CD109 drives pro-tumorigenic cell properties in human non-small cell lung cancer through interaction with desmoglein-2.

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI) anchored cell surface protein, expressed on epithelial and endothelial cells, CD4+ and CD8+ T-cells, and premature lymphocytes. CD109 interacts with different cell surface receptors and thereby modulates intracellular signaling pathways, which ultimately changes cellular functions. One well-studied example is the interaction of CD109 with the TGFß/TGFß-receptor complex at the cell surface. CD109 silences intracellular SMAD2/3 signaling and targets TGFß/TGFß-receptor to the endosomal/lysosomal compartment. In recent years, CD109 emerged as a tumor marker for different tumor entities and expression of CD109 could be linked to adverse outcome in patients. In this study, we show that silencing of CD109 in human non-small cell lung cancer (NSCLC) cells, returns these cells to an epithelial like growth phenotype. On the transcriptional level, we describe changes in cell-cell contact and epithelial-mesenchymal transition associated gene clusters. At the cell surface, we identify desmoglein-2 (DSG2) as a new interaction partner of CD109 and demonstrate CD109 dependent targeting of DSG2 to the apical cell surface, where it forms desmosomes between apical and basal cell poles. Both, CD109 and DSG2 are genetic risk factors, linked to reduced overall survival in lung adenocarcinoma patients (subtype of NSCLC). In this study, we show the expression of both proteins in the same tumor and suggest a new CD109-DSG2 axis in NSCLC patients that could present a targetable therapeutic option in the future.

Oriented display of HIV-1 Env trimers by a novel coupling strategy enhances B cell activation and phagocytosis.

Introduction: Conformationally stabilized Env trimers have been developed as antigens for the induction of neutralizing antibodies against HIV-1. However, the non-glycosylated immunodominant base of these soluble antigens may compete with the neutralizing antibody response. This has prompted attempts to couple Env trimers to organic or inorganic nanoparticles with the base facing towards the carrier. Such a site-directed coupling could not only occlude the base of the trimer, but also enhance B cell activation by repetitive display. Methods: To explore the effect of an ordered display of HIV-1 Env on microspheres on the activation of Env-specific B cells we used Bind&Bite, a novel covalent coupling approach for conformationally sensitive antigens based on heterodimeric coiled-coil peptides. By engineering a trimeric HIV-1 Env protein with a basic 21-aa peptide (Peptide K) extension at the C-terminus, we were able to covalently biotinylate the antigen in a site-directed fashion using an acidic complementary peptide (Peptide E) bearing a reactive site and a biotin molecule. This allowed us to load our antigen onto streptavidin beads in an oriented manner. Results: Microspheres coated with HIV-1 Env through our Bind&Bite system showed i) enhanced binding by conformational anti-HIV Env broadly neutralizing antibodies (bNAbs), ii) reduced binding activity by antibodies directed towards the base of Env, iii) higher Env-specific B cell activation, and iv) were taken-up more efficiently after opsonization compared to beads presenting HIV-1 Env in an undirected orientation. Discussion: In comparison to site-directed biotinylation via the Avi-tag, Bind&Bite, offers greater flexibility with regard to alternative covalent protein modifications, allowing selective modification of multiple proteins via orthogonal coiled-coil peptide pairs. Thus, the Bind&Bite coupling approach via peptide K and peptide E described in this study offers a valuable tool for nanoparticle vaccine design where surface conjugation of correctly folded antigens is required.

Interrelation Between Cerebrospinal Fluid Pressure, Intracranial Morphology and Venous Hemodynamics Studied by 4D Flow MRI.

