Effect of micro- and nanoplastic particles on human macrophages.

Micro- and nanoplastics (MNPs) are ubiquitous in the environment, resulting in the uptake of MNPs by a variety of organisms, including humans, leading to particle-cell interaction. Human macrophages derived from THP-1 cell lines take up Polystyrene (PS), a widespread plastic. The question therefore arises whether primary human macrophages also take up PS micro- and nanobeads (MNBs) and how they react to this stimulation. Major aim of this study is to visualize this uptake and to validate the isolation of macrophages from peripheral blood mononuclear cells (PBMCs) to assess the impact of MNPs on human macrophages. Uptake of macrophages from THP-1 cell lines and PBMCs was examined by transmission electron microscopy (TEM), scanning electron microscopy and live cell imaging. In addition, the reaction of the macrophages was analyzed in terms of metabolic activity, cytotoxicity, production of reactive oxygen species (ROS) and macrophage polarization. This study is the first to visualize PS MNBs in primary human cells using TEM and live cell imaging. Metabolic activity was size- and concentration-dependent, necrosis and ROS were increased. The methods demonstrated in this study outline an approach to assess the influence of MNP exposure on human macrophages and help investigating the consequences of worldwide plastic pollution.

Infrapatellar Fat Pad Modulates Osteoarthritis-Associated Cytokine and MMP Expression in Human Articular Chondrocytes.

Osteoarthritis (OA) most frequently affects the knee joint and is associated with an elevated expression of cytokines and extracellular cartilage matrix (ECM), degrading enzymes such as matrix metalloproteinases (MMPs). Differences in gene expression of the intra-articularly located infrapatellar fat pad (IPFP) and other fatty tissue suggest its autonomous function, yet its role in OA pathogenesis remains unknown. Human IPFPs and articular cartilage were collected from OA patients undergoing total knee arthroplasty, and biopsies from the IPFP of healthy patients harvested during knee arthroscopy served as controls (CO). Isolated chondrocytes were co-cultured with either osteoarthritic (OA) or CO-IPFPs in a transwell system. Chondrocyte expression of MMP1, -3, -13, type 1 and 2 collagens, interleukin IL1ß, IL6, IL10, and tumor necrosis factor TNFα was analyzed by RTD-PCR at day 0 and day 2, and TNFα secretion was analyzed by ELISA. The cytokine release in IPFPs was assessed by an array. Results: Both IPFPs (CO, OA) significantly reduced the expression of type 2 collagen and TNFα in chondrocytes. On the other hand, only CO-IPFP suppressed the expression of type 1 collagen and significantly induced the MMP13 expression. On the contrary, IL1ß and IL6 were significantly induced when exposed to OA-IPFP. Conclusions: The partial loss of the suppressive effect on type 1 collagen gene expression found for OA-IPFP shows the pathological remodeling and dedifferentiation potential of the OA-IPFP on the chondrocytes. However, the significant suppression of TNFα implies that the OA- and CO-IPFP could also exhibit a protective role in the knee joint, preventing the progress of inflammation.

Transient receptor potential channel 3 in human liver and gallbladder - An investigation in body donors.

Since the discovery of TRP proteins in 1969, during studies of the fruit fly Drosophila melanogaster, interest around them and the subfamily of TRPC channels has remained high. TRPC3 was able to be detected in a number of organs in rodents, such as rats and mice, and also in various human tissues. For the most part, these investigations were carried out using gene expression of TRPC3. Further work has already confirmed the relevance of TRPC3 in the context of neurodegenerative diseases, such as spinocerebellar ataxia, and carcinogenic entities, such as ovarian carcinoma. An association with TRPC3 has also been demonstrated for diseases that affect the liver. In order to confirm the expression of TRPC3 in the human liver, this study uses samples taken from eight (n = 8) fixated human body donors and analyzed with immunohistochemistry. In accordance with the macroscopic anatomy of the organs, six samples (n = 6) of liver tissue and three (n = 3) of gallbladder tissue were obtained. TRPC3 was clearly detected in all liver and gallbladder samples examined. Thus, it is not unlikely that TRPC3 plays a role in the extensive metabolic processes of the liver and could also serve as a target for pharmacological interventions in an imbalance of calcium homeostasis.

