Deletion of Talin1 in Myeloid Cells Facilitates Atherosclerosis in Mice.

BACKGROUND: Integrin-regulated monocyte recruitment and cellular responses of monocyte-derived macrophages are critical for the pathogenesis of atherosclerosis. In the canonical model, talin1 controls ligand binding to integrins, a prerequisite for integrins to mediate leukocyte recruitment and induce immune responses. However, the role of talin1 in the development of atherosclerosis has not been studied. Our study investigated how talin1 in myeloid cells regulates the progression of atherosclerosis. METHODS: On an Apoe-/- background, myeloid talin1-deficient mice and the control mice were fed with a high-fat diet for 8 or 12 weeks to induce atherosclerosis. The atherosclerosis development in the aorta and monocyte recruitment into atherosclerotic lesions were analyzed. RESULTS: Myeloid talin1 deletion facilitated the formation of atherosclerotic lesions and macrophage deposition in lesions. Talin1 deletion abolished integrin ß2-mediated adhesion of monocytes but did not impair integrin α4ß1-dependent cell adhesion in a flow adhesion assay. Strikingly, talin1 deletion did not prevent Mn2+- or chemokine-induced activation of integrin α4ß1 to the high-affinity state for ligands. In an in vivo competitive homing assay, monocyte infiltration into inflamed tissues was prohibited by antibodies to integrin α4ß1 but was not affected by talin1 deletion or antibodies to integrin ß2. Furthermore, quantitative polymerase chain reaction and ELISA (enzyme-linked immunosorbent assay) analysis showed that macrophages produced cytokines to promote inflammation and the proliferation of smooth muscle cells. Ligand binding to integrin ß3 inhibited cytokine generation in macrophages, although talin1 deletion abolished the negative effects of integrin ß3. CONCLUSIONS: Integrin α4ß1 controls monocyte recruitment during atherosclerosis. Talin1 is dispensable for integrin α4ß1 activation to the high-affinity state and integrin α4ß1-mediated monocyte recruitment. Yet, talin1 is required for integrin ß3 to inhibit the production of inflammatory cytokines in macrophages. Thus, intact monocyte recruitment and elevated inflammatory responses cause enhanced atherosclerosis in talin1-deficient mice. Our study provides novel insights into the roles of myeloid talin1 and integrins in the progression of atherosclerosis.

Free fatty acids stabilize integrin ß1via S-nitrosylation to promote monocyte-endothelial adhesion.

Hyperlipidemia characterized by high blood levels of free fatty acids (FFAs) is important for the progression of inflammatory cardiovascular diseases. Integrin ß1 is a transmembrane receptor that drives various cellular functions, including differentiation, migration, and phagocytosis. However, the underlying mechanisms modifying integrin ß1 protein and activity in mediating monocyte/macrophage adhesion to endothelium remain poorly understood. In this study, we demonstrated that integrin ß1 protein underwent S-nitrosylation in response to nitrosative stress in macrophages. To examine the effect of elevated levels of FFA on the modulation of integrin ß1 expression, we treated the macrophages with a combination of oleic acid and palmitic acid (2:1) and found that FFA activated inducible nitric oxide synthase/nitric oxide and increased the integrin ß1 protein level without altering the mRNA level. FFA promoted integrin ß1 S-nitrosylation via inducible nitric oxide synthase/nitric oxide and prevented its degradation by decreasing binding to E3 ubiquitin ligase c-Cbl. Furthermore, we found that increased integrin α4ß1 heterodimerization resulted in monocyte/macrophage adhesion to endothelium. In conclusion, these results provided novel evidence that FFA-stimulated N--O stabilizes integrin ß1via S-nitrosylation, favoring integrin α4ß1 ligation to promote vascular inflammation.

Albumin-Binding Lutetium-177-Labeled LLP2A Derivatives as Theranostics for Melanoma.