PURPOSE: To quantify the effects of CSF pressure alterations on intracranial venous morphology and hemodynamics in idiopathic intracranial hypertension (IIH) and spontaneous intracranial hypotension (SIH) and assess reversibility when the underlying cause is resolved. METHODS: We prospectively examined venous volume, intracranial venous blood flow and velocity, including optic nerve sheath diameter (ONSD) as a noninvasive surrogate of CSF pressure changes in 11 patients with IIH, 11 age-matched and sex-matched healthy controls and 9 SIH patients, before and after neurosurgical closure of spinal dural leaks. We applied multiparametric MRI including 4D flow MRI, time-of-flight (TOF) and T2-weighted half-Fourier acquisition single-shot turbo-spin echo (HASTE). RESULTS: Sinus volume overlapped between groups at baseline but decreased after treatment of intracranial hypotension (p = 0.067) along with a significant increase of ONSD (p = 0.003). Blood flow in the middle and dorsal superior sagittal sinus was remarkably lower in patients with higher CSF pressure (i.e., IIH versus controls and SIH after CSF leak closure) but blood flow velocity was comparable cross-sectionally between groups and longitudinally in SIH. CONCLUSION: We were able to demonstrate the interaction of CSF pressure, venous volumetry, venous hemodynamics and ONSD using multiparametric brain MRI. Closure of CSF leaks in SIH patients resulted in symptoms suggestive of increased intracranial pressure and caused a subsequent decrease of intracranial venous volume and of blood flow within the superior sagittal sinus while ONSD increased. In contrast, blood flow parameters from 4D flow MRI did not discriminate IIH, SIH and controls as hemodynamics at baseline overlapped at most vessel cross-sections.

Photon-counting computed tomography - clinical application in oncological, cardiovascular, and pediatric radiology. / Photon-Counting Computertomographie ­ klinische Anwendungen in der onkologischen, kardiovaskulären und pädiatrischen Radiologie.

BACKGROUND: Photon-counting detector computed tomography (PCD-CT) is a promising new technology with the potential to fundamentally change workflows in the daily routine and provide new quantitative imaging information to improve clinical decision-making and patient management. METHOD: The contents of this review are based on an unrestricted literature search of PubMed and Google Scholar using the search terms "photon-counting CT", "photon-counting detector", "spectral CT", "computed tomography" as well as on the authors' own experience. RESULTS: The fundamental difference with respect to the currently established energy-integrating CT detectors is that PCD-CT allows for the counting of every single photon at the detector level. Based on the identified literature, PCD-CT phantom measurements and initial clinical studies have demonstrated that the new technology allows for improved spatial resolution, reduced image noise, and new possibilities for advanced quantitative image postprocessing. CONCLUSION: For clinical practice, the potential benefits include fewer beam hardening artifacts, a radiation dose reduction, and the use of new or combinations of contrast agents. In particular, critical patient groups such as oncological, cardiovascular, lung, and head & neck as well as pediatric patient collectives benefit from the clinical advantages. KEY POINTS: · Photon-counting computed tomography (PCD-CT) is being used for the first time in routine clinical practice, enabling a significant dose reduction in critical patient populations such as oncology, cardiology, and pediatrics.. · Compared to conventional CT, PCD-CT enables a reduction in electronic image noise.. · Due to the spectral data sets, PCD-CT enables fully comprehensive post-processing applications.. CITATION FORMAT: · Hagen F, Soschynski M, Weis M et al. Photon-counting computed tomography - clinical application in oncological, cardiovascular, and pediatric radiology. Fortschr Röntgenstr 2024; 196: 25 - 34.

Chemotherapy-induced release of ADAM17 bearing EV as a potential resistance mechanism in ovarian cancer.

Ovarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell-derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)-itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient-derived EV induce AREG shedding and restore chemoresistance in ADAM17-deficient cells. Confirming that ADAM17-containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.

Epithelial-mesenchymal transition in the lacrimal gland morphogenesis, damage and repair.

Epithelial-mesenchymal transition (EMT) constitutes an important pathway in organ fibrosis seen in the lungs, liver, eye, and salivary glands. This review summarizes the EMT observed within the lacrimal gland during its development, tissue damage and repair along with possible translational implications. Existing animal and human studies have reported the increased expression of EMT regulators i.e., transcription factors like Snail, TGF-ß1 within the lacrimal glands, and a possible role of reactive oxygen species, which might be initiating the cascade of EMT. In these studies, EMT is typically detected by reduced E-cadherin expression in the epithelial cells and increased Vimentin and Snail expression within the lacrimal glands' myoepithelial or ductal epithelial cells. Other than specific markers, electron microscopic evidence of disrupted basal lamina, increased collagen deposition, reorganised cytoskeleton of myoepithelial cells also indicated EMT. Very few studies have shown myoepithelial cells to be the cells transitioning into mesenchymal cells with increased extracellular matrix deposition within the lacrimal glands. EMT in animal models seemed reversible as glands got repaired after damage with IL-1α injection or duct ligation and transiently used the EMT as a means for tissue repair. The EMT cells also expressed nestin, a marker for progenitor cells in a rabbit duct ligation model. However, lacrimal glands of ocular graft versus host disease and IgG4 dacryoadenitis demonstrate irreversible acinar atrophy along with signs of EMT-fibrosis, reduced E-cadherin, and increased Vimentin and Snail expression. Future studies exploring the molecular mechanisms of EMT and thereby developing targeted therapies capable of transforming the mesenchymal cells into epithelial cells or blocking the EMT might help in the restoration of the lacrimal gland function.

