Sialoadhesin (CD169/Siglec-1) is an extended molecule that escapes inhibitory cis-interactions and synergizes with other macrophage receptors to promote phagocytosis.

Sialoadhesin (CD169/Siglec-1, Sn) is a macrophage receptor that interacts with sialic acids on both host cells and pathogens. It is a type 1 membrane protein with an unusually large number of 17 extracellular immunoglobulin (Ig)-like domains, made up of an N-terminal V-set domain that binds sialic acid and 16 adjacent C2-set domains. The potential importance of 17 Ig domains in Sn for mediating cellular interactions has not been investigated experimentally. In the present study, Chinese Hamster Ovary (CHO) cells were stably transfected with full-length or truncated forms of Sn. Using human red blood cells (RBC) as a model system, CHO cells expressing truncated forms of Sn with 4 or less Ig domains were unable to bind RBC in comparison to the full-length protein. Immunoelectron microscopy of the CHO cells indicated that full-length Sn extends ~ 33 nm from the plasma membrane compared with ~ 14 nm for a truncated form with 6 N-terminal Ig domains. Co-expresssion of Sn-expressing CHO cells with heavily glycosylated membrane proteins of differing predicted lengths resulted in selective modulation of Sn-dependent binding to RBC and supported the hypothesis that Sn has evolved 17 Ig domains to escape inhibitory cis-interactions. The functional significance of the extended length of Sn was demonstrated in experiments with macrophages showing that Sn synergizes with phagocytic receptors FcR and TIM-4 to strongly promote uptake of IgG-opsonized and eryptotic RBC respectively.

Proteomic Analysis of Dupuytren's Contracture-Derived Sweat Glands Revealed the Synthesis of Connective Tissue Growth Factor and Initiation of Epithelial-Mesenchymal Transition as Major Pathogenetic Events.

Dupuytren's contracture (DC) is a chronic and progressive fibroproliferative disorder restricted to the palmar fascia of the hands. Previously, we discovered the presence of high levels of connective tissue growth factor in sweat glands in the vicinity of DC nodules and hypothesized that sweat glands have an important role in the formation of DC lesions. Here, we shed light on the role of sweat glands in the DC pathogenesis by proteomic analysis and immunofluorescence microscopy. We demonstrated that a fraction of sweat gland epithelium underwent epithelial-mesenchymal transition illustrated by negative regulation of E-cadherin. We hypothesized that the increase in connective tissue growth factor expression in DC sweat glands has both autocrine and paracrine effects in sustaining the DC formation and inducing pathological changes in DC-associated sweat glands.

Stimulation with THBS4 activates pathways that regulate proliferation, migration and inflammation in primary human keratinocytes.

As in other mammalian tissues, the extracellular matrix (ECM) of skin functions as mechanical support and regulative environment that guides the behavior of the cells. ECM is a gel-like structure that is primarily composed of structural and nonstructural proteins. While the content of structural proteins is stable, the level of nonstructural ECM proteins, such as thrombospondin-4 (THBS4), is dynamically regulated. In a previous work we demonstrated that THBS4 stimulated cutaneous wound healing. In this work we discovered that in addition to proliferation, THBS4 stimulated the migration of primary keratinocytes in 3D. By using a proteotransciptomic approach we found that stimulation of keratinocytes with THBS4 regulated the activity of signaling pathways linked to proliferation, migration, inflammation and differentiation. Interestingly, some of the regulated genes (eg IL37, TSLP) have been associated with the pathogenesis of atopic dermatitis (AD). We concluded that THBS4 is a promising candidate for novel wound healing therapies and suggest that there is a potential convergence of pathways that stimulate cutaneous wound healing with those active in the pathogenesis of inflammatory skin diseases.

Matricellular proteins in cutaneous wound healing.

Cutaneous wound healing is a complex process that encompasses alterations in all aspects of the skin including the extracellular matrix (ECM). ECM consist of large structural proteins such as collagens and elastin as well as smaller proteins with mainly regulative properties called matricellular proteins. Matricellular proteins bind to structural proteins and their functions include but are not limited to interaction with cell surface receptors, cytokines, or protease and evoking a cellular response. The signaling initiated by matricellular proteins modulates differentiation and proliferation of cells having an impact on the tissue regeneration. In this review we give an overview of the matricellular proteins that have been found to be involved in cutaneous wound healing and summarize the information known to date about their functions in this process.

Olfactomedin 4 regulates migration and proliferation of immortalized non-transformed keratinocytes through modulation of the cell cycle machinery and actin cytoskeleton remodelling.

