Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies

INTRODUCTION: Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration. METHODS: Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age. RESULTS: Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells. CONCLUSIONS: Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration

Glycosylated extracellular mucin domains protect against SARS-CoV-2 infection at the respiratory surface

Mucins play an essential role in protecting the respiratory tract against microbial infections while also acting as binding sites for bacterial and viral adhesins. The heavily O-glycosylated gel-forming mucins MUC5AC and MUC5B eliminate pathogens by mucociliary clearance. Transmembrane mucins MUC1, MUC4, and MUC16 can restrict microbial invasion at the apical surface of the epithelium. In this study, we determined the impact of host mucins and mucin glycans on epithelial entry of SARS-CoV-2. Human lung epithelial Calu-3 cells express the SARS-CoV-2 entry receptor ACE2 and high levels of glycosylated MUC1, but not MUC4 and MUC16, on their cell surface. The O-glycan-specific mucinase StcE specifically removed the glycosylated part of the MUC1 extracellular domain while leaving the underlying SEA domain and cytoplasmic tail intact. StcE treatment of Calu-3 cells significantly enhanced infection with SARS-CoV-2 pseudovirus and authentic virus, while removal of sialic acid and fucose from the epithelial surface did not impact viral entry. In Calu-3 cells, the transmembrane mucin MUC1 and ACE2 are located to the apical surface in close proximity and StcE treatment results in enhanced binding of purified spike protein. Both MUC1 and MUC16 are expressed on the surface of human organoid-derived air-liquid interface (ALI) differentiated airway cultures and StcE treatment led to mucin removal and increased levels of SARS-CoV-2 replication. In these cultures, MUC1 was highly expressed in non-ciliated cells while MUC16 was enriched in goblet cells. In conclusion, the glycosylated extracellular domains of different transmembrane mucins might have similar protective functions in different respiratory cell types by restricting SARS-CoV-2 binding and entry

High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine

Phosphatidylserine exists lumenally in the ER, Golgi, and mitochondria but cytoplasmically in the trans-Golgi and at the plasma membrane, which suggests that functionally important flipping may occur during trafficking

Characterization of bovine embryos cultured under conditions appropriate for sustaining human naïve pluripotency.

In mammalian preimplantation development, pluripotent cells are set aside from cells that contribute to extra-embryonic tissues. Although the pluripotent cell population of mouse and human embryos can be cultured as embryonic stem cells, little is known about the pathways involved in formation of a bovine pluripotent cell population, nor how to maintain these cells in vitro. The objective of this study was to determine the transcriptomic profile related to bovine pluripotency. Therefore, in vitro derived embryos were cultured in various culture media that recently have been reported capable of maintaining the naïve pluripotent state of human embryonic cells. Gene expression profiles of embryos cultured in these media were compared using microarray analysis and quantitative RT-PCR. Compared to standard culture conditions, embryo culture in 'naïve' media reduced mRNA expression levels of the key pluripotency markers NANOG and POU5F1. A relatively high percentage of genes with differential expression levels were located on the X-chromosome. In addition, reduced XIST expression was detected in embryos cultured in naïve media and female embryos contained fewer cells with H3K27me3 foci, indicating a delay in X-chromosome inactivation. Whole embryos cultured in one of the media, 5iLA, could be maintained until 23 days post fertilization. Together these data indicate that 'naïve' conditions do not lead to altered expression of known genes involved in pluripotency. Interestingly, X-chromosome inactivation and development of bovine embryos were dependent on the culture conditions

Inhibition and Eradication of Pseudomonas aeruginosa Biofilms by Host Defence Peptides

P. aeruginosa is a notorious biofilm producer that causes a wide variety of acute and chronic infections. In this study the in vitro anti-biofilm activity of 13 Host Defence Peptides from different species was tested against P. aeruginosa biofilms. Most HDPs were able to prevent biofilm attachment, due to their antimicrobial effect on planktonic bacteria in the starting inoculum. Activity of HDPs against pre-formed biofilms was also observed, although mainly at short incubation times. Several HDPs were able to kill bacteria in the biofilm (colony counting of biofilm associated bacteria) but only CRAMP eradicated the whole biofilm (crystal violet staining). These results were quantitatively confirmed by confocal microscopy studies using a live/dead stain of the biofilms. Furthermore, for chicken CATH-2 (one of the more potent HDPs) it was shown that the peptide could indeed penetrate the biofilm structures and kill bacteria within the biofilm. These studies highlight the potency but also the limitations of HDPs as new potential anti-biofilm agents

Inhibition and Eradication of Pseudomonas aeruginosa Biofilms by Host Defence Peptides

P. aeruginosa is a notorious biofilm producer that causes a wide variety of acute and chronic infections. In this study the in vitro anti-biofilm activity of 13 Host Defence Peptides from different species was tested against P. aeruginosa biofilms. Most HDPs were able to prevent biofilm attachment, due to their antimicrobial effect on planktonic bacteria in the starting inoculum. Activity of HDPs against pre-formed biofilms was also observed, although mainly at short incubation times. Several HDPs were able to kill bacteria in the biofilm (colony counting of biofilm associated bacteria) but only CRAMP eradicated the whole biofilm (crystal violet staining). These results were quantitatively confirmed by confocal microscopy studies using a live/dead stain of the biofilms. Furthermore, for chicken CATH-2 (one of the more potent HDPs) it was shown that the peptide could indeed penetrate the biofilm structures and kill bacteria within the biofilm. These studies highlight the potency but also the limitations of HDPs as new potential anti-biofilm agents