Multiparametric magnetic resonance imaging for radiation therapy response monitoring in soft tissue sarcomas: a histology and MRI co-registration algorithm.

Rationale: To establish a spatially exact co-registration procedure between in vivo multiparametric magnetic resonance imaging (mpMRI) and (immuno)histopathology of soft tissue sarcomas (STS) to identify imaging parameters that reflect radiation therapy response of STS. Methods: The mpMRI-Protocol included diffusion-weighted (DWI), intravoxel-incoherent motion (IVIM), and dynamic contrast-enhancing (DCE) imaging. The resection specimen was embedded in 6.5% agarose after initial fixation in formalin. To ensure identical alignment of histopathological sectioning and in vivo imaging, an ex vivo MRI scan of the specimen was rigidly co-registered with the in vivo mpMRI. The deviating angulation of the specimen to the in vivo location of the tumor was determined. The agarose block was trimmed accordingly. A second ex vivo MRI in a dedicated localizer with a 4 mm grid was performed, which was matched to a custom-built sectioning machine. Microtomy sections were stained with hematoxylin and eosin. Immunohistochemical staining was performed with anti-ALDH1A1 antibodies as a radioresistance and anti-MIB1 antibodies as a proliferation marker. Fusion of the digitized microtomy sections with the in vivo mpMRI was accomplished through nonrigid co-registration to the in vivo mpMRI. Co-registration accuracy was qualitatively assessed by visual assessment and quantitatively evaluated by computing target registration errors (TRE). Results: The study sample comprised nine tumor sections from three STS patients. Visual assessment after nonrigid co-registration showed a strong morphological correlation of the histopathological specimens with ex vivo MRI and in vivo mpMRI after neoadjuvant radiation therapy. Quantitative assessment of the co-registration procedure using TRE analysis of different pairs of pathology and MRI sections revealed highly accurate structural alignment, with a total median TRE of 2.25 mm (histology - ex vivo MRI), 2.22 mm (histology - in vivo mpMRI), and 2.02 mm (ex vivo MRI - in vivo mpMRI). There was no significant difference between TREs of the different pairs of sections or caudal, middle, and cranial tumor parts, respectively. Conclusion: Our initial results show a promising approach to obtaining accurate co-registration between histopathology and in vivo MRI for STS. In a larger cohort of patients, the method established here will enable the prospective identification and validation of in vivo imaging biomarkers for radiation therapy response prediction and monitoring in STS patients via precise molecular and cellular correlation.

Proteolysis of CD44 at the cell surface controls a downstream protease network.

The cell surface receptor cluster of differentiation 44 (CD44) is the main hyaluronan receptor of the human body. At the cell surface, it can be proteolytically processed by different proteases and was shown to interact with different matrix metalloproteinases. Upon proteolytic processing of CD44 and generation of a C-terminal fragment (CTF), an intracellular domain (ICD) is released after intramembranous cleavage by the γ-secretase complex. This intracellular domain then translocates to the nucleus and induces transcriptional activation of target genes. In the past CD44 was identified as a risk gene for different tumor entities and a switch in CD44 isoform expression towards isoform CD44s associates with epithelial to mesenchymal transition (EMT) and cancer cell invasion. Here, we introduce meprin ß as a new sheddase of CD44 and use a CRISPR/Cas9 approach to deplete CD44 and its sheddases ADAM10 and MMP14 in HeLa cells. We here identify a regulatory loop at the transcriptional level between ADAM10, CD44, MMP14 and MMP2. We show that this interplay is not only present in our cell model, but also across different human tissues as deduced from GTEx (Gene Tissue Expression) data. Furthermore, we identify a close relation between CD44 and MMP14 that is also reflected in functional assays for cell proliferation, spheroid formation, migration and adhesion.