Very late antigen-4 (VLA-4) is a transmembrane integrin protein that is highly expressed in aggressive forms of metastatic melanoma. A small-molecule peptidomimetic, LLP2A, was found to have a low pM affinity binding to VLA-4. Because LLP2A itself does not inhibit cancer cell proliferation and survival, it is an ideal candidate for the imaging and delivery of therapeutic payloads. An analog of [177Lu]Lu-labeled-LLP2A was previously investigated as a therapeutic agent in melanoma tumor-bearing mice, resulting in only a modest improvement in tumor growth inhibition, likely due to rapid clearance of the agent from the tumor. To improve the pharmacokinetic profile, DOTAGA-PEG4-LLP2A with a 4-(p-iodophenyl)butyric acid (pIBA) albumin binding moiety was synthesized. We demonstrate the feasibility of this albumin binding strategy by comparing in vitro cell binding assays and in vivo biodistribution performance of [177Lu]Lu-DOTAGA-PEG4-LLP2A ([177Lu]Lu-1) to the albumin binding [177Lu]Lu-DOTAGA-pIBA-PEG4-LLP2A ([177Lu]Lu-2). In vitro cell binding assay results for [177Lu]Lu-1 and [177Lu]Lu-2 showed Kd values of 0.40 ± 0.07 and 1.75 ± 0.40 nM, with similar Bmax values of 200 ± 6 and 315 ± 15 fmol/mg, respectively. In vivo biodistribution data for both tracers exhibited specific uptake in the tumor, spleen, thymus, and bone due to endogenous expression of VLA-4. Compound [177Lu]Lu-2 exhibited a much longer blood circulation time compared to [177Lu]Lu-1. The tumor uptake for [177Lu]Lu-1 was highest at 1 h (∼15%ID/g) and that for [177Lu]Lu-2 was highest at 4 h (∼23%ID/g). Significant clearance of [177Lu]Lu-1 from the tumor occurs at 24 h (<5%ID/g) while[177Lu]Lu-2 is retained for greater than 96 h (∼10%ID/g). An efficacy study showed that melanoma tumor-bearing mice receiving compound [177Lu]Lu-2 given in two fractions (2 × 14.8 MBq, 14 days apart) had a greater median survival time than mice administered a single 29.6 MBq dose of compound [177Lu]Lu-1, while a single 29.6 MBq dose of [177Lu]Lu-2 imparted hematopoietic toxicity. The in vitro and in vivo data show addition of pIBA to [177Lu]Lu-DOTAGA-PEG4-LLP2A slows blood clearance for a higher tumor uptake, and there is potential of [177Lu]Lu-2 as a theranostic in fractionated administered doses.

ATG8 inhibited endometriosis formation by regulating Treg cells differentiation via integrin α4ß1 and Talin-1 interaction.

RESEARCH QUESTION: What is the relationship between ATG8 and integrin α4ß1, Talin-1, and Treg cell differentiation, and the effects on endometriosis (EMS)? DESIGN: First, the correlation between the ATG8, Talin-1, integrin α4ß1, and differentiation of Treg cells and EMS was examined in clinical samples. Human peripheral blood mononuclear cells (PBMC) and endometrial stromal cells were extracted and identified, oe-ATG8 and oe-integrin α4ß1 were transfected to overexpress ATG8 and integrin α4ß1, and Tregs cell differentiation and endometrial stromal cells (ESC) function were detected. In addition, the molecular mechanism by which ATG8 inhibited EMS disease progression at the molecular and animal levels was investigated. RESULTS: ATG8 expression was negatively correlated with positive proportion of Tregs cells (P = 0.0463). The expression of Talin-1 and integrin-α4ß1 (both P < 0.0001) in PBMC decreased significantly after oe-ATG8 transfection, whereas the Treg cells' positive rate significantly increased (P = 0.0003). The ESC proliferation, adhesion, migration, and invasion (all P < 0.0001) declined after co-culture with Treg cells that underwent oe-ATG8 transfection. The expression of Talin-1 (P = 0.0025) and integrin-α4ß1 (P = 0.0002) in PBMC increased significantly after oe-integrin α4ß1 and oe-ATG8 transfection. In addition, this transfection reversed the corresponding regulation of oe-ATG8 transfection. Finally, animal experiments in vivo confirmed that ATG8 inhibited EMS disease progression. CONCLUSION: The ATG8 regulated Treg cell differentiation and inhibited EMS formation by influencing the interaction between integrin α4ß1 and Talin-1.

The main e-cigarette component vegetable glycerin enhances neutrophil migration and fibrosis in endotoxin-induced lung injury via p38 MAPK activation.

