Type I Interferon Signaling Increases Versican Expression and Synthesis in Lung Stromal Cells During Influenza Infection.

Versican, a chondroitin sulfate proteoglycan, is an essential component of the extracellular matrix (ECM) in inflammatory lung disease. Versican's potential as an immunomodulatory molecule makes it a promising therapeutic target for controlling host immune responses in the lungs. To establish changes to versican expression and accumulation during influenza A viral pneumonia, we document the temporal and spatial changes to versican mRNA and protein in concert with pulmonary inflammatory cell infiltration. These studies were performed in the lungs of wild-type C57BL6/J mice on days 3, 6, 9, and 12 post-infection with influenza A virus using immunohistochemistry, in situ hybridization, and quantitative digital pathology. Using duplex in situ hybridization, we demonstrate that type I interferon signaling contributes significantly to versican expression in lung stromal cells. Our findings show that versican is a type I interferon-stimulated gene in pulmonary fibroblasts and pericytes in the context of viral pneumonia. These data also provide a guide for future studies to determine the role of versican in the pulmonary immune response to influenza infection.

An anti-ADAMTS1 treatment relieved muscle dysfunction and fibrosis in dystrophic mice.

Duchenne Muscular Dystrophy (DMD) is caused by mutations in the dystrophin gene, accompanied by aberrant extracellular matrix synthesis and muscle damage. ADAMTS1 metalloproteinase was reported increased in dystrophin-deficient mdx mouse. The aim of this study was to explore the role of ADAMTS1 in muscle function, fibrosis and damage, and respiratory function of mdx mice. 102 DMD patients and their mothers were included in this study. Multiplex ligation dependent probe amplification (MLPA) assay and Next-generation sequencing (NGS) were adopted to do genetic diagnosis. Dystrophin-deficient mdx mice were treated with anti-ADAMTS1 antibody (anti-ADAMTS1) for three weeks. The results showed that ADAMTS1 was increased in gastrocnemius muscle of mdx mice and serum of DMD patients. Anti-ADAMTS1 treatment increased Versican transcription but suppressed versican protein expression. Besides, we found anti-ADAMTS1 improved muscle strength, diaphragm and extensor digitorum longus muscles functions in mdx mice. Meanwhile, muscle fibrosis and damage were attenuated in anti-ADAMTS1 treated dystrophic mice. In summary, anti-ADAMTS1 antibody relieved muscle dysfunction and fibrosis in dystrophic mice. It is suggested that ADAMTS1 is a potential target for developing new biological therapies for DMD.

Serum amyloid A-containing HDL binds adipocyte-derived versican and macrophage-derived biglycan, reducing its antiinflammatory properties.

The ability of HDL to inhibit inflammation in adipocytes and adipose tissue is reduced when HDL contains serum amyloid A (SAA) that is trapped by proteoglycans at the adipocyte surface. Because we recently found that the major extracellular matrix proteoglycan produced by hypertrophic adipocytes is versican, whereas activated adipose tissue macrophages produce mainly biglycan, we further investigated the role of proteoglycans in determining the antiinflammatory properties of HDL. The distributions of versican, biglycan, apolipoprotein A1 (the major apolipoprotein of HDL), and SAA were similar in adipose tissue from obese mice and obese human subjects. Colocalization of SAA-enriched HDL with versican and biglycan at the cell surface of adipocyte and peritoneal macrophages, respectively, was blocked by silencing these proteoglycans, which also restored the antiinflammatory property of SAA-enriched HDL despite the presence of SAA. Similar to adipocytes, normal HDL exerted its antiinflammatory function in macrophages by reducing lipid rafts, reactive oxygen species generation, and translocation of Toll-like receptor 4 and NADPH oxidase 2 into lipid rafts, effects that were not observed with SAA-enriched HDL. These findings imply that SAA present in HDL can be trapped by adipocyte-derived versican and macrophage-derived biglycan, thereby blunting HDL's antiinflammatory properties.

