5-Ethynyluridine: A Bio-orthogonal Uridine Variant for mRNA-Based Therapies and Vaccines.

The identification of pseudo- and N1 -methylpseudo-uridine (Ψ and mΨ, respectively) as immunosilent uridine analogues has propelled the development of mRNA-based vaccines and therapeutics. Here, we have characterised another uridine analogue, 5-ethynyluridine (EU), which has an ethynyl moiety. We show that this uridine analogue does not cause immune activation in human macrophages, as it does not induce interleukin-6 secretion or expression of the inflammatory and antiviral genes MX1, PKR, and TAP2. Moreover, EU allows for prolonged expression, as shown with mRNA coding for yellow fluorescent protein (YFP). Side-by-side comparisons of EU with unmodified, Ψ, and mΨ revealed that EU-modified mRNA is expressed at lower levels, but confers similar stability and low immunogenicity to the other uridine analogues. Furthermore, structure analysis of modified mRNAs suggests that the observed phenotype is largely independent of RNA folding. Thus, EU is a potential candidate for RNA-based vaccines and therapeutics.

Atypical cofilin signaling drives dendritic cell migration through the extracellular matrix via nuclear deformation.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-µm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity.

Itaconate promotes a wound resolving phenotype in pro-inflammatory macrophages.

Pathological conditions associated with dysfunctional wound healing are characterized by impaired remodelling of extracellular matrix (ECM), increased macrophage infiltration, and chronic inflammation. Macrophages also play an important role in wound healing as they drive wound closure by secretion of molecules like transforming growth factor beta-1 (TGF-ß). As the functions of macrophages are regulated by their metabolism, local administration of small molecules that alter this might be a novel approach for treatment of wound-healing disorders. Itaconate is a tricarboxylic acid (TCA) cycle-derived metabolite that has been associated with resolution of macrophage-mediated inflammation. However, its effects on macrophage wound healing functions are unknown. In this study, we investigated the effects of the membrane-permeable 4-octyl itaconate (4-OI) derivative on ECM scavenging by cultured human blood monocyte-derived macrophages (hMDM). We found that 4-OI reduced signalling of p38 mitogen-activated protein kinase (MAPK) induced by the canonical immune stimulus lipopolysaccharide (LPS). Likely as a consequence of this, the production of the inflammatory mediators like tumor necrosis factor (TNF)-α and cyclooxygenase (COX)-2 were also reduced. On the transcriptional level, 4-OI increased expression of the gene coding for TGF-ß (TGFB1), whereas expression of the collagenase matrix metalloprotease-8 (MMP8) was reduced. Furthermore, surface levels of the anti-inflammatory marker CD36, but not CD206 and CD11c, were increased in these cells. To directly investigate the effect of 4-OI on scavenging of ECM by macrophages, we developed an assay to measure uptake of fibrous collagen. We observed that LPS promoted collagen uptake and that this was reversed by 4-OI-induced signaling of nuclear factor erythroid 2-related factor 2 (NRF2), a regulator of cellular resistance to oxidative stress and the reduced glycolytic capacity of the macrophage. These results indicate that 4-OI lowers macrophage inflammation, likely promoting a more wound-resolving phenotype.

Cerebral microvascular endothelial cell-derived extracellular vesicles regulate blood - brain barrier function.

Autoreactive T lymphocytes crossing the blood-brain barrier (BBB) into the central nervous system (CNS) play a crucial role in the initiation of demyelination and neurodegeneration in multiple sclerosis (MS). Recently, extracellular vesicles (EV) secreted by BBB endothelial cells (BBB-EC) have emerged as a unique form of cell-to-cell communication that contributes to cerebrovascular dysfunction. However, the precise impact of different size-based subpopulations of BBB-EC-derived EV (BBB-EV) on the early stages of MS remains unclear. Therefore, our objective was to investigate the content and function of distinct BBB-EV subpopulations in regulating BBB integrity and their role in T cell transendothelial migration, both in vitro and in vivo. Our study reveals that BBB-ECs release two distinct size based EV populations, namely small EV (sEV; 30-150 nm) and large EV (lEV; 150-300 nm), with a significantly higher secretion of sEV during inflammation. Notably, the expression patterns of cytokines and adhesion markers differ significantly between these BBB-EV subsets, indicating specific functional differences in the regulation of T cell migration. Through in vitro experiments, we demonstrate that lEV, which predominantly reflect their cellular source, play a major role in BBB integrity loss and the enhanced migration of pro-inflammatory Th1 and Th17.1 cells. Conversely, sEV appear to protect BBB function by inducing an anti-inflammatory phenotype in BBB-EC. These findings align with our in vivo data, where the administration of sEV to mice with experimental autoimmune encephalomyelitis (EAE) results in lower disease severity compared to the administration of lEV, which exacerbates disease symptoms. In conclusion, our study highlights the distinct and opposing effects of BBB-EV subpopulations on the BBB, both in vitro and in vivo. These findings underscore the need for further investigation into the diagnostic and therapeutic potential of BBB-EV in the context of MS.

Assessing the Cervicovaginal Microbiota in the Context of hrHPV Infections: Temporal Dynamics and Therapeutic Strategies.

Cervical cancer is the third leading cause of female cancers globally, resulting in more than 300,000 deaths every year. The majority of all cervical cancers are caused by persistent infections with high-risk human papillomaviruses (hrHPV) that can progress to cancer via a series of premalignant lesions. Most women, however, clear this infection within a year, concomitant with disease regression. Both hrHPV clearance and disease regression have been associated with the composition of the cervicovaginal microenvironment, which is defined by the host immune system and the cervicovaginal microbiome (CVM). A healthy microbiome is generally characterized by a high abundance of Lactobacillus species, and a change in the composition may cause bacterial vaginosis (BV), which is associated with an increased susceptibility to persistent hrHPV infections and disease. In this review, the composition of the CVM is discussed, with emphasis on the possible causes that drive changes in the cervicovaginal microbiota in relation to hrHPV infections, disease progression, and disease regression. The literature search focused on the composition of the CVM and its correlation with hrHPV infections and neoplastic lesions as well as the current efforts to adjust the microbiome against adverse viral outcomes.