We investigated the effects of vegetable glycerin (VG), a main e-cigarette constituent, on endotoxin-induced acute lung injury (ALI). Mice received intratracheal administration of 30% VG in phosphate buffered saline (PBS) vehicle or only PBS (control) for 4 days. On Day 5, mice received an intratracheal instillation of lipopolysaccharide (LPS) (LPS group and VG + LPS group) or PBS (VG group and control group). Lung histopathology, expression of chemokine receptors, and regulatory signaling were analyzed 24 h after the Day 5 treatment. VG significantly increased ALI-associated histopathological and fibrotic changes in both the VG group and LPS-induced ALI mice (VG + LPS group). Immunohistochemistry (IHC) and western blot analyses revealed that VG administration resulted in upregulation of neutrophil markers [lymphocyte antigen 6 complex locus G6D (Ly6G) and myeloperoxidase (MPO)] as well as upregulation of the expression of transforming growth factor-ß (TGF-ß), a central mediator of fibrogenesis, in the lungs of both VG and VG + LPS groups. VG enhanced the expression of adhesion molecules [very late antigen 4 (VLA-4) and vascular cell adhesion molecule 1 (VCAM-1)] and increased activation of p38 mitogen-activated protein kinase (p38 MAPK) to prompt neutrophil recruitment in the lungs of mice with ALI. Intraperitoneal administration of a p38 inhibitor attenuated these histopathological changes significantly as well as VG-induced upregulation in expression of Ly6G, MPO, VLA-4, VCAM-1, TGF-ß, and collagen-1 in mice with ALI. In conclusion, VG enhances neutrophil chemotaxis and fibrosis and it amplifies the inflammatory response associated with LPS-induced ALI in the lungs via enhancement of p38 MAPK activity.

Activation of Protein Tyrosine Phosphatase Receptor Type γ Suppresses Mechanisms of Adhesion and Survival in Chronic Lymphocytic Leukemia Cells.

The regulatory role of protein tyrosine kinases in ß1- and ß2-integrin activation and in the survival of chronic lymphocytic leukemia (CLL) cells is well established. In contrast, the involvement of protein tyrosine phosphatases in CLL biology was less investigated. We show that selective activation of the protein tyrosine phosphatase receptor type γ (PTPRG) strongly suppresses integrin activation and survival in leukemic B cells isolated from patients with CLL. Activation of PTPRG specifically inhibits CXCR4- as well as BCR-induced triggering of LFA-1 and VLA-4 integrins and mediated rapid adhesion. Triggering of LFA-1 affinity is also prevented by PTPRG activity. Analysis of signaling mechanisms shows that activation of PTPRG blocks chemokine-induced triggering of JAK2 and Bruton's tyrosine kinase protein tyrosine kinases and of the small GTP-binding protein RhoA. Furthermore, activated PTPRG triggers rapid and robust caspase-3/7-mediated apoptosis in CLL cells in a manner quantitatively comparable to the Bruton's tyrosine kinase inhibitor ibrutinib. However, in contrast to ibrutinib, PTPRG-triggered apoptosis is insensitive to prosurvival signals generated by CXCR4 and BCR signaling. Importantly, PTPRG activation does not trigger apoptosis in healthy B lymphocytes. The data show that activated PTPRG inhibits, at once, the signaling pathways controlling adhesion and survival of CLL cells, thus emerging as a negative regulator of CLL pathogenesis. These findings suggest that pharmacological potentiation of PTPRG tyrosine-phosphatase enzymatic activity could represent a novel approach to CLL treatment.

Design, Pharmacological Characterization, and Molecular Docking of Minimalist Peptidomimetic Antagonists of α4ß1 Integrin.

Integrin receptors mediate cell-cell interactions via the recognition of cell-adhesion glycoproteins, as well as via the interactions of cells with proteins of the extracellular matrix, and upon activation they transduce signals bi-directionally across the cell membrane. In the case of injury, infection, or inflammation, integrins of ß2 and α4 families participate in the recruitment of leukocytes, a multi-step process initiated by the capturing of rolling leukocytes and terminated by their extravasation. In particular, α4ß1 integrin is deeply involved in leukocyte firm adhesion preceding extravasation. Besides its well-known role in inflammatory diseases, α4ß1 integrin is also involved in cancer, being expressed in various tumors and showing an important role in cancer formation and spreading. Hence, targeting this integrin represents an opportunity for the treatment of inflammatory disorders, some autoimmune diseases, and cancer. In this context, taking inspiration from the recognition motives of α4ß1 integrin with its natural ligands FN and VCAM-1, we designed minimalist α/ß hybrid peptide ligands, with our approach being associated with a retro strategy. These modifications are expected to improve the compounds' stability and bioavailability. As it turned out, some of the ligands were found to be antagonists, being able to inhibit the adhesion of integrin-expressing cells to plates coated with the natural ligands without inducing any conformational switch and any activation of intracellular signaling pathways. An original model structure of the receptor was generated using protein-protein docking to evaluate the bioactive conformations of the antagonists via molecular docking. Since the experimental structure of α4ß1 integrin is still unknown, the simulations might also shed light on the interactions between the receptor and its native protein ligands.