DNA methyltransferase 3A controls intestinal epithelial barrier function and regeneration in the colon.

Genetic variants in the DNA methyltransferase 3 A (DNMT3A) locus have been associated with inflammatory bowel disease (IBD). DNMT3A is part of the epigenetic machinery physiologically involved in DNA methylation. We show that DNMT3A plays a critical role in maintaining intestinal homeostasis and gut barrier function. DNMT3A expression is downregulated in intestinal epithelial cells from IBD patients and upon tumor necrosis factor treatment in murine intestinal organoids. Ablation of DNMT3A in Caco-2 cells results in global DNA hypomethylation, which is linked to impaired regenerative capacity, transepithelial resistance and intercellular junction formation. Genetic deletion of Dnmt3a in intestinal epithelial cells (Dnmt3aΔIEC) in mice confirms the phenotype of an altered epithelial ultrastructure with shortened apical-junctional complexes, reduced Goblet cell numbers and increased intestinal permeability in the colon in vivo. Dnmt3aΔIEC mice suffer from increased susceptibility to experimental colitis, characterized by reduced epithelial regeneration. These data demonstrate a critical role for DNMT3A in orchestrating intestinal epithelial homeostasis and response to tissue damage and suggest an involvement of impaired epithelial DNMT3A function in the etiology of IBD.

Epithelial X-Box Binding Protein 1 Coordinates Tumor Protein p53-Driven DNA Damage Responses and Suppression of Intestinal Carcinogenesis.

BACKGROUND & AIMS: Throughout life, the intestinal epithelium undergoes constant self-renewal from intestinal stem cells. Together with genotoxic stressors and failing DNA repair, this self-renewal causes susceptibility toward malignant transformation. X-box binding protein 1 (XBP1) is a stress sensor involved in the unfolded protein response (UPR). We hypothesized that XBP1 acts as a signaling hub to regulate epithelial DNA damage responses. METHODS: Data from The Cancer Genome Atlas were analyzed for association of XBP1 with colorectal cancer (CRC) survival and molecular interactions between XBP1 and p53 pathway activity. The role of XBP1 in orchestrating p53-driven DNA damage response was tested in vitro in mouse models of chronic intestinal epithelial cell (IEC) DNA damage (Xbp1/H2bfl/fl, Xbp1ΔIEC, H2bΔIEC, H2b/Xbp1ΔIEC) and via orthotopic tumor organoid transplantation. Transcriptome analysis of intestinal organoids was performed to identify molecular targets of Xbp1-mediated DNA damage response. RESULTS: In The Cancer Genome Atlas data set of CRC, low XBP1 expression was significantly associated with poor overall survival and reduced p53 pathway activity. In vivo, H2b/Xbp1ΔIEC mice developed spontaneous intestinal carcinomas. Orthotopic tumor organoid transplantation revealed a metastatic potential of H2b/Xbp1ΔIEC-derived tumors. RNA sequencing of intestinal organoids (H2b/Xbp1fl/fl, H2bΔIEC, H2b/Xbp1ΔIEC, and H2b/p53ΔIEC) identified a transcriptional program downstream of p53, in which XBP1 directs DNA-damage-inducible transcript 4-like (Ddit4l) expression. DDIT4L inhibits mechanistic target of rapamycin-mediated phosphorylation of 4E-binding protein 1. Pharmacologic mechanistic target of rapamycin inhibition suppressed epithelial hyperproliferation via 4E-binding protein 1. CONCLUSIONS: Our data suggest a crucial role for XBP1 in coordinating epithelial DNA damage responses and stem cell function via a p53-DDIT4L-dependent feedback mechanism.

Deficiency in X-linked inhibitor of apoptosis protein promotes susceptibility to microbial triggers of intestinal inflammation.