Olfactomedin 4 (OLFM4), a multifunctional matricellular protein, is involved in regulation of angiogenesis, innate immunity, inflammation, tumorigenesis and metastasis formation via modulation of important cellular processes like adhesion, proliferation, differentiation as well as apoptosis. In our previous work we demonstrated the upregulation of OLFM4 during liver regeneration and cutaneous wound healing. Here we studied the outcomes of OLFM4 downregulation in human immortalized keratinocytes - the HaCaT cells. The suppression of OLFM4 inhibited migration but enhanced the proliferation of these cells. By using proteomic, and phosphoproteomic analysis, we found that OLFM4 downregulation induced changes in the levels of 184 proteins and 348 phosphosites. An integrated pathway analysis suggested that the increased phosphorylation of CDK7 at Ser164 and Thr170 may serve as the key event in the activation of CDK2 and consequent activation of cell cycle progression. Furthermore, the decrease in GIT1 and WAVE2 protein levels were connected to the disorganization of the actin cytoskeleton, reduction of lamellipodia formation at the leading edge of HaCaT cells, and decrease in their migration capacity.

Olfactomedin-4 improves cutaneous wound healing by promoting skin cell proliferation and migration through POU5F1/OCT4 and ESR1 signalling cascades.

Olfactomedin-4 (OLFM4) is an olfactomedin-domain-containing glycoprotein, which regulates cell adhesion, proliferation, gastrointestinal inflammation, innate immunity and cancer metastasis. In the present study we investigated its role in skin regeneration. We found that OLFM4 expression is transiently upregulated in the proliferative phase of cutaneous wound healing in humans as well as in mice. Moreover, a significant increase in OLFM4 expression was detected in the skin of lesional psoriasis, a chronic inflammatory disease characterized by keratinocyte hyperproliferation. In vitro experiments demonstrated that OLFM4 selectively stimulated keratinocyte proliferation and increased both keratinocyte and fibroblast migration. Using proteotranscriptomic pathway analysis we revealed that transcription factors POU5F1/OCT4 and ESR1 acted as hubs for OLFM4-induced signalling in keratinocytes. In vivo experiments utilizing mouse splinted full-thickness cutaneous wound healing model showed that application of recombinant OLFM4 protein can significantly improve wound healing efficacy. Taken together, our results suggest that OLFM4 acts as a transiently upregulated inflammatory signal that promotes wound healing by regulating both dermal and epidermal cell compartments of the skin.

Long-term maintenance of functional primary human hepatocytes in 3D gelatin matrices produced by solution blow spinning.

Solution blow spinning (SBS) has recently emerged as a novel method that can produce nano- and microfiber structures suitable for tissue engineering. Gelatin is an excellent precursor for SBS as it is derived mainly from collagens that are abundant in natural extracellular matrices. Here we report, for the first time the successful generation of 3D thermally crosslinked preforms by using SBS from porcine gelatin. These SBS mats were shown to have three-dimensional fibrous porous structure similar to that of mammalian tissue extracellular matrix. In pharma industry, there is an urgent need for adequate 3D liver tissue models that could be used in high throughput setting for drug screening and to assess drug induced liver injury. We used SBS mats as culturing substrates for human hepatocytes to create an array of 3D human liver tissue equivalents in 96-well format. The SBS mats were highly cytocompatible, facilitated the induction of hepatocyte specific CYP gene expression in response to common medications, and supported the maintenance of hepatocyte differentiation and polarization status in long term cultures for more than 3 weeks. Together, our results show that SBS-generated gelatin scaffolds are a simple and efficient platform for use in vitro for drug testing applications.

Thrombospondin-4 Is a Soluble Dermal Inflammatory Signal That Selectively Promotes Fibroblast Migration and Keratinocyte Proliferation for Skin Regeneration and Wound Healing.

Thrombospondin-4 (THBS4) is a non-structural extracellular matrix molecule associated with tissue regeneration and a variety of pathological processes characterized by increased cell proliferation and migration. However, the mechanisms of how THBS4 regulates cell behavior as well as the pathways contributing to its effects have remained largely unexplored. In the present study we investigated the role of THBS4 in skin regeneration both in vitro and in vivo. We found that THBS4 expression was upregulated in the dermal compartment of healing skin wounds in humans as well as in mice. Application of recombinant THBS4 protein promoted cutaneous wound healing in mice and selectively stimulated migration of primary fibroblasts as well as proliferation of keratinocytes in vitro. By using a combined proteotranscriptomic pathway analysis approach we discovered that ß-catenin acted as a hub for THBS4-dependent cell signaling and likely plays a key role in promoting its downstream effects. Our results suggest that THBS4 is an important contributor to wound healing and its incorporation into novel wound healing therapies may be a promising strategy for treatment of cutaneous wounds.

