Natural killer cells and their exosomes in viral infections and related therapeutic approaches: where are we?

Innate immunity is the first line of the host immune system to fight against infections. Natural killer cells are the innate immunity lymphocytes responsible for fighting against virus-infected and cancerous cells. They have various mechanisms to suppress viral infections. On the other hand, viruses have evolved to utilize different ways to evade NK cell-mediated responses. Viruses can balance the response by regulating the cytokine release pattern and changing the proportion of activating and inhibitory receptors on the surface of NK cells. Exosomes are a subtype of extracellular vesicles that are involved in intercellular communication. Most cell populations can release these nano-sized vesicles, and it was shown that these vesicles produce identical outcomes to the originating cell from which they are released. In recent years, the role of NK cell-derived exosomes in various diseases including viral infections has been highlighted, drawing attention to utilizing the therapeutic potential of these nanoparticles. In this article, the role of NK cells in various viral infections and the mechanisms used by viruses to evade these important immune system cells are initially examined. Subsequently, the role of NK cell exosomes in controlling various viral infections is discussed. Finally, the current position of these cells in the treatment of viral infections and the therapeutic potential of their exosomes are reviewed. Video Abstract.

Bleeding disorder of unknown cause: Results from Iranian study.

BACKGROUND: Definite diagnosis of patients with mild to moderate bleeding is challenging. Some studies reported that even more than 50% of their patients remained undiagnosed which is classified as a Bleeding disorder of unknown cause (BDUC). This study aims to document the clinical characteristics and proportion of patients with BDUC in the Iranian Comprehensive Hemophilia Care Center (ICHCC) one of the referral centers for diagnosis of congenital bleeding disorder in Iran. METHODS: This study was conducted on 397 patients who were referred with a bleeding manifestation to ICHCC from 2019 to 2022. Demographic and laboratory data were documented for all patients. Bleeding questionnaires including ISTH-Bleeding Assessment tool (ISTH-BAT) and the Molecular and Clinical Markers for the Diagnosis and Management of Type 1 (MCMDM-1 (ISTH-BAT, MCMDM-1, and the Pictorial Bleeding Assessment Chart (PBLAC) were filled out for all patients. The data were analyzed by the statistical package for social science (SPSS version 22, SPSS, Chicago, Illinois, USA). RESULTS: BDUC was diagnosed in 200 patients and 197 patients reached the final diagnosis. Hemophilia, von Willebrand disease (VWD), factor (F) VII deficiency, and platelet functional disorders (PFDs) were confirmed in 54, 49, 34, and 15 of the patients, respectively. No significant difference was found in bleeding scores between patients with BDUC and those with confirmed disease. In contrast, after setting cut-off (ISTH-BAT for males ≥ 4 and females ≥ 6 and MCMDM-1 for males ≥ 3 and females ≥ 5) clinically significant difference was found. There was no association between having a positive consanguineous marriage and setting a diagnosis; however, significant associations were seen for having a positive family history of bleeding. Age (OR =0.977, 95% CI.965-0.989), gender (BDUC female, 151/200; final diagnosis female, 95/197) (OR=3.3, 95% CI 2.16-5.06), family history (OR = 3.19, 95% CI 1.99-5.11), and consanguineous marriage (OR=1.59, 95% CI 1.03-2.45) were considered as a risk factor for categorizing the patients with BDUC or final diagnosis. CONCLUSION: The findings are mainly in line with previous studies about BDUC patients. The large number of patients with BDUC underlines the incompleteness of available routine laboratory tests and shows the necessity of progress in the development of reliable diagnostic tools to identify underlying bleeding disorders.

Natural killer cells in COVID-19: from infection, to vaccination and therapy.

Natural killer (NK) cells are among the most important innate immunity members, which are the first cells that fight against infected cells. The function of these cells is impaired in patients with COVID-19 and they are not able to prevent the spread of the disease or destroy the infected cells. Few studies have evaluated the effects of COVID-19 vaccines on NK cells, though it has been demonstrated that DNA vaccines and BNT162b2 can affect NK cell response. In the present paper, the effects of SARS-CoV-2 on the NK cells during infection, the effect of vaccination on NK cells, and the NK cell-based therapies were reviewed.

The Role of NK Cells and Their Exosomes in Graft Versus Host Disease and Graft Versus Leukemia.

