CD4+ and CD8+ regulatory T cells characterization in the rat using a unique transgenic Foxp3-EGFP model.

Background: CD4+ and CD8+ regulatory T cells (Treg) in diverse species include different subsets from different origins. In all species, CD8+ Treg have been poorly characterized. CD4+ and CD8+ Treg in rats have only partially been characterized and there is no rat model in which FOXP3+ Treg are genetically tagged. Results: We generated a rat transgenic line using the CRISPR/Cas9 system in which EGFP was inserted in frame on the 3 end of the Foxp3 gene using a 2A self-cleaving peptide. EGFP was exclusively expressed by CD4+ and CD8+ T cells in similar proportion as observed with anti-FOXP3 antibodies. CD4+EGFP+ Treg were 5-10 times more frequent than CD8+EGFP+ Treg. CD4+ and CD8+ EGFP+ Treg expressed both the CD25highCD127lowCD45RClow/- markers. The suppressive activity of CD4+ and CD8+ Treg was largely confined to EGFP+ cells. RNAseq analyses showed similarities but also differences among CD4+ and CD8+ EGFP+ cells and provided the first description of the natural FOXP3+ CD8+ Treg transcriptome. In vitro culture of CD4+ and CD8+ EGFP- cells with TGFbeta and IL-2 resulted in the induction of EGFP+ Treg. Preferential expansion of CD4+ and CD8+ EGFP+ Treg could be detected upon in vivo administration of a low dose of IL-2. Conclusions: This new and unique Foxp3-EGFP rat line constitutes a useful model to identify and isolate viable natural and induced CD4+ and CD8+ Treg. Additionally, it allows to identify new molecules expressed in CD8+ Treg that may allow to better define their phenotype and function not only in rats but also in other species.

Inhibiting ACSL1 related ferroptosis restrains MHV-A59 infection

Murine hepatitis virus strain A59 (MHV-A59) belongs to the β-coronavirus and is considered as a representative model for studying coronavirus infection. MHV-A59 was shown to induce pyroptosis, apoptosis and necroptosis of infected cells, especially the murine macrophages. However, whether ferroptosis, a recently identified form of lytic cell death, was involved in the pathogenicity of MHV-A59, is unknown. Here, we demonstrate inhibiting ferroptosis suppresses MHV-A59 infection. MHV-A59 infection upregulates the expression of Acsl1, a novel ferroptosis inducer. MHV-A59 upregulates Acsl1 expression depending on the NF-kB activation, which is TLR4-independent. Ferroptosis inhibitor inhibits viral propagation, inflammatory cytokines release and MHV-A59 infection induced cell syncytia formation. ACSL1 inhibitor Triacsin C suppresses MHV-A59 infection induced syncytia formation and viral propagation. In vivo administration of liproxstatin-1 ameliorates lung inflammation and tissue injuries caused by MHV-A59 infection. Collectively, these results indicate that ferroptosis inhibition protects hosts from MHV-A59 infection. Targeting ferroptosis may serves as a potential treatment approach for dealing with hyper-inflammation induced by coronavirus infection.

Preconditioned Mesenchymal Stromal Cells to Improve Allotransplantation Outcome

Mesenchymal stromal cells (MSCs) are tissue-derived progenitor cells with immunomodulatory as well as multilineage differentiation capacities, and have been widely applied as cellular therapeutics in different disease systems in both preclinical models and clinical studies. Although many studies have applied MSCs in different types of allotransplantation, the efficacy varies. It has been demonstrated that preconditioning MSCs prior to in vivo administration may enhance their efficacy. In the field of organ/tissue allotransplantation, many recent studies have shown that preconditioning of MSCs with (1) pretreatment with bioactive factors or reagents such as cytokines, or (2) specific gene transfection, could prolong allotransplant survival and improve allotransplant function. Herein, we review these preconditioning strategies and discuss potential directions for further improvement.

