; Gilead Sciences: Consultancy, Research Funding; Bristol-Meyers Squibb: Research Funding; Aptose Biosciences: Research Funding; Bayer Oncology: Consultancy, Research Funding; Celgene: Consultancy; Curis: Consultancy; Abbvie: Consultancy; Pharmacyclics: Consultancy; Janssen: Consultancy.