Binding of myeloperoxidase to the extracellular matrix of smooth muscle cells and subsequent matrix modification.

The extracellular matrix (ECM) of tissues is susceptible to modification by inflammation-associated oxidants. Considerable data support a role for hypochlorous acid (HOCl), generated by the leukocyte-derived heme-protein myeloperoxidase (MPO) in these changes. HOCl can modify isolated ECM proteins and cell-derived matrix, with this resulting in decreased cell adhesion, modulated proliferation and gene expression, and phenotypic changes. Whether this arises from free HOCl, or via site-specific reactions is unresolved. Here we examine the mechanisms of MPO-mediated changes to human coronary smooth muscle cell ECM. MPO is shown to co-localize with matrix fibronectin as detected by confocal microscopy, and bound active MPO can initiate ECM modification, as detected by decreased antibody recognition of fibronectin, versican and type IV collagen, and formation of protein carbonyls and HOCl-mediated damage. These changes are recapitulated by a glucose/glucose oxidase/MPO system where low continuous fluxes of H2O2 are generated. HOCl-induced modifications enhance MPO binding to ECM proteins as detected by ELISA and MPO activity measurements. These data demonstrate that MPO-generated HOCl induces ECM modification by interacting with ECM proteins in a site-specific manner, and generates alterations that increase MPO adhesion. This is proposed to give rise to an increasing cycle of alterations that contribute to tissue damage.

Versican is produced by Trif- and type I interferon-dependent signaling in macrophages and contributes to fine control of innate immunity in lungs.

Growing evidence suggests that versican is important in the innate immune response to lung infection. Our goal was to understand the regulation of macrophage-derived versican and the role it plays in innate immunity. We first defined the signaling events that regulate versican expression, using bone marrow-derived macrophages (BMDMs) from mice lacking specific Toll-like receptors (TLRs), TLR adaptor molecules, or the type I interferon receptor (IFNAR1). We show that LPS and polyinosinic-polycytidylic acid [poly(I:C)] trigger a signaling cascade involving TLR3 or TLR4, the Trif adaptor, type I interferons, and IFNAR1, leading to increased expression of versican by macrophages and implicating versican as an interferon-stimulated gene. The signaling events regulating versican are distinct from those for hyaluronan synthase 1 (HAS1) and syndecan-4 in macrophages. HAS1 expression requires TLR2 and MyD88. Syndecan-4 requires TLR2, TLR3, or TLR4 and both MyD88 and Trif. Neither HAS1 nor syndecan-4 is dependent on type I interferons. The importance of macrophage-derived versican in lungs was determined with LysM/Vcan-/- mice. These studies show increased recovery of inflammatory cells in the bronchoalveolar lavage fluid of poly(I:C)-treated LysM/Vcan-/- mice compared with control mice. IFN-ß and IL-10, two important anti-inflammatory molecules, are significantly decreased in both poly(I:C)-treated BMDMs from LysM/Vcan-/- mice and bronchoalveolar lavage fluid from poly(I:C)-treated LysM/Vcan-/- mice compared with control mice. In short, type I interferon signaling regulates versican expression, and versican is necessary for type I interferon production. These findings suggest that macrophage-derived versican is an immunomodulatory molecule with anti-inflammatory properties in acute pulmonary inflammation.

Versican-Derived Matrikines Regulate Batf3-Dendritic Cell Differentiation and Promote T Cell Infiltration in Colorectal Cancer.