Soluble CD40L activates soluble and cell-surface integrin αvß3, α5ß1, and α4ß1 by binding to the allosteric ligand-binding site (site 2).

CD40L is a member of the TNF superfamily that participates in immune cell activation. It binds to and signals through several integrins, including αvß3 and α5ß1, which bind to the trimeric interface of CD40L. We previously showed that several integrin ligands can bind to the allosteric site (site 2), which is distinct from the classical ligand-binding site (site 1), raising the question of if CD40L activates integrins. In our explorations of this question, we determined that integrin α4ß1, which is prevalently expressed on the same CD4+ T cells as CD40L, is another receptor for CD40L. Soluble (s)CD40L activated soluble integrins αvß3, α5ß1, and α4ß1 in cell-free conditions, indicating that this activation does not require inside-out signaling. Moreover, sCD40L activated cell-surface integrins in CHO cells that do not express CD40. To learn more about the mechanism of binding, we determined that sCD40L bound to a cyclic peptide from site 2. Docking simulations predicted that the residues of CD40L that bind to site 2 are located outside of the CD40L trimer interface, at a site where four HIGM1 (hyper-IgM syndrome type 1) mutations are clustered. We tested the effect of these mutations, finding that the K143T and G144E mutants were the most defective in integrin activation, providing support that this region interacts with site 2. We propose that allosteric integrin activation by CD40L also plays a role in CD40L signaling, and defective site 2 binding may be related to the impaired CD40L signaling functions of these HIGM1 mutants.

VCAM1/VLA4 interaction mediates Ly6Clow monocyte recruitment to the brain in a TNFR signaling dependent manner during fungal infection.

Monocytes exist in two major populations, termed Ly6Chi and Ly6Clow monocytes. Compared to Ly6Chi monocytes, less is known about Ly6Clow monocyte recruitment and mechanisms involved in the recruitment of this subset. Furthermore, the role of Ly6Clow monocytes during infections is largely unknown. Here, using intravital microscopy, we demonstrate that Ly6Clow monocytes are predominantly recruited to the brain vasculature following intravenous infection with Cryptococcus neoformans, a fungal pathogen causing meningoencephalitis. The recruitment depends primarily on the interaction of VCAM1 expressed on the brain endothelium with VLA4 expressed on Ly6Clow monocytes. Furthermore, TNFR signaling is essential for the recruitment through enhancing VLA4 expression on Ly6Clow monocytes. Interestingly, the recruited Ly6Clow monocytes internalized C. neoformans and carried the organism while crawling on and adhering to the luminal wall of brain vasculature and migrating to the brain parenchyma. Our study reveals a substantial recruitment of Ly6Clow monocytes to the brain and highlights important properties of this subset during infection.

Visualizing γδ T cells by very late antigen-4-targeted positron emission tomography.

PURPOSE: γδ T cell-based immunotherapy has been rolled out as a promising treatment strategy for malignant tumors due to their potent anti-tumor cytotoxicity, ease of expansion, and unrestricted MHC feature. However, the dynamics and outcomes of γδ T cells in tumor sites are poorly understood. Reported strategies rely on ex vivo biolabeling, significantly limiting the application of γδ T cell molecular imaging. Herein, we investigated whether VLA-4 (very late antigen-4), a crucial component in the effective trafficking of lymphocytes, could serve as a biomarker to non-invasively visualize γδ T cells. METHODS: VLA-4-targeted tracer, 68 Ga-LLP2A, was evaluated in MDA-MB-231- and A549-bearing mice with adoptive transfer of γδ T cells by longitudinal PET/CT imaging. Imaging data were verified by ex vivo biodistribution studies, and the co-localization of CD3 and VLA-4 was validated by immunohistochemistry studies. RESULTS: 68 Ga-LLP2A showed high specificity to VLA-4-expressing γδ T cells in both in vitro and tumor-bearing mice with adoptive transfer of γδ T cells. Longitudinal PET imaging of 68 Ga-LLP2A in tumor-bearing mice with adoptive transfer of γδ T cells showed an increasing tumor tracer uptake, revealing the tumor-specific homing of γδ T cells. The presence of VLA-4-expressing γδ T cells in tumors was confirmed via histological analysis. CONCLUSION: To the best of our knowledge, we reported the first molecular probe, 68 Ga-LLP2A, for in vivo imaging of γδ T cells in live tumors, which advances PET imaging of γδ T cells and supports the translation of imaging agents for immunotherapeutic monitoring.