Blocking Jak/STAT signalling using tofacitinib inhibits angiogenesis in experimental arthritis

Objective During rheumatoid arthritis (RA), the angiogenic processes, occurring with pannus-formation, may be a therapeutic target. JAK/STAT-pathway may play a role and the aim of this work was to investigate the inhibiting role of a JAK-inhibitor, tofacitinib, on the angiogenic mechanisms occurring during RA. Methods After ethical approval, JAK-1, JAK-3, STAT-1, STAT-3 and VEGF expression was evaluated on RA-synovial-tissues. In vitro, endothelial cells (ECs), stimulated with 20 ng/ml of VEGF and/or 1 μM of tofacitinib, were assessed for tube formation, migration and proliferation, by Matrigel, Boyden chamber assay and ki67 gene-expression. In vivo, 32 mice received collagen (collagen-induced arthritis (CIA)) and 32 mice PBS (control). At day 19, CIA and controls mice were divided: 16 mice receiving vehicle and 16 mice receiving tofacitinib. At day 35, the arthritis score, the thickness of paw joints and the serum levels of VEGF and Ang-2 were evaluated. Results The expression of JAK-1, JAK-3, STAT-1, STAT-3 and VEGF in synovial tissue of RA-patients were significantly higher than healthy controls. In vitro, tofacitinib inhibited the ECs ability to form vessels, to proliferate and to migrate. In vivo, administration of tofacitinib prevented the increase of the arthritis score, the paw thickness, the synovial vessels and VEGF and Ang-2 serum-accumulation, when compared to CIA without tofacitinib. Conclusions We explored the anti-angiogenic role of tofacitinib, reporting its ability to inhibit in vitro the angiogenic mechanisms of ECs and in vivo the formation of new synovial vessels, occurring in CIA model. These findings suggest that the therapeutic effect of tofacitinib during RA may be also related to its anti-angiogenic activity.

Unraveling the In Vivo Protein Corona

Understanding the behavior of nanoparticles upon contact with a physiological environment is of urgent need in order to improve their properties for a successful therapeutic application. Most commonly, the interaction of nanoparticles with plasma proteins are studied under in vitro conditions. However, this has been shown to not reflect the complex situation after in vivo administration. Therefore, here we focused on the investigation of magnetic nanoparticles with blood proteins under in vivo conditions. Importantly, we observed a radically different proteome in vivo in comparison to the in vitro situation underlining the significance of in vivo protein corona studies. Next to this, we found that the in vivo corona profile does not significantly change over time. To mimic the in vivo situation, we established an approach, which we termed “ex vivo” as it uses whole blood freshly prepared from an animal. Overall, we present a comprehensive analysis focusing on the interaction between nanoparticles and blood proteins under in vivo conditions and how to mimic this situation with our ex vivo approach. This knowledge is needed to characterize the true biological identity of nanoparticles.

Aging Process of Polyamidoamine Dendrimers: Effect of pH and Shaking in the Fluorescence Emission and Aggregation-State

In the last years, it has been discovered and intensely studied the non-traditional intrinsic fluorescence of PAMAM dendrimers. Nevertheless, their aging process in aqueous suspension is scarcely studied, being unknown the causes of the observed changes in their fluorescence properties. Hence, this work aims to characterize the PAMAM dendrimers of generation 4.0 (DG4.0) and 4.5 (DG4.5) through the aging process at three different pH conditions, stored with or without shaking. We studied, up to 16 days, the UV-Vis absorption, the fluorescence emission, and the size of dendrimers/aggregates. In a different way than the already published work, we demonstrated that there is no chemical change in dendrimers through the aging process, even though changes in fluorescence emission were observed. Besides, we have put in evidence that changes in the agglomeration patterns of dendrimers would not be related to change in the fluorescence emission thought aging. Moreover, we demonstrated that DG4.5 formed large aggregates in water that need to be disrupted by shaking previous to an in vivo administration. <br>

Aging Process of Polyamidoamine Dendrimers: Effect of pH and Shaking in the Fluorescence Emission and Aggregation-State

In the last years, it has been discovered and intensely studied the non-traditional intrinsic fluorescence of PAMAM dendrimers. Nevertheless, their aging process in aqueous suspension is scarcely studied, being unknown the causes of the observed changes in their fluorescence properties. Hence, this work aims to characterize the PAMAM dendrimers of generation 4.0 (DG4.0) and 4.5 (DG4.5) through the aging process at three different pH conditions, stored with or without shaking. We studied, up to 16 days, the UV-Vis absorption, the fluorescence emission, and the size of dendrimers/aggregates. In a different way than the already published work, we demonstrated that there is no chemical change in dendrimers through the aging process, even though changes in fluorescence emission were observed. Besides, we have put in evidence that changes in the agglomeration patterns of dendrimers would not be related to change in the fluorescence emission thought aging. Moreover, we demonstrated that DG4.5 formed large aggregates in water that need to be disrupted by shaking previous to an in vivo administration. <br>