Kelen Cristina Ribeiro Malmegrim

The COVID-19 pandemic, caused by SARS-CoV-2, has significantly affected global health, with severe inflammatory responses leading to tissue damage and persistent symptoms. Macrophage-derived extracellular vesicles (EVs) are involved in the modulation of immune responses, but their involvement in SARS-CoV-2-induced inflammation and senescence remains unclear. Triggering receptors expressed on myeloid cell-1 (TREM-1) are myeloid cell receptors that amplify inflammation, described as a biomarker of the severity and mortality of COVID-19. This study investigated the composition and effects of macrophage-derived EVs stimulated by SARS-CoV-2 (MφV-EVs) on the recipient cell response. Our results, for the first time, show that SARS-CoV-2 stimulation modifies the cargo profile of MφV-EVs, enriching them with TREM-1 and miRNA-155 association, along with MMP-9 and IL-8/CXCL8. These EVs carry senescence-associated secretory phenotype (SASP) components, promote cellular senescence, and compromise antibacterial defenses upon internalization. Our findings provide evidence that MφV-EVs are key drivers of inflammation and immune dysfunction, underscoring their potential as therapeutic targets in COVID-19.

Over the last three decades, autologous hematopoietic stem cell transplantation (AHSCT) has emerged as a significant therapeutic strategy for patients with various refractory autoimmune diseases. Globally, more than 3000 AHSCT procedures have been performed for severe autoimmune diseases. The rationale behind this treatment is abrogation of autoreactivity; renovation of the immune system from the infused hematopoietic stem cells and establishment of a balanced, long-lasting, and self-tolerant immune system. Thymic rebound is a central event in the mechanisms of action of transplantation, promoting the generation of a new repertoire of T cells and reinstating self-tolerance. Here, we critically review the immunological mechanisms of AHSCT in various autoimmune disease settings, with a central focus on thymic rejuvenation. We understand that elucidating the mechanisms of action of the transplant and conducting immunological monitoring studies are crucial for comprehending the risks, benefits, and long-term efficacy of the treatment, thereby promoting functional improvements in patients with autoimmune diseases refractory to conventional treatment.

Extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) have shown increasing therapeutic potential in the last years. However, large production of EV is required for therapeutic purposes. Thereby, scaling up MSC cultivation in bioreactors is essential to allow culture parameters monitoring. In this study, we reported the establishment of a scalable bioprocess to produce MSC‐EV in suspension cultures using spinner flasks and human collagen‐coated microcarriers (3D culture system). We compared the EV production in this 3D culture system with the standard static culture using T‐flasks (2D culture system). The EV produced in both systems were characterized and quantify by western blotting and nanoparticle tracking analysis. The presence of the typical protein markers CD9, CD63, and CD81 was confirmed by western blotting analyses for EV produced in both culture systems. The cell fold‐increase was 5.7‐fold for the 3D culture system and 4.6‐fold for the 2D culture system, signifying a fold‐change of 1.2 (calculated as the ratio of fold‐increase 3D to fold‐increase 2D). Furthermore, it should be noted that the total cell production in the spinner flask cultures was 4.8 times higher than that in T‐flask cultures. The total cell production in the spinner flask cultures was 5.2‐fold higher than that in T‐flask cultures. While the EV specific production (particles/cell) in T‐flask cultures (4.40 ± 1.21 × 10 ⁸ particles/mL, p < 0.05) was higher compared to spinner flask cultures (2.10 ± 0.04 × 10 ⁸ particles/mL, p < 0.05), the spinner flask culture system offers scalability, making it capable of producing enough MSC‐EV at a large scale for clinical applications. Therefore, we concluded that 3D culture system evaluated here serves as an efficient transitional platform that enables the scaling up of MSC‐EV production for therapeutic purposes by utilizing stirred tank bioreactors and maintaining xeno‐free conditions.

Objectives: Allogeneic haematopoietic stem cell transplantation (allo‐HSCT) is the only currently available curative treatment for sickle cell disease (SCD). Here, we comprehensively evaluated the reconstitution of T‐ and B‐cell compartments in 29 SCD patients treated with allo‐HSCT and how it correlated with the development of acute graft‐versus‐host disease (aGvHD).Methods: T‐cell neogenesis was assessed by quantification of signal‐joint and β‐chain TCR excision circles. B‐cell neogenesis was evaluated by quantification of signal‐joint and coding‐joint K‐chain recombination excision circles. T‐ and B‐cell peripheral subset numbers were assessed by flow cytometry.Results: Before allo‐HSCT (baseline), T‐cell neogenesis was normal in SCD patients compared with age‐, gender‐ and ethnicity‐matched healthy controls. Following allo‐HSCT, T‐cell neogenesis declined but was fully restored to healthy control levels at one year post‐transplantation. Peripheral T‐cell subset counts were fully restored only at 24 months post‐transplantation. Occurrence of acute graft‐versus‐host disease (aGvHD) transiently affected T‐ and B‐cell neogenesis and overall reconstitution of T‐ and B‐cell peripheral subsets. B‐cell neogenesis was significantly higher in SCD patients at baseline than in healthy controls, remaining high throughout the follow‐up after allo‐HSCT. Notably, after transplantation SCD patients showed increased frequencies of IL‐10‐producing B‐regulatory cells and IgM⁺memory B‐cell subsets compared with baseline levels and with healthy controls.Conclusion: Our findings revealed that the T‐ and B‐cell compartments were normally reconstituted in SCD patients after allo‐HSCT. In addition, the increase of IL‐10‐producing B‐regulatory cells may contribute to improve immune regulation and homeostasis after transplantation.