A Monoclonal Human Alveolar Epithelial Cell Line ("Arlo") with Pronounced Barrier Function for Studying Drug Permeability and Viral Infections.

In the development of orally inhaled drug products preclinical animal models regularly fail to predict pharmacological as well as toxicological responses in humans. Models based on human cells and tissues are potential alternatives to animal experimentation allowing for the isolation of essential processes of human biology and making them accessible in vitro. Here, the generation of a novel monoclonal cell line "Arlo," derived from the polyclonal human alveolar epithelium lentivirus immortalized cell line hAELVi via single-cell printing, and its characterization as a model for the human alveolar epithelium as well as a building block for future complex in vitro models is described. "Arlo" is systematically compared in vitro to primary human alveolar epithelial cells (hAEpCs) as well as to the polyclonal hAELVi cell line. "Arlo" cells show enhanced barrier properties with high transepithelial electrical resistance (TEER) of ≈3000 Ω cm2 and a potential difference (PD) of ≈30 mV under air-liquid interface (ALI) conditions, that can be modulated. The cells grow in a polarized monolayer and express genes relevant to barrier integrity as well as homeostasis as is observed in hAEpCs. Successful productive infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a proof-of-principle study offers an additional, attractive application of "Arlo" beyond biopharmaceutical experimentation.

TGF-Beta Modulates the Integrity of the Blood Brain Barrier In Vitro, and Is Associated with Metabolic Alterations in Pericytes.

The blood-brain barrier (BBB) is a selectively permeable boundary that separates the circulating blood from the extracellular fluid of the brain and is an essential component for brain homeostasis. In glioblastoma (GBM), the BBB of peritumoral vessels is often disrupted. Pericytes, being important to maintaining BBB integrity, can be functionally modified by GBM cells which induce proliferation and cell motility via the TGF-ß-mediated induction of central epithelial to mesenchymal transition (EMT) factors. We demonstrate that pericytes strengthen the integrity of the BBB in primary endothelial cell/pericyte co-cultures as an in vitro BBB model, using TEER measurement of the barrier integrity. In contrast, this effect was abrogated by TGF-ß or conditioned medium from TGF-ß secreting GBM cells, leading to the disruption of a so far intact and tight BBB. TGF-ß notably changed the metabolic behavior of pericytes, by shutting down the TCA cycle, driving energy generation from oxidative phosphorylation towards glycolysis, and by modulating pathways that are necessary for the biosynthesis of molecules used for proliferation and cell division. Combined metabolomic and transcriptomic analyses further underscored that the observed functional and metabolic changes of TGF-ß-treated pericytes are closely connected with their role as important supporting cells during angiogenic processes.

Impaired bidirectional communication between interneurons and oligodendrocyte precursor cells affects social cognitive behavior.

Cortical neural circuits are complex but very precise networks of balanced excitation and inhibition. Yet, the molecular and cellular mechanisms that form the balance are just beginning to emerge. Here, using conditional γ-aminobutyric acid receptor B1- deficient mice we identify a γ-aminobutyric acid/tumor necrosis factor superfamily member 12-mediated bidirectional communication pathway between parvalbumin-positive fast spiking interneurons and oligodendrocyte precursor cells that determines the density and function of interneurons in the developing medial prefrontal cortex. Interruption of the GABAergic signaling to oligodendrocyte precursor cells results in reduced myelination and hypoactivity of interneurons, strong changes of cortical network activities and impaired social cognitive behavior. In conclusion, glial transmitter receptors are pivotal elements in finetuning distinct brain functions.

miRNATissueAtlas2: an update to the human miRNA tissue atlas.