Inflammatory bowel disease (IBD) is characterized by inappropriate immune responses to the microbiota in genetically susceptible hosts, but little is known about the pathways that link individual genetic alterations to microbiota-dependent inflammation. Here, we demonstrated that the loss of X-linked inhibitor of apoptosis protein (XIAP), a gene associated with Mendelian IBD, rendered Paneth cells sensitive to microbiota-, tumor necrosis factor (TNF)­, receptor-interacting protein kinase 1 (RIPK1)­, and RIPK3-dependent cell death. This was associated with deficiency in Paneth cell­derived antimicrobial peptides and alterations in the stratification and composition of the microbiota. Loss of XIAP was not sufficient to elicit intestinal inflammation but provided susceptibility to pathobionts able to promote granulomatous ileitis, which could be prevented by administration of a Paneth cell­derived antimicrobial peptide. These data reveal a pathway critical for host-microbial cross-talk, which is required for intestinal homeostasis and the prevention of inflammation and which is amenable to therapeutic targeting.

Characterization of the Cancer-Associated Meprin Βeta Variants G45R and G89R.

Meprin ß is a metalloprotease associated with neurodegeneration, inflammation, extracellular matrix homeostasis, transendothelial cell migration, and cancer. In this study, we investigated two melanoma-associated variants of meprin ß, both exhibiting a single amino acid exchange, namely, meprin ß G45R and G89R. Based on the structural data of meprin ß and with regard to the position of the amino acid exchanges, we hypothesized an increase in proteolytic activity in the case of the G45R variant due to the induction of a potential new activation site and a decrease in proteolytic activity from the G89R variant due to structural instability. Indeed, the G89R variant showed, overall, a reduced expression level compared to wild-type meprin ß, accompanied by decreased activity and lower cell surface expression but strong accumulation in the endoplasmic reticulum. This was further supported by the analysis of the shedding of the interleukin-6 receptor (IL-6R) by meprin ß and its variants. In transfected HEK cells, the G89R variant was found to generate less soluble IL-6R, whereas the expression of meprin ß G45R resulted in increased shedding of the IL-6R compared to wild-type meprin ß and the G89R variant. A similar tendency of the induced shedding capacity of G45R was seen for the well-described meprin ß substrate CD99. Furthermore, employing an assay for cell migration in a collagen IV matrix, we observed that the transfection of wild-type meprin ß and the G45R variant resulted in increased migration of HeLa cells, while the G89R variant led to diminished mobility.

Cell Surface Processing of CD109 by Meprin ß Leads to the Release of Soluble Fragments and Reduced Expression on Extracellular Vesicles.

Cluster of differentiation 109 (CD109) is a glycosylphosphatidylinositol (GPI)-anchored protein expressed on primitive hematopoietic stem cells, activated platelets, CD4+ and CD8+ T cells, and keratinocytes. In recent years, CD109 was also associated with different tumor entities and identified as a possible future diagnostic marker linked to reduced patient survival. Also, different cell signaling pathways were proposed as targets for CD109 interference including the TGFß, JAK-STAT3, YAP/TAZ, and EGFR/AKT/mTOR pathways. Here, we identify the metalloproteinase meprin ß to cleave CD109 at the cell surface and thereby induce the release of cleavage fragments of different size. Major cleavage was identified within the bait region of CD109 residing in the middle of the protein. To identify the structural localization of the bait region, homology modeling and single-particle analysis were applied, resulting in a molecular model of membrane-associated CD109, which allows for the localization of the newly identified cleavage sites for meprin ß and the previously published cleavage sites for the metalloproteinase bone morphogenetic protein-1 (BMP-1). Full-length CD109 localized on extracellular vesicles (EVs) was also identified as a release mechanism, and we can show that proteolytic cleavage of CD109 at the cell surface reduces the amount of CD109 sorted to EVs. In summary, we identified meprin ß as the first membrane-bound protease to cleave CD109 within the bait region, provide a first structural model for CD109, and show that cell surface proteolysis correlates negatively with CD109 released on EVs.

Lipid-Iron Nanoparticle with a Cell Stress Release Mechanism Combined with a Local Alternating Magnetic Field Enables Site-Activated Drug Release.