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is the only currently available curative treatment for sickle cell disease (SCD). However, the effects of HSCT on SCD pathophysiology are poorly elucidated. Here, we assessed red blood cell (RBC) adhesiveness, intensity of hemolysis, vascular tone markers and systemic inflammation, in SCD patients treated with allogeneic HSCT. Thirty-two SCD patients were evaluated before and on long-term follow-up after HSCT. Overall survival was 94% with no severe (grade III-IV) graft-vs-host disease and a 22% rejection rate (graft failure). Hematological parameters, reticulocyte counts, and levels of lactate dehydrogenase (LDH), endothelin-1 and VCAM-1 normalized in SCD patients post-HSCT. Expression of adhesion molecules on reticulocytes and RBC was lower in patients with sustained engraftment. Levels of IL-18, IL-15 and LDH were higher in patients that developed graft failure. Increased levels of plasma pro-inflammatory cytokines, mainly TNF-α, were found in SCD patients long-term after transplantation. SCD patients with sustained engraftment after allo-HSCT showed decreased reticulocyte counts and adhesiveness, diminished hemolysis, and lower levels of vascular tonus markers. Nevertheless, systemic inflammation persists for at least five years after transplantation, indicating that allo-HSCT does not equally affect all aspects of SCD pathophysiology.

The rationale of autologous haematopoietic stem cell transplantation (AHSCT) for autoimmune diseases is that high-dose immunosuppression eradicates autoreactive T and B cells and the infused autologous haematopoietic stem cells promote reconstitution of a naïve and self-tolerant immune system. The aim of this study was to evaluate the reconstitution of different B cell subsets, both quantitatively and functionally, in SSc patients treated with AHSCT. Peripheral blood was harvested from 22 SSc patients before transplantation and at 30, 60, 120, 180 and 360 days post-AHSCT. Immunophenotyping of B cell subsets, B cell cytokine production, signalling pathways and suppressive capacity of regulatory B cells (Bregs) were assessed by flow cytometry. Naïve B cell frequencies increased from 60 to 360 days post-AHSCT compared with pre-transplantation. Conversely, memory B cell frequencies decreased during the same period. Plasma cell frequencies transiently decreased at 60 days post-AHSCT. IL-10-producing Bregs CD19⁺CD24^hiCD38^hi and CD19⁺CD24^hiCD27⁺ frequencies increased at 180 days. Moreover, the phosphorylation of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase increased in B cells reconstituted post-AHSCT. Notably, CD19⁺CD24^hiCD38^hi Bregs recovered their ability to suppress production of Th1 cytokines by CD4⁺ T cells at 360 days post-AHSCT. Finally, IL-6 and TGF-β1-producing B cells decreased following AHSCT. Taken together, these results suggest improvements in immunoregulatory and anti-fibrotic mechanisms after AHSCT for SSc, which may contribute to re-establishment of self-tolerance and clinical remission.

Mesenchymal stromal cells (MSCs) constitute a heterogeneous population of stromal cells with immunomodulatory and regenerative properties that support their therapeutic use. MSCs isolated from many tissue sources replicate vigorously in vitro and maintain their main biological properties allowing their widespread clinical application. To date, most MSC-based preclinical and clinical trials targeted immune-mediated and inflammatory diseases. Nevertheless, MSCs have antiviral properties and have been used in the treatment of various viral infections in the last years. Here, we revised in detail the biological properties of MSCs and their preclinical and clinical applications in viral diseases, including the disease caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection (COVID-19). Notably, rapidly increasing numbers of MSC-based therapies for COVID-19 have recently been reported. MSCs are theoretically capable of reducing inflammation and promote lung regeneration in severe COVID-19 patients. We critically discuss the rationale, advantages and disadvantages of MSC-based therapies for viral infections and also specifically for COVID-19 and point out some directions in this field. Finally, we argue that MSC-based therapy may be a promising therapeutic strategy for severe COVID-19 and other emergent respiratory tract viral infections, beyond the viral infection diseases in which MSCs have already been clinically applied. Graphical Abstract