Colorectal cancer originates within immunologically complex microenvironments. To date, the benefits of immunotherapy have been modest, except in neoantigen-laden mismatch repair-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. In this article, we report that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN) strongly correlated with CD8+ T cell infiltration in colorectal cancer, regardless of mismatch repair status. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T cell infiltration. Tumor-intrinsic WNT pathway activation was associated with CD8+ T cell exclusion and VCAN accumulation. In addition to regulating VCAN levels at the tumor site, VCAN proteolysis results in the generation of bioactive fragments with novel functions (VCAN-derived matrikines). Versikine, a VCAN-derived matrikine, enhanced the generation of CD103+CD11chiMHCIIhi conventional dendritic cells (cDCs) from Flt3L-mobilized primary bone marrow-derived progenitors, suggesting that VCAN proteolysis may promote differentiation of tumor-seeding DC precursors toward IRF8- and BATF3-expressing cDCs. Intratumoral BATF3-dependent DCs are critical determinants for T cell antitumor immunity, effector T cell trafficking to the tumor site, and response to immunotherapies. Our findings provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker and VCAN-derived matrikines as novel immunotherapy agents.

Gonadotropin and tumorigenesis: Direct and indirect effects on inflammatory and immunosuppressive mediators and invasion.

Human chorionic gonadotropin (hCG), a hormone essential for pregnancy, is also ectopically expressed by a variety of cancers and is associated with poor prognosis; molecular mechanisms which may contribute to tumor progression remain ill-defined. Exogenous hCG enhanced the viability of human colorectal and lung cancer cells and promoted the growth of syngeneic tumors in mice. It induced the synthesis of VEGF, IL-8, matrix metalloprotease (MMP)-2 and MMP-9, and increased invasiveness in an MMP-dependent manner. While inducing the secretion of the tumor-associated extra-cellular matrix proteoglycan versican from tumor cells, hCG consequently caused the TLR-2-mediated generation of the inflammatory, tumor-associated cytokines TNF-α and IL-6 from peripheral blood adherent cells. The molecule up-modulated the Treg-associated transcription factor FOXP3 in tumor cells and increased the secretion of TGFß and IL-10, thereby inhibiting T cell proliferation and inducing the differentiation FOXP3- CD4+ CD25- cells into functional FOXP3+ CD4+ CD25+ suppressor cells. Co-culture of hCG-treated tumor cells with mature bone-marrow derived dendritic cells induced the generation of active indoleamine deoxygenase. While anti-hCG antibodies restricted the growth of implanted tumor cells in nude mice, immunization of immune competent mice with a ßhCG-TT conjugate supplemented with Mycobacterium indicus pranii provided synergistic survival benefit in animals implanted with syngeneic, hCG-responsive tumor cells. These studies elucidate the pathways by which hCG can promote tumorigenesis, providing further rationale for anti-hCG vaccination in the treatment of gonadotropin-sensitive tumors. © 2016 Wiley Periodicals, Inc.

Crosstalk Between T Lymphocytes and Lung Fibroblasts: Generation of a Hyaluronan-Enriched Extracellular Matrix Adhesive for Monocytes.

In immunity and inflammation, T cells are often associated with stromal mesenchymal cells such as fibroblasts. Hyaluronan and proteins that associate with hyaluronan such as versican and tumor necrosis factor-inducible gene-6 (TSG-6) are extracellular matrix (ECM) components that promote leukocyte adhesion, accumulation, and activation. However, the factors responsible for producing this specialized ECM and its impact on inflammatory events are not well understood. In this study, we explored the role of T cells in stimulating lung fibroblasts to produce an ECM that impacts monocyte adhesion. We found that CD3/CD28-activated human CD4+ T cells when co-cultured with human lung fibroblasts stimulated the expression of mRNA for hyaluronan synthase 2 (HAS2) and decreased the expression of hyaluronidase 2 (HYAL2). This led to an increase in the deposition of hyaluronan that formed cable-like structures within the ECM. Co-culturing activated T cells with fibroblasts also led to increased expression and accumulation of TSG-6. Surprisingly, addition of activated CD4+ T cells to the fibroblasts reduced the expression of mRNA for versican, and increased the expression of enzymes that degrade versican, such as ADAMTS4 and ADAMTS9 (a disintegrin and metalloproteinase with a thrombospondin type-1 motif) leading to a decrease in versican in the ECM of the co-cultures. Furthermore, addition of human monocytes to these co-cultures resulted in elevated monocyte adhesion to the cable-like structures in the ECM when compared to controls. These results illustrate the importance of crosstalk between T cells and fibroblasts in promoting the generation of a matrix that is adhesive for monocytes. J. Cell. Biochem. 118: 2118-2130, 2017. © 2016 Wiley Periodicals, Inc.