Most available cancer chemotherapies are based on systemically administered small organic molecules, and only a tiny fraction of the drug reaches the disease site. The approach causes significant side effects and limits the outcome of the therapy. Targeted drug delivery provides an alternative to improve the situation. However, due to the poor release characteristics of the delivery systems, limitations remain. This report presents a new approach to address the challenges using two fundamentally different mechanisms to trigger the release from the liposomal carrier. We use an endogenous disease marker, an enzyme, combined with an externally applied magnetic field, to open the delivery system at the correct time only in the disease site. This site-activated release system is a novel two-switch nanomachine that can be regulated by a cell stress-induced enzyme at the cellular level and be remotely controlled using an applied magnetic field. We tested the concept using sphingomyelin-containing liposomes encapsulated with indocyanine green, fluorescent marker, or the anticancer drug cisplatin. We engineered the liposomes by adding paramagnetic beads to act as a receiver of outside magnetic energy. The developed multifunctional liposomes were characterized in vitro in leakage studies and cell internalization studies. The release system was further studied in vivo in imaging and therapy trials using a squamous cell carcinoma tumor in the mouse as a disease model. In vitro studies showed an increased release of loaded material when stress-related enzyme and magnetic field was applied to the carrier liposomes. The theranostic liposomes were found in tumors, and the improved therapeutic effect was shown in the survival studies.

Functional Characterization of Colon-Cancer-Associated Variants in ADAM17 Affecting the Catalytic Domain.

Although extensively investigated, cancer is still one of the most devastating and lethal diseases in the modern world. Among different types, colorectal cancer (CRC) is most prevalent and mortal, making it an important subject of research. The metalloprotease ADAM17 has been implicated in the development of CRC due to its involvement in signaling pathways related to inflammation and cell proliferation. ADAM17 is capable of releasing membrane-bound proteins from the cell surface in a process called shedding. A deficiency of ADAM17 activity has been previously shown to have protective effects against CRC in mice, while an upregulation of ADAM17 activity is suspected to facilitate tumor development. In this study, we characterize ADAM17 variants found in tissue samples of cancer patients in overexpression studies. We here focus on point mutations identified within the catalytic domain of ADAM17 and could show a functional dysregulation of the CRC-associated variants. Since the catalytic domain of ADAM17 is the only region structurally determined by crystallography, we study the effect of each point mutation not only to learn more about the role of ADAM17 in cancer, but also to investigate the structure-function relationships of the metalloprotease.

Liposomal Encapsulated Curcumin Effectively Attenuates Neuroinflammatory and Reactive Astrogliosis Reactions in Glia Cells and Organotypic Brain Slices.

INTRODUCTION: The polyphenolic spice and food coloring ingredient curcumin has beneficial effects in a broad variety of inflammatory diseases. Amongst them, curcumin has been shown to attenuate microglia reaction and prevent from glial scar formation in spinal cord and brain injuries. METHODS: We developed a protocol for the efficient encapsulation of curcumin as a model for anti-inflammatory drugs yielding long-term stable, non-toxic liposomes with favorable physicochemical properties. Subsequently, we evaluate the effects of liposomal curcumin in experimental models for neuroinflammation and reactive astrogliosis. RESULTS: We could show that liposomal curcumin can efficiently reduce the reactivity of human microglia and astrocytes and preserve tissue integrity of murine organotypic cortex slices. DISCUSSION AND PERSPECTIVE: In perspective, we want to administer this curcumin formulation in brain implant coatings to prevent neuroinflammation and glial scar formation as foreign body responses of the brain towards implanted materials.

Meprin ß cleaves TREM2 and controls its phagocytic activity on macrophages.

The triggering receptor expressed on myeloid cells 2 (TREM2) is a multifunctional surface protein that affects survival, migration, and phagocytic capacity of myeloid cells. Soluble TREM2 levels were found to be increased in early stages of sporadic and familial Alzheimer's disease (AD) probably reflecting a defensive microglial response to some initial brain damage. The disintegrin and metalloproteases (ADAM) 10 and 17 were identified as TREM2 sheddases. We demonstrate that meprin ß is a direct TREM2 cleaving enzyme using ADAM10/17 deficient HEK293 cells. LC-MS/MS analysis of recombinant TREM2 incubated with meprin ß revealed predominant cleavage between Arg136 and Asp137, distant to the site identified for ADAM10/17. We further demonstrate that the metalloprotease meprin ß cleaves TREM2 on macrophages concomitant with decreased levels of soluble TREM2 in the serum of Mep1b-/- mice compared to WT controls. Isolated BMDMs from Mep1b-/- mice showed significantly increased full-length TREM2 levels and enhanced phagocytosis efficiency compared to WT cells. The diminished constitutive shedding of TREM2 on meprin ß deficient macrophages could be rescued by ADAM stimulation through LPS treatment. Our data provide evidence that meprin ß is a TREM2 sheddase on macrophages and suggest that multiple proteases may be involved in the generation of soluble TREM2.

