A Novel CD206 Targeting Peptide Inhibits Bleomycin-Induced Pulmonary Fibrosis in Mice.

Activated M2-polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios, including Idiopathic Pulmonary Fibrosis (IPF). In this study, we investigated the effects of targeting the CD206 receptor in M2-like macrophages with a novel synthetic analogue of a naturally occurring Host Defense Peptide (HDP), RP-832c, to decrease profibrotic cytokines. RP-832c selectively binds to CD206 on M2-polarized bone marrow-derived macrophages (BMDM) in vitro, resulting in a time-dependent decrease in CD206 expression and a transient increase in M1-macrophage marker TNF-α. To elucidate the antifibrotic effects of RP-832c, we used a murine model of bleomycin (BLM)-induced early-stage pulmonary fibrosis. RP-832c significantly reduced fibrosis in a dose-dependent manner, and decreased CD206, TGF-ß1, and α-SMA expression in mouse lungs. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased lung fibrosis and significantly decreased inflammatory cytokines TNF-α, IL-6, IL-10, IFN-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with the FDA-approved drugs Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed. In summary, our findings showed that inhibiting the profibrotic alternatively activated M2-like macrophages using a novel peptide, RP-832c, could reduce BLM-induced pulmonary fibrosis in mice, warranting the therapeutic potential of this peptide for patients with pulmonary fibrosis.

Matrigel: history/background, uses, and future applications.

Basement membranes are thin sheets of extracellular matrix with many diverse roles in the body. Those in normal tissue are also highly insoluble and resist attempts to extract and characterize their components. A mouse tumor, the EHS tumor, has provided large amounts of basement membrane material, which has facilitated the structural and functional characterization of its components. An extract of the tumor, known as Matrigel, contains components which reconstitute into a solid gel at 37°. This solid basement membrane matrix has been used in both cell culture and in vivo. Matrigel has been utilized in some 12,000-plus publications for a variety of studies with embryonic, normal, and stem or malignant cells. Evidence presented in this Commentary suggests that Matrigel isolated from tumors grown in diverse hosts could exert unique effects that could be helpful in analyzing the causes of various pathologies and for screening possible therapeutic agents.

A Novel CD206 Targeting Peptide Inhibits Bleomycin Induced Pulmonary Fibrosis in Mice.

Activated M2 polarized macrophages are drivers of pulmonary fibrosis in several clinical scenarios such as Acute Respiratory Disease Syndrome (ARDS) and Idiopathic Pulmonary Fibrosis (IPF), through the production of inflammatory and fibrosis-inducing cytokines. In this study, we investigated the effect of targeting the CD206 receptor with a novel fragment of a Host Defense Peptide (HDP), RP-832c to decrease cytokines that cause fibrosis. RP-832c selectively binds to CD206 on M2 polarized bone marrow derived macrophages (BMDM) in vitro , resulting in a time-dependent decrease in CD206 expression, and a transient increase in M1 marker TNFα, which resolves over a 24hr period. To elucidate the antifibrotic effect of RP-832c, we used a murine model of bleomycin (BLM) -induced early-stage pulmonary fibrosis. RP-832c significantly reduced bleomycin-induced fibrosis in a dosage dependent manner, as well as decreased CD206, TGF-ß1 and α-SMA expression in mouse lungs. Interestingly we did not observe any changes in the resident alveolar macrophage marker CD170 expression. Similarly, in an established model of lung fibrosis, RP-832c significantly decreased fibrosis in the lung, as well as significantly decreased inflammatory cytokines TNFα, IL-6, IL-10, INF-γ, CXCL1/2, and fibrosis markers TGF-ß1 and MMP-13. In comparison with FDA approved drugs, Nintedanib and Pirfenidone, RP-832c exhibited a similar reduction in fibrosis compared to Pirfenidone, and to a greater extent than Nintedanib, with no apparent toxicities observed on body weight or blood chemistry. In summary, RP-832c is a potential agent to mitigate the overactivity of M2 macrophages in pathogenesis several pulmonary fibrotic diseases, including SARS-CoV-2 induced lung fibrosis.

Zika virus-like particle (VLP) based vaccine.

The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development.

Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration.

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

A simplified laminin nomenclature.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis.

INTRODUCTION: Clinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients. METHODS: Dermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors. RESULTS: Luminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator. CONCLUSIONS: Our data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.

Collagen fibril diameter and alignment promote the quiescent keratocyte phenotype.

In this study, we investigated how matrix nanotopography affects corneal fibroblast phenotype and matrix synthesis. To this end, corneal fibroblasts isolated from bovine corneas were grown on collagen nanofiber scaffolds of different diameters and alignment--30 nm aligned fibrils (30A), 300 nm or larger aligned fibrils (300A), and 30 nm nonaligned fibrils (30NA) in comparison with collagen coated flat glass substrates (FC). Cell morphology was visualized using confocal microscopy. Quantitative PCR was used to measure expression levels of six target genes: the corneal crystallin-transketolase (TKT), the myofibroblast marker-α-smooth muscle actin (SMA), and four matrix proteins-collagen 1 (COL1), collagen 3 (COL3), fibronectin (FN), and biglycan. It was found that SMA expression was down-regulated and TKT expression was increased on all three collagen nanofiber substrates, compared with the FC control substrates. However, COL3 and biglycan expression was also significantly increased on 300A, compared with the FC substrates. Thus matrix nanotopography down-regulates the fibrotic phenotype, promotes formation of the quiescent keratocyte phenotype, and influences matrix synthesis. These results have significant implications for the engineering of corneal replacements and for promoting regenerative healing of the cornea after disease and/or injury.

Matrigel: basement membrane matrix with biological activity.

The basement membrane extracellular matrix contacts epithelial, endothelial, fat and smooth muscle cells. Because this extracellular matrix is so thin, it had been hard to study its composition, structure, and function. An extract of a tumor was found to contain all of the components present in basement and to be very biologically active. This extract, termed Matrigel, Cultrex, or EHS matrix, promotes cell differentiation, and is used to measure the invasive activity of tumor cells. In vivo, it is used for measuring angiogenic inhibitors and stimulators, to improve graft survival, repair damaged tissues, and increase tumor growth.

Future research directions in idiopathic pulmonary fibrosis: summary of a National Heart, Lung, and Blood Institute working group.

Idiopathic pulmonary fibrosis (IPF) is an insidious inflammatory fibroproliferative disease whose cause and course before diagnosis are unknown, and for which existing treatments are of limited benefit. The National Heart, Lung, and Blood Institute convened a working group to develop specific recommendations for future IPF research. Inflammatory and immune processes are involved in IPF pathogenesis, and current therapeutic strategies are aimed at suppressing the inflammation. Recent data suggest that the molecular processes underlying the fibrogenesis may provide new opportunities for therapeutic intervention. Specific areas of future research recommended by the working group include studies to elucidate the etiology of IPF, to develop novel diagnostic techniques and molecular diagnostics, to establish a program for identification of molecular targets for IPF treatment and identification and generation of agonists or antagonists that inhibit fibrogenesis, to foster investigations that couple the use of new technologies (e.g., laser capture microdissection, microarrays, and mass spectroscopic analysis of proteins) with data from the human genome project, to establish a national consortium of Clinical Centers of Excellence to conduct coordinated clinical and laboratory studies of well-characterized patients and patient-derived materials, and to stimulate research to develop animal models of persistent and progressive pulmonary fibrosis for evaluation of new intervention approaches.