Natural killer (NK) cells are one of the innate immune cells that play an important role in preventing and controlling tumors and viral diseases, but their role in hematopoietic stem cell transplantation (HCT) is not yet fully understood. However, according to some research, these cells can prevent infections and tumor relapse without causing graft versus host disease (GVHD). In addition to NK cells, several studies are about the anti-leukemia effects of NK cell-derived exosomes that can highlight their roles in graft-versus-leukemia (GVL). In this paper, we intend to investigate the results of various articles on the role of NK cells in allogeneic hematopoietic cell transplantation and also their exosomes in GVL. Also, we have discussed the antiviral effects of these cells in post-HCT cytomegalovirus infection.

Wharton's Jelly Mesenchymal Stem Cells Exosomes and Conditioned Media Increased Neutrophil Lifespan and Phagocytosis Capacity.

Neutrophils are the first cells involved in inflammation and pathogen elimination, but they have a short lifespan. So, strategies for enhancing neutrophil lifespan and activities can be useful in many situations such as patients with immunodeficiencies. Previous researches demonstrated that mesenchymal stem cell (MSC) has anti-apoptotic effects on neutrophils. These multipotent cells have immunomodulatory properties and can be isolated from different tissues. MSCs isolated from Wharton's jelly (WJ-MSCs), a mucosal connective tissue of the umbilical cord, may be better candidates than MSCs obtained from bone marrow or adipose tissue, because WJ-MSCs are younger and protected from damages that are resulted from aging, environmental toxins, and diseases. In addition, they have high proliferative capacity, easier accessibility, and more abundance. It was shown that following in vitro expansion, they are more effective than other sources of MSCs. Cell to cell contact or secretion of soluble factors and exosomes are the main approaches of MSCs in applying their effects. Exosomes and conditioned media (CM) were prepared from WJ-MSCs. Then, neutrophils were isolated and cultured with medium, CM, or exosomes. Then, neutrophil respiratory burst, apoptosis, and phagocytosis capacity were assessed by NBT assay, Annexin V-PI method, and Giemsa staining, respectively. Both treatments improved neutrophil lifespan and phagocytosis. Only MSC-CM could enhance neutrophil respiratory burst. This research demonstrated that MSC-exosomes and CM have protective effects on neutrophil function and lifespan. It can be concluded that MSC mediators can be responsible factors for protective functions of MSCs on neutrophils.

Improving the function of neutrophils from chronic granulomatous disease patients using mesenchymal stem cells' exosomes.

In chronic granulomatous disease (CGD) patients, reactive oxygen species (ROS) production by neutrophils is impaired. So, they are susceptible to infections. Studies showed that, mesenchymal stem cells (MSCs) have protective effects on the function of neutrophils and an approach that MSCs use to apply their effects, is secreting soluble factors and exosomes. So, we investigated the effects of MSC-exosomes and MSC-conditioned media (MSC-CM) on the function and apoptosis of neutrophils in CGD patients. In this study, neutrophils were isolated from healthy donors and CGD patients and then incubated with exosomes or CM that were prepared from MSCs. Then, neutrophil respiratory burst, apoptosis and phagocytosis capacity were evaluated by NBT assay, Annexin V-PI method and Giemsa staining. It was demonstrated that both MSC-exosomes and CM could improve the phagocytosis capacity and ROS production of neutrophils in CGD patients and healthy donors. In contrast to the healthy group, in CGD patients, exosomes significantly reduced the percentage of viable neutrophils. This report indicated that MSC exosomes and CM could increase the function of the neutrophils isolated from CGD patients. But decreasing the number of the living cells is one of the limitations of them. However, it is hoped that this intervention will be developed in future studies to minimize its limitations.

Hypothesis for the management and treatment of the COVID-19-induced acute respiratory distress syndrome and lung injury using mesenchymal stem cell-derived exosomes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a member of the coronaviridae that causes respiratory disorders. After infection, large amounts of inflammatory cytokines are secreted, known as the cytokine storm. These cytokines can cause pulmonary damage induced by inflammation resulting in acute respiratory distress syndrome (ARDS), and even death. One of the therapeutic approaches for treatment of ARDS is a mesenchymal stem cell (MSC). MSCs suppress inflammation and reduce lung injury through their immunomodulatory properties. MSCs also have the potential to prevent apoptosis of the lung cells and regenerate them. But our suggestion is using MSCs-derived exosomes. Because these exosomes apply the same immunomodulatory and tissue repair effects of MSCs and they don't have problems associated to cell maintenance and injections. For investigation the hypothesis, MSCs should be isolated from tissues and characterized. Then, the exosomes should be isolated from the supernatants and characterized. These exosomes should be injected into a transgenic animal for COVID-19. In the final section, lung function assessment, histological examination, micro-CT, differential leukocyte, viral load analysis, cytokine assay, and CRP level analysis can be investigated. COVID-19 treatment is currently focused on supportive therapies and no vaccine has been developed for it. So, numerous researches are needed to find potential therapies. Since the pathogenesis of this disease was identified in previous studies and can cause lung injury with ARDS, investigation of the therapeutic approaches that can suppress inflammation, cytokine storm and ARDS can be helpful in finding a novel therapeutic approach for this disease.

