Pancreatic stellate cells: the top managers of the pancreatic tumor microenvironment.

Background Stellate pancreatocytes, being cells - producers of stromal components, actively interact with cancer cells, determine the formation of a stromal barrier between the latter and thereby provide tumor chemoresistance. Objective The review is devoted to the analysis of recent data on the role of stellate pancreatocytes in the formation of the stromal microenvironment of pancreatic tumors, molecular mechanisms through which the regulation and realization of stellate cell functions is carried out. Methods Data processing was carried out by the method of complex material analysis. Results. Stellate pancreatocytes (PSC) exhibit phenotypically and functionally two states: inactive and active. PSC activation is carried out by cells of the developing tumor through a variety of molecular mediators. Activation triggers for PSC are Yes-associated protein, TGF-β1, miRNA let-7d, IL-8, MCP1, TGF-β2, IGFBP2, and others. 10 actively expressed genes were identified: TP53, SRC, IL6, JUN, ISG15, CAD, STAT1, OAS3, OAS1, VIM during co-cultivation of a cancer cell line (PCC) with PSC. PSC deactivation is associated with speckle-type mediator POZ (SPOP) acting through nuclear factor-kappaB, transretinoic acid (ATRA). Exhibiting their activity, PSCs express several stem cell markers, α-SMA (α-actin of smooth muscle cells), vimentin, α ITGA 11 (collagen type I receptor), α5 integrin receptor ITGA5 (fibronectin receptor), hyaluronic acid, hyaluronan synthase 2 (HAS2), hyaluronidase 1 (HYAL1), BAG3 , matrix metallopeptidase 2 (MMP2), Nodal protein, miR-1246 and miR-1290, miR-210, CCN2 (connective tissue growth factor), TRPV1, SP and CGRP (Calcitonin gene-related peptide) and many other factors. Сonclusion. Stellate pancreatocytes, being producers of the interacinar stroma, are activated by various factors (TNF-α, IL-6, MCP-1, ATP, and HMGB1, etc.), including factors produced by tumor cells of the pancreas, and act as regulators of proliferation, migration, and suppression apoptosis of the latter. An increase in the expression of α ITGA 11 (type I collagen receptor), α5 integrin receptor ITGA5 (fibronectin receptor), metallopeptidases, Nodal protein, miR-1246, miR-1290, and miR-210 is observed in tumor tissue, that indicates the activation of these cells. The maintenance of the active state of PSC is provided by tumor cells, for which stellate pancreatocytes are partners in the progression of the neoplastic process. Further study of the mechanisms of interaction in the PSC-tumor cell system creates the prospect of revealing levers of influence on the pathogenesis of pancreatic tumors.

An alternate covalent form of platelet αIIbβ3 integrin that resides in focal adhesions and has altered function

The αIIbβ3 integrin receptor coordinates platelet adhesion, activation and mechanosensing in thrombosis and haemostasis. Using differential cysteine alkylation and mass spectrometry, we have identified a disulfide bond in the αIIb subunit linking cysteines 490 and 545 that is missing in about one in three integrin molecules on the resting and activated human platelet surface. This alternate covalent form of αIIbβ3 is pre-determined as it is also produced by human megakaryoblasts and baby hamster kidney fibroblasts (BHK) transfected with recombinant integrin. From co-immunoprecipitation experiments, the alternate form selectively partitions into focal adhesions on the activated platelet surface. Its function was evaluated in BHK cells expressing a mutant integrin with an ablated C490-C545 disulfide bond. The disulfide mutant integrin has functional outside-in signalling but extended residency time in focal adhesions due to reduced rate of clathrin-mediated integrin internalisation and recycling, which is associated with enhanced affinity of the αIIb subunit for clathrin adaptor protein-2. Molecular dynamics simulations indicate that the alternate covalent form of αIIb requires higher forces to transition from bent to open conformational states that is in accordance with reduced affinity for fibrinogen and activation by manganese ions. These findings indicate that the αIIbβ3 integrin receptor is produced in different covalent forms that have different cell surface distribution and function. The C490, C545 cysteine pair is conserved across all 18 integrin α subunits and the disulfide bond in the αV and α2 subunits in cultured cells is similarly missing, suggesting that this alternate integrin form and function is also conserved.