Elastin MIcrofibriL INterfacer1 (EMILIN-1) is an alternative prosurvival VLA-4 ligand in chronic lymphocytic leukemia.

CD49d, the α4 chain of the VLA-4 integrin, is a negative prognosticator in chronic lymphocytic leukemia (CLL) with a key role in CLL cell-microenvironment interactions mainly occurring via its ligands VCAM-1 and fibronectin. In the present study, we focused on EMILIN-1 (Elastin-MIcrofibriL-INterfacer-1), an alternative VLA-4 ligand whose role has been so far reported only in non-hematological settings, by investigating: i) the distribution of EMILIN-1 in CLL-involved tissues; ii) the capability of EMILIN-1 to operate, via its globular C1q (gC1q) domain, as additional adhesion ligand in CLL; iii) the functional meaning of EMILIN-1 gC1q/VLA-4 interactions in CLL. EMILIN-1 is widely present in the CLL-involved areas of bone marrow biopsies (BMBs) without difference between CD49d negative and positive cases, displaying at least three different expression patterns: "fibrillar", "dot-like" and "mixed". The lack in CLL-BMB of neutrophil elastase, whose proteolytic activity degrades EMILIN-1 and impairs EMILIN-1 function, suggests full functional EMILIN-1 in CLL independently of its expression pattern. Functionally, EMILIN-1 gC1q domain promotes adhesion of CLL cells through specific interaction with VLA-4, and releases pro-survival signals for CLL cells, as demonstrated by enhanced ERK and AKT phosphorylation and impairment of in-vitro-induced apoptosis. EMILIN-1/VLA-4 interaction can efficiently contribute to the maintenance of the neoplastic clone in CLL.

Increased Levels of VCAM-1 in Sera and VLA-4 Expression on Neutrophils in Dermatomyositis with Interstitial Lung Disease.

Vascular cell adhesion molecule-1 (VCAM-1) and its ligand very late antigen (VLA-4) play important roles in many autoimmune diseases. Our study aimed to investigate the serum level of VCAM-1 and VLA-4 expression on peripheral blood neutrophil surface in patients with dermatomyositis (DM), especially focusing on patients with interstitial lung disease (ILD). Blood specimens of 42 patients with DM and 42 healthy controls matched for age and gender were recruited. Total serum VCAM-1 level was measured using commercial enzyme-linked immunosorbent assay (ELISA) and the percentages of VLA-4 expression on neutrophils were analyzed by flow cytometry. We divided patients into subgroups according to whether they had ILD and whether they exhibited diffuse alveolar damage (DAD) via high-resolution computed tomography (HRCT). sVCAM-1 was increased in classical DM (cDM) and clinical amyopathic dermatomyositis (CADM) compared with healthy controls (both p < .01). DM-ILD had higher sVCAM-1 levels than the none-ILD group (p < .01). sVCAM-1 was also significantly increased in the DAD group compared to the none-DAD group (p < .01). The percentages of VLA-4 expression on neutrophils in cDM and CADM patients were significantly elevated than that in healthy controls (both p < .01). The percentage of VLA-4 expression on neutrophils in DM patients with ILD was higher than none-ILD group (p < .01). In the patients with ILD, DAD group had a higher percentage of VLA-4 expression on neutrophils than none-DAD group (p < .01). Our findings indicated that serum VCAM-1 levels combined with VLA-4 expression on neutrophils might be useful for detecting the severity of lung disease in patients with DM.

An Allosteric Shift in CD11c Affinity Activates a Proatherogenic State in Arrested Intermediate Monocytes.