The Bacterial Component Flagellin Induces Anti-Sepsis Protection Through TLR-5, IL-1RN and VCAN During Polymicrobial Sepsis in Mice.

BACKGROUND: The present study was designed to observe the effects of the bacterial component flagellin on anti-sepsis protection through TLR-5, VCAN and IL-1RN. METHODS: A clinically relevant model of sepsis was induced by cecal ligation and puncture (CLP). An in vitro culture of endothelial cells was analyzed. RESULTS: Flagellin induced anti-sepsis protection through inhibition of inflammation and induction of endothelial proliferation by down-regulating the expression of TLR 3, TLR 4, and IL-1RN and promoting the expression of VCAN in mice 24 h post-CLP. In vitro, flagellin promoted the proliferation of endothelial cells. These effects could be inhibited by transfection of endothelial cells with VCAN siRNA or IL-1RN over-expression constructs. VCAN expression decreased after transfection of the cells with an IL-1RN over-expression construct and increased after transfection of the cells with an IL-1RN siRNA construct. IL-1RN expression remained unchanged after transfection of the cells with VCAN over-expression or siRNA constructs. CONCLUSIONS: These data suggest that flagellin pretreatment promoted anti-sepsis protection through the TLR-5, IL-1RN and VCAN pathway. This pathway is necessary to mediate endothelial repair and thereby promote survival following sepsis challenge.

Could Ex Vivo Lung Perfusion Be a Platform to Decrease the Incidence of Chronic Lung Allograft Dysfunction?

The number of patients requiring lung transplantation is increasing, with a significant unmet demand for grafts. Ex vivo lung perfusion has been developed to increase graft recruitment. The major complications of lung transplantation include chronic allograft dysfunction (CLAD) whose cumulative incidence ranges from 43-80% within the first 5 years of transplantation. Many risk factors are listed for development of CLAD and almost all of those risk factors would involve activation of Toll-like receptors. This paper represents the author's overview regarding the development of CLAD as a complication of lung transplantation and the possible protective potential of ex vivo lung perfusion in this regard.

Expression of versican V3 by arterial smooth muscle cells alters tumor growth factor ß (TGFß)-, epidermal growth factor (EGF)-, and nuclear factor κB (NFκB)-dependent signaling pathways, creating a microenvironment that resists monocyte adhesion.

Monocyte/macrophage accumulation plays a critical role during progression of cardiovascular diseases, such as atherosclerosis. Our previous studies demonstrated that retrovirally mediated expression of the versican V3 splice variant (V3) by arterial smooth muscle cells (ASMCs) decreases monocyte adhesion in vitro and macrophage accumulation in a model of lipid-induced neointimal formation in vivo. We now demonstrate that V3-expressing ASMCs resist monocyte adhesion by altering the composition of the microenvironment surrounding the cells by affecting multiple signaling pathways. Reduction of monocyte adhesion to V3-expressing ASMCs is due to the generation of an extracellular matrix enriched in elastic fibers and depleted in hyaluronan, and reduction of the proinflammatory cell surface vascular cell adhesion molecule 1 (VCAM1). Blocking these changes reverses the protective effect of V3 on monocyte adhesion. The enhanced elastogenesis induced by V3 expression is mediated by TGFß signaling, whereas the reduction in hyaluronan cable formation induced by V3 expression is mediated by the blockade of epidermal growth factor receptor and NFκB activation pathways. In addition, expression of V3 by ASMCs induced a marked decrease in NFκB-responsive proinflammatory cell surface molecules that mediate monocyte adhesion, such as VCAM1. Overall, these results indicate that V3 expression by ASMCs creates a microenvironment resistant to monocyte adhesion via differentially regulating multiple signaling pathways.