Structure of the integrin receptor M2 headpiece in complex with a function-modulating nanobody

The integrin receptor M2 mediates phagocytosis of complement-opsonized objects, adhesion to the extracellular matrix and trans-endothelial migration of leukocytes. Here we present the first atomic structure of the human M2 headpiece fragment in complex with the nanobody hCD11bNb1 determined at a resolution of 3.2 Å. The receptor headpiece adopts the closed conformation expected to have low ligand affinity. The crystal structure advocates that in the R77H M variant associated with systemic lupus erythematosus, the modified allosteric coupling between ligand coupling and integrin outside-inside signalling is due to subtle conformational effects transmitted over 40 Å. The nanobody binds to the I domain of the M subunit in an Mg2+ independent manner with low nanomolar affinity. Biochemical and biophysical experiments with purified proteins argue that the nanobody acts as a competitive inhibitor through steric hindrance exerted on the thioester domain of iC3b attempting to bind the M subunit. Surprisingly, the nanobody stimulates the interaction of cell-bound M2 with iC3b suggesting that it represents a novel high-affinity proteinaceous M2 specific agonist. We propose a model based on the conformational spectrum of the receptor to reconcile these conflicting observations regarding the functional consequences of hCD11bNb1 binding to M2. Furthermore, our data suggest that the iC3b-M2 complex may be more dynamic than predicted from the crystal structure of the core complex.

Pericyte-derived vitronectin regulates blood-CNS barrier function via integrin signaling

Blood-central nervous system (CNS) barriers are physiological interfaces separating the neural tissue from circulating blood and are essential for neuronal function and cellular homeostasis. Endothelial cells that form the walls of CNS blood vessels constitute these barriers but barrier properties are not intrinsic to these cells; rather they are actively induced and maintained by the surrounding CNS microenvironment. Notably, the abluminal surface of CNS capillary endothelial cells is ensheathed by pericytes and astrocytic endfeet. However, the specific extrinsic factors from these perivascular cells that regulate barrier integrity are largely unknown. Here, we establish vitronectin, an extracellular matrix protein secreted specifically by CNS pericytes as an essential factor in regulating blood-CNS barrier function via interactions with its integrin receptor in adjacent endothelial cells. Genetic ablation of vitronectin results in leaky blood-CNS barriers, despite having normal pericyte coverage and vascular patterning. Electron microscopy reveals increased transcytosis in endothelial cells of Vtn−/− mice without functional defects in tight-junctions. We further demonstrate that vitronectin binding to integrin receptors is essential for barrier function, as mice harboring a point mutation in vitronectin that specifically abolishes integrin binding, VtnRGE, phenocopy the barrier defects in Vtn−/− mice. Furthermore, endothelial-specific deletion of integrin α5, an RGD-ligand binding integrin receptor that is expressed in CNS endothelial cells, also results in similar blood-CNS barrier defects as observed in Vtn−/− and VtnRGE mice. Finally, integrin α5 activation by vitronectin inhibits transcytosis in endothelial cells and vitronectin-integrin α5 signaling regulates barrier function independent of the caveolae pathway. These results demonstrate that signaling from perivascular cells to endothelial cells via ligand-receptor interactions is a key mechanism to regulate barrier permeability.SummaryVitronectin-integrin signaling between pericytes and CNS endothelial cells regulates blood-CNS barrier function

Natalizumab Pharmacokinetics and -Dynamics and Serum Neurofilament in Patients With Multiple Sclerosis