Small non-coding RNAs (sncRNAs) are pervasive regulators of physiological and pathological processes. We previously developed the human miRNA Tissue Atlas, detailing the expression of miRNAs across organs in the human body. Here, we present an updated resource containing sequencing data of 188 tissue samples comprising 21 organ types retrieved from six humans. Sampling the organs from the same bodies minimizes intra-individual variability and facilitates the making of a precise high-resolution body map of the non-coding transcriptome. The data allow shedding light on the organ- and organ system-specificity of piwi-interacting RNAs (piRNAs), transfer RNAs (tRNAs), microRNAs (miRNAs) and other non-coding RNAs. As use case of our resource, we describe the identification of highly specific ncRNAs in different organs. The update also contains 58 samples from six tissues of the Tabula Muris collection, allowing to check if the tissue specificity is evolutionary conserved between Homo sapiens and Mus musculus. The updated resource of 87 252 non-coding RNAs from nine non-coding RNA classes for all organs and organ systems is available online without any restrictions (https://www.ccb.uni-saarland.de/tissueatlas2).

First Responders to Hyperosmotic Stress in Murine Astrocytes: Connexin 43 Gap Junctions Are Subject to an Immediate Ultrastructural Reorganization.

In a short-term model of hyperosmotic stress, primary murine astrocytes were stimulated with a hyperosmolar sucrose solution for five minutes. Astrocytic gap junctions, which are mainly composed of Connexin (Cx) 43, displayed immediate ultrastructural changes, demonstrated by freeze-fracture replica immunogold labeling: their area, perimeter, and distance of intramembrane particles increased, whereas particle numbers per area decreased. Ultrastructural changes were, however, not accompanied by changes in Cx43 mRNA expression. In contrast, transcription of the gap junction regulator zonula occludens (ZO) protein 1 significantly increased, whereas its protein expression was unaffected. Phosphorylation of Serine (S) 368 of the Cx43 C-terminus has previously been associated with gap junction disassembly and reduction in gap junction communication. Hyperosmolar sucrose treatment led to enhanced phosphorylation of Cx43S368 and was accompanied by inhibition of gap junctional intercellular communication, demonstrated by a scrape loading-dye transfer assay. Taken together, Cx43 gap junctions are fast reacting elements in response to hyperosmolar challenges and can therefore be considered as one of the first responders to hyperosmolarity. In this process, phosphorylation of Cx43S368 was associated with disassembly of gap junctions and inhibition of their function. Thus, modulation of the gap junction assembly might represent a target in the treatment of brain edema or trauma.

IL-17C and IL-17RE Promote Wound Closure in a Staphylococcus aureus-Based Murine Wound Infection Model.

The epithelial cytokine interleukin-17C (IL-17C) mediates inflammation through the interleukin 17 receptor E (IL-17RE). Prior studies showed a detrimental role of IL-17C in the pathogenesis of immune-mediated skin diseases (e.g., psoriasis). Here, we examined the role of IL-17C/IL-17RE in wound closure in a Staphylococcus aureus wound infection model. We demonstrate that wound closure is significantly delayed in IL-17RE (Il-17re-/-)- and 17C (Il-17c-/-)-deficient mice. There was no significant difference between WT, Il-17re-/-, and Il-17c-/- mice in the absence of infection. Deficiency for IL-17RE and IL-17C did not significantly affect the elimination of bacteria. IL-17C expression was increased in the epidermis of human S. aureus-infected skin. Our results indicate that the IL-17C/IL-17RE axis contributes to the closure of infected wounds but does not contribute to the elimination of S. aureus.

siRNA delivery to macrophages using aspherical, nanostructured microparticles as delivery system for pulmonary administration.