Case Report: Unravelling the Mysterious Lichtenberg Figure Skin Response in a Patient With a High-Voltage Electrical Injury.

We describe a case of Lichtenberg Figures (LFs) following an electrical injury from a high-voltage switchgear in a 47 year-old electrician. LFs, also known as ferning pattern or keraunographic markings, are a pathognomonic skin sign for lightning strike injuries. Their true pathophysiology has remained a mystery and only once before described following an electical injury. The aim was to characterise the tissue response of LFs by performing untargeted non-labelled proteomics and immunohistochemistry on paraffin-embedded sections of skin biopsies taken from the area of LFs at presentation and at 3 months follow-up. Our results demonstrated an increase in dermal T-cells and greatly increased expression of the iron-binding glycoprotein lactoferrin by keratinocytes and lymphocytes. These changes in the LF-affected skin were associated with extravasation of red blood cells from dermal vessels. Our results provide an initial molecular and cellular insight into the tissue response associated with LFs.

Endogenous beta-galactosidase activity marks a TREM2-expressing Kupffer cell population in injured livers of Lgr5-LacZ and wild-type mice.

Lgr5-LacZ mice harbor the Escherichia coli LacZ gene encoding ß-galactosidase (ß-gal) under the control of the Lgr5 promoter, a stem/progenitor cell marker. In injured livers of Lgr5-LacZ mice, cells expressing ß-galactosidase (ß-gal) are considered as potential bipotent liver progenitors; however, their origin and identity remain unknown. Unexpectedly, using lineage tracing, we demonstrate that the ß-gal+ cells do not originate from liver parenchymal cells. Instead, ß-gal+ cells, isolated from injured livers of both Lgr5-LacZ and wild-type mice, are positive for markers of Kupffer cells, liver-resident macrophages. The ß-gal expression in these cells is a result of elevated expression of the endogenous beta-galactosidase Glb1. In injured livers of Lgr5-LacZ mice, bacterial ß-gal expression is very low, suggesting transgene silencing. The gene expression profile of the ß-gal+ Kupffer cells from injured livers suggests a role in liver regeneration.

Discovery of increased epidermal DNAH10 expression after regeneration of dermis in a randomized with-in person trial - reflections on psoriatic inflammation.

Because molecular memories of past inflammatory events can persist in epidermal cells, we evaluated the long-term epidermal protein expression landscapes after dermal regeneration and in psoriatic inflammation. We first characterized the effects of two dermal regeneration strategies on transplants of indicator split-thickness skin grafts (STSGs) in ten adult patients with deep burns covering more than 20% of their body surface area. After fascial excision, three adjacent areas within the wound were randomized to receive a permanent dermal matrix, a temporary granulation-tissue-inducing dressing or no dermal component as control. Control areas were covered with STSG immediately, and treated areas after two-weeks of dermis formation. Epidermis-dermis-targeted proteomics of one-year-follow-up samples were performed for protein expression profiling. Epidermal expression of axonemal dynein heavy chain 10 (DNAH10) was increased 20-fold in samples having had regenerating dermis vs control. Given the dermal inflammatory component found in our dermal regeneration samples as well as in early psoriatic lesions, we hypothesized that DNAH10 protein expression also would be affected in psoriatic skin samples. We discovered increased DNAH10 expression in inflammatory lesions when compared to unaffected skin. Our results associate DNAH10 expression with cell proliferation and inflammation as well as with the epidermal memory resulting from the previous regenerative signals of dermis. This study (ISRCTN14499986) was funded by the Finnish Ministry of Defense and by government subsidies for medical research.

A label-retaining but unipotent cell population resides in biliary compartment of mammalian liver.

Cells with slow proliferation kinetics that retain the nuclear label over long time periods-the label-retaining cells (LRCs)-represent multipotent stem cells in a number of adult tissues. Since the identity of liver LRCs (LLRCs) had remained elusive we utilized a genetic approach to reveal LLRCs in normal non-injured livers and characterized their regenerative properties in vivo and in culture. We found that LLRCs were located in biliary vessels and participated in the regeneration of biliary but not hepatocyte injury. In culture experiments the sorted LLRCs displayed an enhanced self-renewal capacity but a unipotent biliary differentiation potential. Transcriptome analysis revealed a unique set of tumorigenesis- and nervous system-related genes upregulated in LLRCs when compared to non-LRC cholangiocytes. We conclude that the LLRCs established during the normal morphogenesis of the liver do not represent a multipotent primitive somatic stem cell population but act as unipotent biliary progenitor cells.

The alterations in the extracellular matrix composition guide the repair of damaged liver tissue.