Intermediate monocytes (iMo; CD14+CD16+) increase in number in the circulation of patients with unstable coronary artery disease (CAD), and their recruitment to inflamed arteries is implicated in events leading to mortality following MI. Monocyte recruitment to inflamed coronary arteries is initiated by high affinity ß2-integrin (CD11c/CD18) that activates ß1-integrin (VLA-4) to bind endothelial VCAM-1. How integrin binding under shear stress mechanosignals a functional shift in iMo toward an inflammatory phenotype associated with CAD progression is unknown. Whole blood samples from patients treated for symptomatic CAD including non-ST elevation MI, along with healthy age-matched subjects, were collected to assess chemokine and integrin receptor levels on monocytes. Recruitment on inflamed human aortic endothelium or rVCAM-1 under fluid shear stress was assessed using a microfluidic-based artery on a chip (A-Chip). Membrane upregulation of high affinity CD11c correlated with concomitant activation of VLA-4 within focal adhesive contacts was required for arrest and diapedesis across inflamed arterial endothelium to a greater extent in non-ST elevation MI compared with stable CAD patients. The subsequent conversion of CD11c from a high to low affinity state under fluid shear activated phospho-Syk- and ADAM17-mediated proteolytic cleavage of CD16. This marked the conversion of iMo to an inflammatory phenotype associated with nuclear translocation of NF-κB and production of IL-1ß+ We conclude that CD11c functions as a mechanoregulator that activates an inflammatory state preferentially in a majority of iMo from cardiac patients but not healthy patients.

Effector T-cell trafficking between the leptomeninges and the cerebrospinal fluid.

In multiple sclerosis, brain-reactive T cells invade the central nervous system (CNS) and induce a self-destructive inflammatory process. T-cell infiltrates are not only found within the parenchyma and the meninges, but also in the cerebrospinal fluid (CSF) that bathes the entire CNS tissue. How the T cells reach the CSF, their functionality, and whether they traffic between the CSF and other CNS compartments remains hypothetical. Here we show that effector T cells enter the CSF from the leptomeninges during Lewis rat experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. While moving through the three-dimensional leptomeningeal network of collagen fibres in a random Brownian walk, T cells were flushed from the surface by the flow of the CSF. The detached cells displayed significantly lower activation levels compared to T cells from the leptomeninges and CNS parenchyma. However, they did not represent a specialized non-pathogenic cellular sub-fraction, as their gene expression profile strongly resembled that of tissue-derived T cells and they fully retained their encephalitogenic potential. T-cell detachment from the leptomeninges was counteracted by integrins VLA-4 and LFA-1 binding to their respective ligands produced by resident macrophages. Chemokine signalling via CCR5/CXCR3 and antigenic stimulation of T cells in contact with the leptomeningeal macrophages enforced their adhesiveness. T cells floating in the CSF were able to reattach to the leptomeninges through steps reminiscent of vascular adhesion in CNS blood vessels, and invade the parenchyma. The molecular/cellular conditions for T-cell reattachment were the same as the requirements for detachment from the leptomeningeal milieu. Our data indicate that the leptomeninges represent a checkpoint at which activated T cells are licensed to enter the CNS parenchyma and non-activated T cells are preferentially released into the CSF, from where they can reach areas of antigen availability and tissue damage.

CC16 Binding to α4ß1 Integrin Protects against Mycoplasma pneumoniae Infection.

Rationale CC16 (club cell secretory protein) is a pneumoprotein produced predominantly by pulmonary club cells. Circulating CC16 is associated with protection from the inception and progression of the two most common obstructive lung diseases (asthma and chronic obstructive pulmonary disease). Objectives Although exact mechanisms remain elusive, studies consistently suggest a causal role of CC16 in mediating antiinflammatory and antioxidant functions in the lung. We sought to determine any novel receptor systems that could participate in CC16's role in obstructive lung diseases. Methods Protein alignment of CC16 across species led to the discovery of a highly conserved sequence of amino acids, leucine-valine-aspartic acid (LVD), a known integrin-binding motif. Recombinant CC16 was generated with and without the putative integrin-binding site. A Mycoplasma pneumoniae mouse model and a fluorescent cellular adhesion assay were used to determine the impact of the LVD site regarding CC16 function during live infection and on cellular adhesion during inflammatory conditions. Measurements and Main Results CC16 bound to integrin α4ß1), also known as the adhesion molecule VLA-4 (very late antigen 4), dependent on the presence of the LVD integrin-binding motif. During infection, recombinant CC16 rescued lung function parameters both when administered to the lung and intravenously but only when the LVD integrin-binding site was intact; likewise, neutrophil recruitment during infection and leukocyte adhesion were both impacted by the loss of the LVD site. Conclusions We discovered a novel receptor for CC16, VLA-4, which has important mechanistic implications for the role of CC16 in circulation as well as in the lung compartment.

Neutrophils promote VLA-4-dependent B cell antigen presentation and accumulation within the meninges during neuroinflammation.