SV40 Transfected Human Anterior Cruciate Ligament Derived Ligamentocytes-Suitable as a Human in Vitro Model for Ligament Reconstruction?

Cultured human primary cells have a limited lifespan undergoing dedifferentiation or senescence. Anterior cruciate ligaments (ACL) are hypocellular but tissue engineering (TE) requires high cell numbers. Simian virus (SV) 40 tumor (T) antigen expression could extend the lifespan of cells. This study aimed to identify cellular changes induced by SV40 expression in human ACL ligamentocytes by comparing them with non-transfected ligamentocytes and tissue of the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40-year-old female donor after ACL rupture) were either transfected with a SV40 plasmid or remained non-transfected (control) before monitored for SV40 expression, survival, and DNA content. Protein expression of cultured ligamentocytes was compared with the donor tissue. Ligamentocyte spheroids were seeded on scaffolds embroidered either from polylactic acid (PLA) threads solely or combined PLA and poly (L-lactide-co-ε-caprolactone) (P(LA-CL)) threads. These scaffolds were further functionalized with fluorination and fibrillated collagen foam. Cell distribution and survival were monitored for up to five weeks. The transfected cells expressed the SV40 antigen throughout the entire observation time, but often exhibited random and incomplete cell divisions with significantly more dying cells, significantly more DNA and more numerous nucleoli than controls. The expression profile of non-transfected and SV40-positive ligamentocytes was similar. In contrast to controls, SV40-positive cells formed larger spheroids, produced less vimentin and focal adhesions and died on the scaffolds after 21 d. Functionalized scaffolds supported human ligamentocyte growth. SV40 antigen expressing ligamentocytes share many properties with their non-transfected counterparts suggesting them as a model, however, applicability for TE is limited.

Prdx4 limits caspase-1 activation and restricts inflammasome-mediated signaling by extracellular vesicles.

Inflammasomes are cytosolic protein complexes, which orchestrate the maturation of active IL-1ß by proteolytic cleavage via caspase-1. Although many principles of inflammasome activation have been described, mechanisms that limit inflammasome-dependent immune responses remain poorly defined. Here, we show that the thiol-specific peroxidase peroxiredoxin-4 (Prdx4) directly regulates IL-1ß generation by interfering with caspase-1 activity. We demonstrate that caspase-1 and Prdx4 form a redox-sensitive regulatory complex via caspase-1 cysteine 397 that leads to caspase-1 sequestration and inactivation. Mice lacking Prdx4 show an increased susceptibility to LPS-induced septic shock. This effect was phenocopied in mice carrying a conditional deletion of Prdx4 in the myeloid lineage (Prdx4-ΔLysMCre). Strikingly, we demonstrate that Prdx4 co-localizes with inflammasome components in extracellular vesicles (EVs) from inflammasome-activated macrophages. Purified EVs are able to transmit a robust IL-1ß-dependent inflammatory response in vitro and also in recipient mice in vivo. Loss of Prdx4 boosts the pro-inflammatory potential of EVs. These findings identify Prdx4 as a critical regulator of inflammasome activity and provide new insights into remote cell-to-cell communication function of inflammasomes via macrophage-derived EVs.

The cancer-associated meprin ß variant G32R provides an additional activation site and promotes cancer cell invasion.

The extracellular metalloprotease meprin ß is expressed as a homodimer and is primarily membrane bound. Meprin ß can be released from the cell surface by its known sheddases ADAM10 and ADAM17. Activation of pro-meprin ß at the cell surface prevents its shedding, thereby stabilizing its proteolytic activity at the plasma membrane. We show that a single amino acid exchange variant (G32R) of meprin ß, identified in endometrium cancer, is more active against a peptide substrate and the IL-6 receptor than wild-type meprin ß. We demonstrate that the change to an arginine residue at position 32 represents an additional activation site used by furin-like proteases in the Golgi, which consequently leads to reduced shedding by ADAM17. We investigated this meprin ß G32R variant to assess cell proliferation, invasion through a collagen IV matrix and outgrowth from tumor spheroids. We found that increased meprin ß G32R activity at the cell surface reduces cell proliferation, but increases cell invasion.