Background: Natalizumab (NAT) is a high-efficacy treatment for relapsing remitting multiple sclerosis (RRMS). However, it is associated with an increased risk of progressive multifocal leukoencephalopathy that sometimes requires treatment cessation with a risk of returning disease activity. The aim of this study was to characterize the pharmacokinetics and -dynamics as well as neurodestruction marker serum neurofilament light chain (sNfL) in patients with RRMS and secondary progressive MS (SPMS) stopping NAT in correlation to clinical data.Methods: In this study, 50 RRMS and 9 SPMS patients after NAT cessation were included. Five RRMS patients on NAT treatment holiday were evaluated. Clinical and radiological disease activity were systemically assessed by frequent exams after NAT stop. Free NAT concentration, cell bound NAT, α4-integrin expression and α4-integrin-receptor saturation as well as immune cell frequencies were measured for up to 4 months after NAT withdrawal. Additionally, sNfL levels were observed up to 12 months in RRMS and up to 4 months in SPMS patients.Results: NAT cessation was associated with a return of disease activity in 38% of the RRMS and 33% of the SPMS patients within 12 and 7 months, respectively. Concentration of free and cell bound NAT as well as α4-integrin-receptor saturation decreased in the RRMS and SPMS patients whereas α4-integrin expression increased over time. NAT induced increase of lymphocytes and its subsets normalized and a non-significant drop of NK and Th17 T-cells counts could be detected. All RRMS patients showed physiological sNfL levels <8pg/ml 1 month after last NAT infusion. During follow-up period sNfL levels peaked up to 16-fold and were linked to return of disease activity in 19 of the 37 RRMS patients. Treatment holiday was also associated with a return of disease activity in 4 of 5 patients and with an increase of sNfL at an individual level.Conclusions: We demonstrate the reversibility of NAT pharmacodynamic and -kinetic markers. sNfL levels are associated with the recurrence of disease activity and can also serve as an early marker to predict present before onset of clinical or radiological disease activity on the individual level.

Mycobacterium tuberculosis PPiA interacts with host integrin receptor to exacerbate disease progression

Attenuated intracellular survival of Mycobacterium tuberculosis (Mtb) secretory gene mutants exemplifies their role as virulence factors. Mtb peptidyl prolyl isomerase A (PPiA) assists in protein folding through cis/trans isomerization of prolyl bonds. Here, we show that PPiA abets Mtb survival and aids in the disease progression by exploiting host-associated factors. While the deletion of PPiA has no discernable effect on the bacillary survival in a murine infection model, it compromises the formation of granuloma-like lesions and promotes host cell death through ferroptosis. Overexpression of PPiA enhances the bacillary load and exacerbates pathology in mice lungs. Importantly, PPiA interacts with the integrin α5β1 receptor through a conserved surface-exposed RGD motif. The secretion of PPiA as well as interaction with integrin contributes to the disease progression by upregulating multiple host matrix metalloproteinases. Collectively, we identified a novel non-chaperone role of PPiA that is critical in facilitating host-pathogen interaction ensuing disease progression.

Fibrin is a critical regulator of neutrophil effector function at mucosal barrier sites

Tissue-specific cues are critical for homeostasis at mucosal barriers. Here, we document that the clotting factor fibrin is a critical regulator of neutrophil function at mucosal barriers. We demonstrate that fibrin engages neutrophils through the αMβ2 integrin receptor and activates effector functions, including the production of reactive oxygen species and NET formation. These immune-protective neutrophil functions become tissue damaging in the context of impaired plasmin-mediated fibrinolysis. Indeed, the accumulation of fibrin due to Mendelian genetic defects in plasmin leads to severe oral mucosal immunopathology in mice and humans. Concordantly, genetic polymorphisms in the human PLG gene, encoding plasminogen, are associated with common forms of the oral mucosal disease periodontitis. Our work uncovers fibrin as a critical regulator of neutrophil effector function within the mucosal tissue microenvironment and suggests fibrin-neutrophil engagement as a pathogenic instigator and therapeutic target in common mucosal disease.