The success of B cell depletion therapies and identification of leptomeningeal ectopic lymphoid tissue (ELT) in patients with multiple sclerosis (MS) has renewed interest in the antibody-independent pathogenic functions of B cells during neuroinflammation. The timing and location of B cell antigen presentation during MS and its animal model experimental autoimmune encephalomyelitis (EAE) remain undefined. Using a new EAE system that incorporates temporal regulation of MHCII expression by myelin-specific B cells, we observed the rapid formation of large B cell clusters in the spinal cord subarachnoid space. Neutrophils preceded the accumulation of meningeal B cell clusters, and inhibition of CXCR2-mediated granulocyte trafficking to the central nervous system reduced pathogenic B cell clusters and disease severity. Further, B cell-restricted very late antigen-4 (VLA-4) deficiency abrogated EAE dependent on B cell antigen presentation. Together, our findings demonstrate that neutrophils coordinate VLA-4-dependent B cell accumulation within the meninges during neuroinflammation, a key early step in the formation of ELT observed in MS.

Truncated O-Glycan-Bearing MUC16 Enhances Pancreatic Cancer Cells Aggressiveness via α4ß1 Integrin Complexes and FAK Signaling.

Elevated levels of Mucin-16 (MUC16) in conjunction with a high expression of truncated O-glycans is implicated in playing crucial roles in the malignancy of pancreatic ductal adenocarcinoma (PDAC). However, the mechanisms by which such aberrant glycoforms present on MUC16 itself promote an increased disease burden in PDAC are yet to be elucidated. This study demonstrates that the CRISPR/Cas9-mediated genetic deletion of MUC16 in PDAC cells decreases tumor cell migration. We found that MUC16 enhances tumor malignancy by activating the integrin-linked kinase and focal adhesion kinase (ILK/FAK)-signaling axis. These findings are especially noteworthy in truncated O-glycan (Tn and STn antigen)-expressing PDAC cells. Activation of these oncogenic-signaling pathways resulted in part from interactions between MUC16 and integrin complexes (α4ß1), which showed a stronger association with aberrant glycoforms of MUC16. Using a monoclonal antibody to functionally hinder MUC16 significantly reduced the migratory cascades in our model. Together, these findings suggest that truncated O-glycan containing MUC16 exacerbates malignancy in PDAC by activating FAK signaling through specific interactions with α4 and ß1 integrin complexes on cancer cell membranes. Targeting these aberrant glycoforms of MUC16 can aid in the development of a novel platform to study and treat metastatic pancreatic cancer.

Intravenous infusion of iPSC-derived neural precursor cells increases acid ß-glucosidase function in the brain and lessens the neuronopathic phenotype in a mouse model of Gaucher disease.

Gaucher disease (GD) is caused by GBA1 mutations leading to functional deficiency of acid-ß-glucosidase (GCase). No effective treatment is available for neuronopathic GD (nGD). A subclass of neural stem and precursor cells (NPCs) expresses VLA4 (integrin α4ß1, very late antigen-4) that facilitates NPC entry into the brain following intravenous (IV) infusion. Here, the therapeutic potential of IV VLA4+NPCs was assessed for nGD using wild-type mouse green fluorescent protein (GFP)-positive multipotent induced pluripotent stem cell (iPSC)-derived VLA4+NPCs. VLA4+NPCs successfully engrafted in the nGD (4L;C*) mouse brain. GFP-positive cells differentiated into neurons, astrocytes and oligodendrocytes in the brainstem, midbrain and thalamus of the transplanted mice and significantly improved sensorimotor function and prolonged life span compared to vehicle-treated 4L;C* mice. VLA4+NPC transplantation significantly decreased levels of CD68 and glial fibrillary acidic protein, as well as TNFα mRNA levels in the brain, indicating reduced neuroinflammation. Furthermore, decreased Fluoro-Jade C and NeuroSilver staining suggested inhibition of neurodegeneration. VLA4+NPC-engrafted 4L;C* midbrains showed 35% increased GCase activity, reduced substrate [glucosylceramide (GC, -34%) and glucosylsphingosine (GS, -11%)] levels and improved mitochondrial oxygen consumption rates in comparison to vehicle-4L;C* mice. VLA4+NPC engraftment in 4L;C* brain also led to enhanced expression of neurotrophic factors that have roles in neuronal survival and the promotion of neurogenesis. This study provides evidence that iPSC-derived NPC transplantation has efficacy in an nGD mouse model and provides proof of concept for autologous NPC therapy in nGD.

