SARS-CoV-2 is responsible for the development of coronavirus disease 2019 (COVID-19) in infected individuals, who can either exhibit mild symptoms or progress toward a life-threatening acute respiratory distress syndrome (ARDS). Exacerbated inflammation and dysregulated immune responses involving T and myeloid cells occur in COVID-19 patients with severe clinical progression. However, the differential contribution of specific subsets of dendritic cells and monocytes to ARDS is still poorly understood. In addition, the role of CD8⁺ T cells present in the lung of COVID-19 patients and relevant for viral control has not been characterized. Here, we have studied the frequencies and activation profiles of dendritic cells and monocytes present in the blood and lung of COVID-19 patients with different clinical severity in comparison with healthy individuals. Furthermore, these subpopulations and their association with antiviral effector CD8⁺ T cell subsets were also characterized in lung infiltrates from critical COVID-19 patients. Our results indicate that inflammatory transitional and nonclassical monocytes and CD1c⁺ conventional dendritic cells preferentially migrate from blood to lungs in patients with severe COVID-19. Thus, this study increases the knowledge of specific myeloid subsets involved in the pathogenesis of COVID-19 disease and could be useful for the design of therapeutic strategies for fighting SARS-CoV-2 infection.

Extracellular vesicles (EVs) are emerging as potent non‐invasive biomarkers. However, current methodologies are time consuming and difficult to translate to clinical practice. To analyse EV‐encapsulated circulating miRNA, we searched for a quick, easy and economic method to enrich frozen human serum samples for EV. We compared the efficiency of several protocols and commercial kits to isolate EVs. Different methods based on precipitation, columns or filter systems were tested and compared with ultracentrifugation, which is the most classical protocol to isolate EVs. EV samples were assessed for purity and quantity by nanoparticle tracking analysis and western blot or cytometry against major EV protein markers. For biomarker validation, levels of a set of miRNAs were determined in EV fractions and compared with their levels in total serum. EVs isolated with precipitation‐based methods were enriched for a subgroup of miRNAs that corresponded to miRNAs described to be encapsulated into EVs (miR‐126, miR‐30c and miR‐143), while the detection of miR‐21, miR‐16‐5p and miR‐19a was very low compared with total serum. Our results point to precipitation using polyethylene glycol (PEG) as a suitable method for an easy and cheap enrichment of serum EVs for miRNA analyses. The overall performance of PEG was very similar, or better than other commercial precipitating reagents, in both protein and miRNA yield, but in comparison to them PEG is much cheaper. Other methods presented poorer results, mostly when assessing miRNA by qPCR analyses. Using PEG precipitation in a longitudinal study with human samples, we demonstrated that miRNA could be assessed in frozen samples up to 8 years of storage. We report a method based on a cut‐off value of mean of fold EV detection versus serum that provides an estimate of the degree of encapsulation of a given miRNA.

Accumulating evidence on the role of Thrombospondin-1 (TSP-1) in the immune response has emerged during the last years. In spite of the importance of TSP-1 not only as anti-angiogenic factor but also as an immunomodulatory molecule, studies on the role of TSP-1 in psoriasis have been neglected. TSP-1 and CD47 expression were analyzed in skin samples from psoriasis patients and control subjects using RT-PCR and immunofluorescence. Expression of these molecules was also evaluated in peripheral blood CD4+ T cells, moDCs, and circulating primary DCs. The functional role of TSP-1/CD47 signaling axis in psoriasis was assessed in Th17 and Treg differentiation assays. Additionally, small interfering RNA assays specific to TSP-1 were performed in CD4+ T cells and monocyte derived DC to specifically evaluate the function of this protein. Lesional skin of psoriasis patients expressed lower TSP-1 and CD47 mRNA levels compared to non-lesional skin or skin from controls. Immunofluorescence staining revealed decreased expression of CD47 in CD45+ dermal cells from psoriasis samples compared to control subjects. Peripheral CD4+ T cells and circulating primary DCs from psoriasis also expressed lower levels of CD47 compared to controls. Although no significant differences were detected in TSP-1 expression in CD4+ T cells and moDCs between patients and controls, TSP-1 expression in psoriasis patients inversely correlated with disease activity evaluated by the Psoriasis Area and Index Activity. Furthermore, exogenous TSP-1 inhibited Th17 differentiation and stimulated the differentiation of CD4+ T cells toward Treg cells. Furthermore, RNA interference specific for TSP-1 confirmed the role of this molecule as a negative regulator of T cell activation. Because of the impact of TSP-1/CD47 signaling axis in Th17 and Treg differentiation, a dysregulated expression of these molecules in the immune cells from psoriasis patients may favor the exacerbated inflammatory response in this disease.

The interplay between T cells, dendritic cells and keratinocytes is crucial for the development and maintenance of inflammation in psoriasis. GADD45 proteins mediate DNA repair in different cells including keratinocytes. In the immune system, GADD45a and GADD45b regulate the function and activation of both T lymphocytes and dendritic cells and GADD45a links DNA repair and epigenetic regulation through its demethylase activity. Here, we analyzed the expression of GADD45a and GADD45b in the skin, dendritic cells and circulating T cells in a cohort of psoriasis patients and their regulation by inflammatory signals. Thirty patients (17 male/13 female) with plaque psoriasis and 15 controls subjects (7 male/8 female), were enrolled. Psoriasis patients exhibited a lower expression of GADD45a at the epidermis but a higher expression in dermal infiltrating T cells in lesional skin. The expression of GADD45a and GADD45b was also higher in peripheral T cells from psoriasis patients, although no differences were observed in p38 activation. The expression and methylation state of the GADD45a target UCHL1 were evaluated, revealing a hypermethylation of its promoter in lesional skin compared to controls. Furthermore, reduced levels of GADD45a correlated with a lower expression UCHL1 in lesional skin. We propose that the demethylase function of GADD45a may account for its pleiotropic effects, and the complex and heterogeneous pattern of expression observed in psoriatic disease.