Changes of early post-traumatic osteoarthritis in an ovine model of simulated ACL reconstruction are associated with transient acute post-injury synovial inflammation and tissue catabolism.

The study described here tested the hypothesis that early intra-articular inflammation is associated with the development of post-traumatic osteoarthritis (PTOA) in a sheep model. We extended previously published work in which we investigated joint gross morphology and synovial mRNA expression of inflammatory and catabolic molecules 2 weeks after anatomic Anterior cruciate ligament (ACL) autograft reconstructive surgery (ACL-R). The same variables have been analyzed at 20 weeks post surgery together with new experimental variables at both time points. Animals were sacrificed at 20 weeks post ACL-R surgery and their joints graded for signs of PTOA. Synovial samples were harvested for histological grading plus mRNA and protein analysis for a panel of inflammatory and catabolic molecules. The mRNA expression levels for this panel plus connective tissue matrix turnover molecules were also investigated in cartilage samples. Results of gross morphological assessments at 20 weeks post surgery showed some changes consistent with early OA, but indicated little progression of damage from the 2 week time point. While significant alterations in mRNA levels for synovial inflammatory and catabolic molecules were detected at 2 weeks, values had normalized by 20 weeks. Similarly, all mRNA expression levels for inflammatory and catabolic molecules in articular cartilage had returned to normal levels by 20 weeks post ACL-R surgery. We conclude that synovial inflammatory processes are initiated very early after ACL-R surgery and may instigate events that lead to the gross cartilage and joint abnormalities observed as early as 2 weeks. However, the absence of sustained inflammation and joint instability may prevent OA progression.

T-cell responses to versican in ankylosing spondylitis.

The objective of our study was to undertake a systematic analysis of the T-cell response to the proteoglycan versican G1-globular domain (VG1) in ankylosing spondylitis (AS) as immunity to VG1 in mice can induce a pathology closely resembling AS. Peripheral blood lymphocytes from 36 AS patients and 33 healthy controls were incubated with recombinant human VG1 in culture for 6 h. T-cell responses were assessed by FACS analyses using mAb against surface expression of the activation marker CD69 and against the intracellular cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha. T cells activated by exposure to versican were determined by assessing the percentage of CD4+ or CD8+ T cells that were CD69/cytokine double-positive cells as compared to isotype control staining. In the AS patients, exposure to VG1 resulted in increased expression by CD4+ T cells of IFN-gamma in 55.6% of patients and of TNF-alpha in 52.8% of patients. In the controls, only 36.4% of subjects demonstrated an IFN-gamma response and 36.4% demonstrated a TNF-alpha response (P value 0.148, 0.227, respectively). With respect to CD8+ T-cell responses, versican stimulation enhanced IFN-gamma expression in 44.4% of AS patients and 39.4% of controls, and enhanced TNF-alpha response in 50.0% of AS patients and 39.4% of controls (P value 0.620, 0.327, respectively). Although, there was no statistically significant difference in the magnitude of the IFN-γ or TNF secretion by CD4+ T cells and CD8+ T cells between AS and controls, our results demonstrate an enhanced T-cell response to VG1 in AS.

Immunohistochemical aspects of the fibrogenic pathway in nephrogenic systemic fibrosis.