Evaluation of the effects of mesenchymal stem cells on neutrophils isolated from severe congenital neutropenia patients.

Neutrophils are the most abundant, yet with the shortest lifespan among the circulating leukocytes. These cells are produced in the bone marrow during granulopoiesis process. Severe congenital neutropenia (SCN) is a hematological disorder with disturbance in granulopoiesis process, in which the neutrophils apoptosis rate is escalated. Previous reports indicated that mesenchymal stem cells (MSCs), as an immunomodulator cell, could increase neutrophil lifespan in addition to the supportive effects on cardiomyocytes or the neuroprotective effects. In this study, MSCs were co-cultured with neutrophils isolated from SCN patients and healthy individuals. Then, we evaluated the MSC co-culture effects on neutrophils survival (annexin V/PI assay), reactive oxygen species (ROS) production (colorimetric NBT assay), and phagocytic activity (Giemsa staining after exposure to yeasts). It was demonstrated that MSC co-culture could increase neutrophil lifespan and phagocytic activity of the neutrophils isolated from SCN patients. Regarding healthy donors' neutrophils, only phagocytic activity improvement was seen. It could be concluded that MSCs could be considered as novel candidates for treatment of SCN patients.

Exosomes derived from mesenchymal stem cells improved function and survival of neutrophils from severe congenital neutropenia patients in vitro.

Severe congenital neutropenia (SCN) is described by the absolute neutrophil counts less than 500 cells/mm3, bacterial infections, and an arrest of neutrophil differentiation. So, effective strategies for improving the function and lifespan of the existing neutrophils in these patients are necessary. Mesenchymal stem cells (MSCs) have supportive effects on neutrophils. Recently, it was determined that MSCs exert their effects, mostly by secreting soluble factors and exosomes. So, in this study, neutrophils were isolated from the bloodstream of healthy donors and SCN patients and cultured with medium, MSC-exosomes or MSC-conditioned media (MSC-CM). Then, the effects of the two treatments on neutrophil respiratory burst, apoptosis and phagocytosis percentage were assessed using nitro blue tetrazolium (NBT) assay, annexin V-propidium iodide (PI) and Giemsa staining, respectively. Both treatments could significantly augment respiratory burst of neutrophils from SCN patients and healthy donors. But, only CM could significantly enhance phagocytosis index. About the lifespan of neutrophils, only exosomes could significantly enhance it in both groups. Based on these results, both exosomes and CM derived from MSCs could be attractive candidates for rescuing SCN patients from serious infections.

Comparison of the effects of adipose tissue mesenchymal stromal cell-derived exosomes with conditioned media on neutrophil function and apoptosis.

BACKGROUND: Neutrophils are short-lived cells of the innate immune system that have an important role in defending against pathogens by producing reactive oxygen species (ROS). Therefore, effective strategies for increasing neutrophil's viability and function may be beneficial, especially in many conditions such as infections and immunodeficiency diseases. Some studies suggest using multipotent mesenchymal stromal cells (MSCs) and MSC-conditioned media (MSC-CM) for this aim. But, there is no study on using MSC-derived exosomes for improving neutrophil's viability and function. So, we examined the effects of MSC-exosomes and also MSC-CM on neutrophil's function and survival and compared them with each other. METHODS: Exosomes and CM were isolated from human adipose tissue MSCs. Exosomes were characterized, and the protein content of them was determined. Neutrophils were isolated from five healthy donors, and the effects of the two independent treatments (exosomes and conditioned media) on neutrophil's apoptosis were measured by Annexin V-PI method, then neutrophil's function was evaluated using NBT and phagocytosis assays. RESULTS: It was recognized that exosomes decreased neutrophils apoptosis and increased their phagocytosis capacity. The conditioned media augmented neutrophil's phagocytosis and reactive oxygen species (ROS) production, but it couldn't decrease neutrophil's apoptosis. DISCUSSION: Briefly, we concluded that MSC-exosomes augment neutrophil's viability more than their function while MSC-CM increase neutrophil's function more than the survival. This report showed that the use of MSC-exosomes and CM might be useful for increasing immunity by improving neutrophil's function and survival.