The delivery of oligonucleotides such as siRNA to the lung is a major challenge, as this group of drugs has difficulties to overcome biological barriers due to its polyanionic character and the associated hydrophilic properties, resulting in inefficient delivery. Especially in diseases such as asthma, chronic obstructive pulmonary disease and cystic fibrosis, where increased proinflammation is present, a targeted RNA therapy is desirable due to the high potency of these oligonucleotides. To address these problems and to ensure efficient uptake of siRNA in macrophages, a microparticulate, cylindrical delivery system was developed. In the first step, this particle system was tested for its aerodynamic characteristics to evaluate the aerodynamic properties to optimize lung deposition. The mass median aerodynamic diameter of 2.52 ± 0.23 µm, indicates that the desired target should be reached. The inhibition of TNF-α release, as one of the main mediators of proinflammatory reactions, was investigated. We could show that our carrier system can be loaded with siRNA against TNF-α. Gel electrophoreses allowed to demonstrate that the load can be incorporated and released without being degraded. The delivery system was found to transport a mass fraction of 0.371% [%w/w] as determined by inductively coupled plasma mass spectroscopy. When investigating the release kinetics, the results showed that several days are necessary to release a major amount of the siRNA indicating a sustained release. The cylindrical microparticles with an aspect ratio of 3.3 (ratio of length divided by width) were then tested in vitro successfully reducing TNF-α release from human macrophages significantly by more than 30%. The developed formulation presents a possible oligonucleotide delivery system allowing due to its internal structure to load and protect siRNA.

Lesions in the lungs of fatal corona virus disease Covid-19.

The corona virus outbreak in Wuhan, China, at the end of 2019 has rapidly evolved into a pandemic which is still virulent in many countries. An infection with SARS-CoV-2 can lead to corona virus disease (Covid-19). This paper presents an overview of the knowledge gained so far with regard to histopathological lung lesions in fatal courses of Covid-19. The main findings were diffuse alveolar damage and micro-angiopathies. These included the development of hyaline membranes, thrombi, endothelial inflammation, haemorrhages and angiogenesis. Overall, the vessel lesions seemed to be more lethal than the diffuse alveolar damage. There was obvious hyperreactivity and hyperinflammation of the cellular immune system. An expanded T-cell memory may explain the increased risk of a severe course in the elderly.

A hydrogel-based in vitro assay for the fast prediction of antibiotic accumulation in Gram-negative bacteria.

The pipeline of antibiotics has been for decades on an alarmingly low level. Considering the steadily emerging antibiotic resistance, novel tools are needed for early and easy identification of effective anti-infective compounds. In Gram-negative bacteria, the uptake of anti-infectives is especially limited. We here present a surprisingly simple in vitro model of the Gram-negative bacterial envelope, based on 20% (w/v) potato starch gel, printed on polycarbonate 96-well filter membranes. Rapid permeability measurements across this polysaccharide hydrogel allowed to correctly predict either high or low accumulation for all 16 tested anti-infectives in living Escherichia coli. Freeze-fracture TEM supports that the macromolecular network structure of the starch hydrogel may represent a useful surrogate of the Gram-negative bacterial envelope. A random forest analysis of in vitro data revealed molecular mass, minimum projection area, and rigidity as the most critical physicochemical parameters for hydrogel permeability, in agreement with reported structural features needed for uptake into Gram-negative bacteria. Correlating our dataset of 27 antibiotics from different structural classes to reported MIC values of nine clinically relevant pathogens allowed to distinguish active from nonactive compounds based on their low in vitro permeability specifically for Gram-negatives. The model may help to identify poorly permeable antimicrobial candidates before testing them on living bacteria.

Degeneration of Lumbar Intervertebral Discs: Characterization of Anulus Fibrosus Tissue and Cells of Different Degeneration Grades.

Intervertebral disc (IVD) herniation and degeneration is a major source of back pain. In order to regenerate a herniated and degenerated disc, closure of the anulus fibrosus (AF) is of crucial importance. For molecular characterization of AF, genome-wide Affymetrix HG-U133plus2.0 microarrays of native AF and cultured cells were investigated. To evaluate if cells derived from degenerated AF are able to initiate gene expression of a regenerative pattern of extracellular matrix (ECM) molecules, cultivated cells were stimulated with bone morphogenetic protein 2 (BMP2), transforming growth factor ß1 (TGFß1) or tumor necrosis factor-α (TNFα) for 24 h. Comparative microarray analysis of native AF tissues showed 788 genes with a significantly different gene expression with 213 genes more highly expressed in mild and 575 genes in severe degenerated AF tissue. Mild degenerated native AF tissues showed a higher gene expression of common cartilage ECM genes, whereas severe degenerated AF tissues expressed genes known from degenerative processes, including matrix metalloproteinases (MMP) and bone associated genes. During monolayer cultivation, only 164 differentially expressed genes were found. The cells dedifferentiated and altered their gene expression profile. RTD-PCR analyses of BMP2- and TGFß1-stimulated cells from mild and severe degenerated AF tissue after 24 h showed an increased expression of cartilage associated genes. TNFα stimulation increased MMP1, 3, and 13 expression. Cells derived from mild and severe degenerated tissues could be stimulated to a comparable extent. These results give hope that regeneration of mildly but also strongly degenerated disc tissue is possible.