Nephrogenic systemic fibrosis (NSF) is a rare gadolinium-dependent disorder of the skin and viscera. The aim of this study was to revisit some immunopathologic clues of NSF, including the characterization of glycosaminoglycans, cell tensegrity, and cell proliferation in the dermis. Immunohistochemistry was done using antibodies directed to vimentin, CD34, Factor XIIIa, calprotectin, α-smooth muscle actin, Ulex europaeus agglutinin-1 (UEA-1), and MIB1/Ki67 and to glycosaminoglycans, including CD44 var3, versican, and perlecan. The vimentin+ cell density was markedly increased. The vast majority of them corresponded to CD34+ or Factor XIIIa+ dermal dendrocytes (DD) showing distinct cell tensegrity. CD34+DD were slender, elongated, and usually scattered in the dermis but focally clustered in nodular collections. By contrast, Factor XIIIa+ was plump with squat dendrites showing no evidence for being under mechanical stress. Cells in the vicinity of the microvasculature were rounded and exhibited calprotectin immunoreactivity typical for monocyte/macrophages. The microvasculature highlighted by UEA-1 and α-smooth muscle actin looked unremarkable. The cell proliferation highlighted by the MIB/Ki67 immunoreactivity was unusually high (>20%) in the interstitial stromal cells. Stromal cells enriched in versican were plump, abundant, and seemed interconnected each other by a dense network of dendrites. By contrast, the immunolabeling for perlecan and CD44 var 3 was unremarkable. In conclusion, the cell population involved in NSF seemed phenotypically heterogeneous, and its growth fraction was clearly boosted in the skin. The intracellular load in versican was prominent. The aspect of cell tensegrity did not suggest the influence of mechanical stress putting stromal cells under tension in the dermis.

Ligation of TLR2 by versican: a link between inflammation and metastasis.

Versican, a large extracellular matrix proteoglycan, accumulates both in tumor stroma and cancer cells. It participates in cell adhesion, migration, and angiogenesis, all features of invasion and metastasis. However, the mechanism(s) whereby versican promotes cancer invasion and metastasis is not yet fully understood. A recent study has documented that versican can activate tumor-infiltrating myeloid cells through toll-like receptor (TLR) 2 and its co-receptors TLR6 and CD14 and elicit the production of proinflammatory cytokines including TNF-alpha that enhance tumor metastasis. As both resident fibroblasts and endothelial cells (ECs) also express functional TLR2 and its co-receptors, we hypothesized that, in addition to myeloid cells, versican may trigger the activation of both fibroblasts and ECs. Of interest, TLR2-mediated activation of EC and fibroblast has been observed to increase the secretion of interleukin-8, a proinflammatory CXC chemokine that potentiates neutrophil infiltration and angiogenesis, as well as metastatic growth. Ligation of TLR2 by versican appears to be directly involved in the activation of multiple types of cells in tumor stroma and the induction of inflammatory cytokine secretion, providing a link between inflammation and cancer metastasis. Accordingly, antagonists of versican and TLR2 restrain the activation of tumor stromal cells, which may offer a novel approach to cancer therapy by targeting tumor microenvironment.

Versican, a major hyaluronan-binding component in the dermis, loses its hyaluronan-binding ability in solar elastosis.

Versican interacts with hyaluronan (HA) at its N-terminus and with fibrillin-1 at its C terminus. As versican in the dermis connects microfibrils to the HA-rich matrix for viscoelasticity, dermal diseases may involve destruction of these complexes. A recombinant versican protein, rVN, covering the HA binding region (HABR) of human versican and a polyclonal antibody, 6084, against rVN were prepared and characterized. Blotting analyses of skin extracts with 6084 and biotin-conjugated HA revealed that versican was a major HA-binding component in the dermis. Matrix metalloprotease-12, which is expressed in areas of solar elastosis, degraded versican and abrogated its HA-binding ability. Immunohistochemical analyses revealed that the elastic materials in solar elastosis lesions were negative for 6084, but positive for 2B1, an antibody recognizing the C-terminus of versican, indicating loss of the HABR in the aggregated elastic fibers. This loss of the HA-binding ability of versican followed by HA exclusion may be responsible for the pathological and phenotypical changes observed in solar elastosis.