While the cellular mechanisms of liver regeneration have been thoroughly studied, the role of extracellular matrix (ECM) in liver regeneration is still poorly understood. We utilized a proteomics-based approach to identify the shifts in ECM composition after CCl4 or DDC treatment and studied their effect on the proliferation of liver cells by combining biophysical and cell culture methods. We identified notable alterations in the ECM structural components (eg collagens I, IV, V, fibronectin, elastin) as well as in non-structural proteins (eg olfactomedin-4, thrombospondin-4, armadillo repeat-containing x-linked protein 2 (Armcx2)). Comparable alterations in ECM composition were seen in damaged human livers. The increase in collagen content and decrease in elastic fibers resulted in rearrangement and increased stiffness of damaged liver ECM. Interestingly, the alterations in ECM components were nonhomogenous and differed between periportal and pericentral areas and thus our experiments demonstrated the differential ability of selected ECM components to regulate the proliferation of hepatocytes and biliary cells. We define for the first time the alterations in the ECM composition of livers recovering from damage and present functional evidence for a coordinated ECM remodelling that ensures an efficient restoration of liver tissue.

Pre-administration of PepFect6-microRNA-146a nanocomplexes inhibits inflammatory responses in keratinocytes and in a mouse model of irritant contact dermatitis.

The skin is a difficult to access tissue for efficient delivery of large and/or charged macromolecules, including therapeutic DNA and RNA oligonucleotides. Cell-penetrating peptide PepFect6 (PF6) has been shown to be suitable transport vehicle for siRNAs in cell culture and systemically in vivo in mice. MiR-146a is known as anti-inflammatory miRNA that inhibits multiple factors from the nuclear factor (NF)-κB pathway in various cell types, including keratinocytes. In this study, PF6 was shown to form unimodal nanocomplexes with miR-146a mimic that entered into human primary keratinocytes, where miR-146a inhibited the expression of its direct targets from the NF-κB pathway and the genes known to be activated by NF-κB, C-C motif ligand (CCL)5 and interleukin (IL)-8. The transfection of miR-146a mimic with PF6 was more efficient in sub-confluent keratinocyte cultures, affected keratinocyte proliferation less and had similar effect on cell viability when compared with a lipid based agent. Subcutaneous pre-administration of PF6-miR-146a nanocomplexes attenuated ear-swelling and reduced the expression of pro-inflammatory cytokines and chemokines IL-6, CCL11, CCL24 and C-X-C motif ligand 1 (CXCL1) in a mouse model of irritant contact dermatitis. Our data demonstrates that PF6-miR-146a nanoparticles might have potential in the development of therapeutics to target inflammatory skin diseases.

Sialoadhesin promotes rapid proinflammatory and type I IFN responses to a sialylated pathogen, Campylobacter jejuni.

Sialoadhesin (Sn) is a macrophage (Mφ)-restricted receptor that recognizes sialylated ligands on host cells and pathogens. Although Sn is thought to be important in cellular interactions of Mφs with cells of the immune system, the functional consequences of pathogen engagement by Sn are unclear. As a model system, we have investigated the role of Sn in Mφ interactions with heat-killed Campylobacter jejuni expressing a GD1a-like, sialylated glycan. Compared to Sn-expressing bone marrow-derived macrophages (BMDM) from wild-type mice, BMDM from mice either deficient in Sn or expressing a non-glycan-binding form of Sn showed greatly reduced phagocytosis of sialylated C. jejuni. This was accompanied by a strong reduction in MyD88-dependent secretion of TNF-α, IL-6, IL-12, and IL-10. In vivo studies demonstrated that functional Sn was required for rapid TNF-α and IFN-ß responses to i.v.-injected sialylated C. jejuni. Bacteria were captured within minutes after i.v. injection and were associated with Mφs in both liver and spleen. In the spleen, IFN-ß-reactive cells were localized to Sn⁺ Mφs and other cells in the red pulp and marginal zone. Together, these studies demonstrate that Sn plays a key role in capturing sialylated pathogens and promoting rapid proinflammatory cytokine and type I IFN responses.

Sialoadhesin in recognition of self and non-self.

The immune system is tightly regulated to maintain an appropriate balance between immune activation and tolerance. Macrophages play a key role in this process since they express many pathogen recognition molecules as well as receptors for 'self'. Sialoadhesin is a major macrophage receptor that specifically recognizes sialic acid, an abundant component of host glycoconjugates but which can also be found on several human pathogens. In recent years, several studies have demonstrated that sialoadhesin can contribute to the uptake and processing of sialylated pathogens as well as playing an important role in regulating inflammatory and autoimmune responses via recognition of self.