Intercellular communication between alveolar epithelial cells and macrophages.

BACKGROUND: The alveolus in the lung tissue is an extremely vulnerable site. Alveolar macrophages control this micro-environment both in states of health and illnesssuch as acute lung injury and infection. It has been reported in mice in vivo that intercellular communication between alveolar macrophages and alveolar epithelial cells is mediated by gap junctions. However, little is known about thismicro-environment in human cells. METHODS: Since this gap junctional intercellular communication is hard to investigate in human tissues, a co-culture model of two human cell lines, one of epithelial and one of macrophage origin, was used. Immunoblot analysis, freeze fracture replica immunolabeling and electron microscopy were performed. RESULTS: Connexin (Cx) 43 protein expression as well as ultrastructurally defined Cx43 gap junctions were detected in co-cultures, yielding evidence of intercellular gap junctions between human alveolar cells of two distinct entities. CONCLUSION: Alveolar macrophages possibly have direct access to the alveolar epithelium via gap junctions in humans, enabling the orchestration of the microenvironment in physiology and disease states.

The Phagocytosis of Blood Leukocytes from Cystic Fibrosis Patients is not Impaired in General.

Impaired phagocytosis of Pseudomonas aeruginosa was found in isolated monocytes of peripheral blood of cystic fibrosis patients, but not in their neutrophils, as reported some years ago. In the present study, we analysed the phagocytic capacity of peripheral blood neutrophils and monocytes of cystic fibrosis patients and of healthy controls. Phagocytosis was determined using a commercial phagocytosis "in whole blood" assay on the basis of fluorescence-labelled opsonized Escherichia coli bacteria and flow cytometry. Venous blood of cystic fibrosis patients and of healthy controls was collected and the phagocytosis assay was performed. No differences in the percentage of phagocytic cells or in the overall phagocytic capacity were found between samples of cystic fibrosis patients and healthy controls either in monocytes or in neutrophils. Thus, our results did not support the hypothesis of a generally reduced phagocytic ability in the peripheral blood immune cells of cystic fibrosis patients.

Discerning the spatio-temporal disease patterns of surgically induced OA mouse models.

Osteoarthritis (OA) is the most common cause of disability in ageing societies, with no effective therapies available to date. Two preclinical models are widely used to validate novel OA interventions (MCL-MM and DMM). Our aim is to discern disease dynamics in these models to provide a clear timeline in which various pathological changes occur. OA was surgically induced in mice by destabilisation of the medial meniscus. Analysis of OA progression revealed that the intensity and duration of chondrocyte loss and cartilage lesion formation were significantly different in MCL-MM vs DMM. Firstly, apoptosis was seen prior to week two and was narrowly restricted to the weight bearing area. Four weeks post injury the magnitude of apoptosis led to a 40-60% reduction of chondrocytes in the non-calcified zone. Secondly, the progression of cell loss preceded the structural changes of the cartilage spatio-temporally. Lastly, while proteoglycan loss was similar in both models, collagen type II degradation only occurred more prominently in MCL-MM. Dynamics of chondrocyte loss and lesion formation in preclinical models has important implications for validating new therapeutic strategies. Our work could be helpful in assessing the feasibility and expected response of the DMM- and the MCL-MM models to chondrocyte mediated therapies.

Facets of Communication: Gap Junction Ultrastructure and Function in Cancer Stem Cells and Tumor Cells.