Cholesterol shuttling is important for RNA replication of coxsackievirus B3 and encephalomyocarditis virus

Picornaviruses are a family of positive-strand RNA viruses that represent important human and animal pathogens. Upon infection, picornaviruses induce an extensive remodeling of host cell membranes into replication organelles, which is critical for replication. Membrane lipids and lipid remodeling processes are at the base of RO formation, yet their involvement remains largely obscure. Recently, phosphatidylinositol-4-phosphate (PI4P) was the first lipid discovered to be important for the replication of a number of picornaviruses. Here, we investigate the role of the lipid cholesterol in picornavirus replication. We show that two picornaviruses from distinct genera that rely on different host factors for replication, namely the enterovirus coxsackievirus B3 (CVB3) and the cardiovirus encephalomyocarditis virus (EMCV), both recruited cholesterol to their ROs. Although CVB3 and EMCV both required cholesterol for efficient genome replication, the viruses appeared to rely on different cellular cholesterol pools. Treatments that altered the distribution of endosomal cholesterol inhibited replication of both CVB3 and EMCV, showing the importance of endosomal cholesterol shuttling for the replication of these viruses. Summarizing, we here demonstrate the importance of cholesterol homeostasis for efficient replication of CVB3 and EMCV

Tissue Microarrays to Visualize Influenza D Attachment to Host Receptors in the Respiratory Tract of Farm Animals

The trimeric hemagglutinin-esterase fusion protein (HEF) of influenza D virus (IDV) binds 9-O-acetylated sialic acid receptors, which are expressed in various host species. While cattle are the main reservoir for IDV, the viral genome has also been detected in domestic pigs. In addition, antibodies against IDV have been detected in other farm animals such as sheep, goats, and horses, and even in farmers working with IDV positive animals. Viruses belonging to various IDV clades circulate, but little is known about their differences in host and tissue tropism. Here we used recombinantly produced HEF proteins (HEF S57A) from the major clades D/Oklahoma (D/OK) and D/Oklahoma/660 (D/660) to study their host and tissue tropism and receptor interactions. To this end, we developed tissue microarrays (TMA) composed of respiratory tissues from various farm animals including cattle, domestic pigs, sheep, goats, and horses. Protein histochemical staining of farm animal respiratory tissue-microarrays with HEF proteins showed that cattle have receptors present over the entire respiratory tract while receptors are only present in the nasal and pharyngeal epithelium of pigs, sheep, goats, and horses. No differences in tropism for tissues and animals were observed between clades, while hemagglutination assays showed that D/OK has a 2-fold higher binding affinity than D/660 for receptors on red blood cells. The removal of O-acetylation from receptors via saponification treatment confirmed that receptor-binding of both clades was dependent on O-acetylated sialic acids

Fungicidal mechanisms of cathelicidins LL-37 and CATH-2 revealed by live-cell imaging

Antifungal mechanisms of action of two cathelicidins, chicken CATH-2 and human LL-37, were studied and compared with the mode of action of the salivary peptide histatin 5 (Hst5). Candida albicans was used as a model organism for fungal pathogens. Analysis by live-cell imaging showed that the peptides kill C. albicans rapidly. CATH-2 is the most active peptide and kills C. albicans within 5 min. Both cathelicidins induce cell membrane permeabilization and simultaneous vacuolar expansion. Minimal fungicidal concentrations (MFC) are in the same order of magnitude for all three peptides, but the mechanisms of antifungal activity are very different. The activity of cathelicidins is independent of the energy status of the fungal cell, unlike Hst5 activity. Live-cell imaging using fluorescently labeled peptides showed that both CATH-2 and LL-37 quickly localize to the C. albicans cell membrane, while Hst5 was mainly directed to the fungal vacuole. Small amounts of cathelicidins internalize at sub-MFCs, suggesting that intracellular activities of the peptide could contribute to the antifungal activity. Analysis by flow cytometry indicated that CATH-2 significantly decreases C. albicans cell size. Finally, electron microscopy showed that CATH-2 affects the integrity of the cell membrane and nuclear envelope. It is concluded that the general mechanisms of action of both cathelicidins are partially similar (but very different from that of Hst5). CATH-2 has unique features and possesses antifungal potential superior to that of LL-37

Dendritic cells release exosomes together with phagocytosed pathogen; potential implications for the role of exosomes in antigen presentation

Dendritic cells (DC) have the unique capacity to activate naïve T cells by presenting T cell receptor specific peptides from exogenously acquired antigens bound to Major Histocompatibility Complex (MHC) molecules. MHC molecules are displayed on the DC plasma membrane as well as on extracellular vesicles (EV) that are released by DC, and both have antigen-presenting capacities. However, the physiological role of antigen presentation by EV is still unclear. We here demonstrate that the release of small EV by activated DC is strongly stimulated by phagocytic events. We show that, concomitant with the enhanced release of EV, a significant proportion of phagocytosed bacteria was expulsed back into the medium. High-resolution fluorescence microscopic images revealed that bacteria in phagosomes were surrounded by EV marker-proteins. Moreover, expulsed bacteria were often found associated with clustered HLA II and CD63. Together, these observations suggest that exosomes may be formed by the inward budding into phagosomes, whereupon they are secreted together with the phagosomal content. These findings may have important implications for selective loading of peptides derived from phagocytosed pathogens onto exosome associated HLA molecules, and have important implications for vaccine design