Small-molecule antagonist of VLA-4 (GW559090) attenuated neuro-inflammation by targeting Th17 cell trafficking across the blood-retinal barrier in experimental autoimmune uveitis.

BACKGROUND: The integrin VLA-4 (α4ß1) plays an important role in leukocyte trafficking. This study investigated the efficacy of a novel topical α4ß1 integrin inhibitor (GW559090, GW) in a mouse model for non-infectious posterior uveitis (experimental autoimmune uveitis; EAU) and its effect on intraocular leukocyte subsets. METHODS: Mice (female; B10.RIII or C57Bl/6; aged 6-8 weeks) were immunized with specific interphotoreceptor retinoid-binding protein (IRBP) peptides to induce EAU. Topically administered GW (3, 10, and 30 mg/ml) were given twice daily either therapeutically once disease was evident, or prophylactically, and compared with vehicle-treated (Veh) and 0.1% dexamethasone-treated (Dex) controls. Mice were sacrificed at peak disease. The retinal T cell subsets were investigated by immunohistochemistry and immunofluorescence staining. The immune cells within the retina, blood, and draining lymph nodes (dLNs) were phenotyped by flow cytometry. The effect of GW559090 on non-adherent, adherent, and migrated CD4+ T cell subsets across a central nervous system (CNS) endothelium was further assayed in vitro and quantitated by flow cytometry. RESULTS: There was a significant reduction in clinical and histological scores in GW10- and Dex-treated groups as compared to controls either administered therapeutically or prophylactically. There were fewer CD45+ leukocytes infiltrating the retinae and vitreous fluids in the treated GW10 group (P < 0.05). Immunofluorescence staining and flow cytometry data identified decreased levels of retinal Th17 cells (P ≤ 0.001) in the GW10-treated eyes, leaving systemic T cell subsets unaffected. In addition, fewer Ly6C+ inflammatory monocyte/macrophages (P = 0.002) and dendritic cells (P = 0.017) crossed the BRB following GW10 treatment. In vitro migration assays confirmed that Th17 cells were selectively suppressed by GW559090 in adhering to endothelial monolayers. CONCLUSIONS: This α4ß1 integrin inhibitor may exert a modulatory effect in EAU progression by selectively blocking Th17 cell migration across the blood-retinal barrier without affecting systemic CD4+ T cell subsets. Local α4ß1 integrin-directed inhibition could be clinically relevant in treating a Th17-dominant form of uveitis.

The tellurium-based immunomodulator, AS101 ameliorates adjuvant-induced arthritis in rats.

Despite undeniable improvement in the management of rheumatoid arthritis (RA), the discovery of more effective, less toxic and, ideally, less immune suppressive drugs are much needed. In the current study, we set to explore the potential anti-rheumatic activity of the non-toxic, tellurium-based immunomodulator, AS101 in an experimental animal model of RA. The effect of AS101 was assessed on adjuvant-induced arthritis (AIA) rats. Clinical signs of arthritis were assessed. Histopathological examination was used to assess inflammation, synovial changes and tissue lesions. Very late antigen-4 (VLA-4)+ cellular infiltration was detected using immunohistochemical staining. Enzyme-linked immunosorbent assay (ELISA) was used to measure circulating anti-cyclic citrullinated-peptide autoantibody (ACPA) and real-time polymerase chain reaction (PCR) was used to measure the in-vitro effect of AS101 on interleukin (IL)-6 and IL-1ß expression in activated primary human fibroblasts. Prophylactic treatment with intraperitoneal AS101 reduced clinical arthritis scores in AIA rats (P < 0·01). AS101 abrogated the migration of active chronic inflammatory immune cells, particularly VLA-4+ cells, into joint cartilage and synovium, reduced the extent of joint damage and preserved joint architecture. Compared to phosphate-buffered saline (PBS)-treated AIA rats, histopathological inflammatory scores were significantly reduced (P < 0·05). Furthermore, AS101 resulted in a marked reduction of circulating ACPA in comparison to PBS-treated rats (P < 0·05). Importantly, AS101 significantly reduced mRNA levels of proinflammatory mediators such as IL-6 (P < 0·05) and IL-1ß (P < 0·01) in activated primary human fibroblasts. Taken together, we report the first demonstration of the anti-rheumatic/inflammatory activity of AS101 in experimental RA model, thereby supporting an alternative early therapeutic intervention and identifying a promising agent for therapeutic intervention.