Gap junction proteins are expressed in cancer stem cells and non-stem cancer cells of many tumors. As the morphology and assembly of gap junction channels are crucial for their function in intercellular communication, one focus of our review is to outline the data on gap junction plaque morphology available for cancer cells. Electron microscopic studies and freeze-fracture analyses on gap junction ultrastructure in cancer are summarized. As the presence of gap junctions is relevant in solid tumors, we exemplarily outline their role in glioblastomas and in breast cancer. These were also shown to contain cancer stem cells, which are an essential cause of tumor onset and of tumor transmission into metastases. For these processes, gap junctional communication was shown to be important and thus we summarize, how the expression of gap junction proteins and the resulting communication between cancer stem cells and their surrounding cells contributes to the dissemination of cancer stem cells via blood or lymphatic vessels. Based on their importance for tumors and metastases, future cancer-specific therapies are expected to address gap junction proteins. In turn, gap junctions also seem to contribute to the unattainability of cancer stem cells by certain treatments and might thus contribute to therapeutic resistance.

Oxygen-Glucose Deprivation in Mouse Astrocytes is Associated with Ultrastructural Changes in Connexin 43 Gap Junctions.

In the intact brain, astrocytes play an important role in a number of physiological functions like spatial buffering of potassium, maintenance of calcium homeostasis, neurotransmitter release, regulation of the cerebral blood flow, and many more. As pathophysiological events upon hypoxic-ischemic brain injury include excitotoxicity by glutamate release as well as oxidative stress, astrocytes and their gap junction-based syncytium are of major relevance for regulating the extent of resulting brain damage. The gap junction protein Connexin (Cx) 43 contributes mainly to the astrocytic intercellular communication. As little is known about the ultrastructural assemblage of Cx43 and its changes in response to hypoxic events, we chose temporary oxygen and glucose deprivation with subsequent reoxygenation (OGD-R) as a metabolic inhibition model of hypoxia in primary murine astrocytes. Gap junction morphology and assembly/disintegration were analyzed at the ultrastructural level using freeze-fracture replica immunolabeling. The exposure of cultured astrocytes to short-term OGD-R resulted in the activation of ERK1/2 (p44/p42), downregulation of Cx43 protein expression, and the rearrangement of Cx43 particles within the cell membrane and within gap junctions. These changes in gap junction morphology were associated with phosphorylation of Cx43 at Serine 368. Analysis of the nearest-neighbor distance within gap junction plaques revealed the loosening of Cx43 particle clusters. Together with the observation of additional connexons being present in the vicinity of gap junction plaques after OGD-R treatment, our study indicates that changes in gap junction assembly are associated with the early phase of hypoxic cell damage.

Spatial and molecular changes of mouse brain metabolism in response to immunomodulatory treatment with teriflunomide as visualized by MALDI-MSI.

Multiple sclerosis (MS) is an immune-mediated neurodegenerative disease of the central nervous system (CNS). One of the most promising recent medications for MS is teriflunomide. Its primary mechanism of action is linked to effects on the peripheral immune system by inhibiting dihydroorotate dehydrogenase (DHODH)-catalyzed de novo pyrimidine synthesis and reducing the expansion of lymphocytes in the peripheral immune system. Some in vitro studies suggested, however, that it can also have a direct effect on the CNS compartment. This potential alternative mode of action depends on the drug's capacity to traverse the blood-brain barrier (BBB) and to exert an effect on the complex network of brain biochemical pathways. In this paper, we demonstrate the application of high-resolution/high-accuracy matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry for molecular imaging of the mouse brain coronal sections from animals treated with teriflunomide. Specifically, in order to assess the effect of teriflunomide on the mouse CNS compartment, we investigated the feasibility of teriflunomide to traverse the BBB. Secondly, we systematically evaluated the spatial and semi-quantitative brain metabolic profiles of 24 different endogenous compounds after 4-day teriflunomide administration. Even though the drug was not detected in the examined cerebral sections (despite the high detection sensitivity of the developed method), in-depth study of the endogenous metabolic compartment revealed noticeable alterations as a result of teriflunomide administration compared to the control animals. The observed differences, particularly for purine and pyrimidine nucleotides as well as for glutathione and carbohydrate metabolism intermediates, shed some light on the potential impact of teriflunomide on the mouse brain metabolic networks. Graphical Abstract.