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

Abstract

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.

Introduction

Chronic granulomatous disease (CGD) is a rare hereditary immunodeficiency that is resulted from the faults in the subunits of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase of including gp91phox, p22phox, p47phox, p67phox, and p40phox [1], [2], [3]. Several genes are involved in encoding these subunits, and in them may cause different forms of CGD [4]. Since NADPH oxidase has crucial role in reactive oxygen species (ROS) production, in CGD patients, the ROS production by phagocytes such as neutrophils is impaired [5].

Neutrophils are phagocytic components of the native immunity and the first cells involving in pathogen elimination. They release lytic enzymes and generate reactive oxygen intermediates with a powerful antimicrobial capacity for killing of the pathogens [6], [7]. In CGD patients, one of the mechanisms that have pivotal role in pathogen killing of neutrophils is impaired. So, due to impaired neutrophil function, CGD patients are very vulnerable to severe and sometimes fatal infections. Therefore, effective strategies for improving neutrophil function are necessary in these individuals. A common treatment for them is interferon gamma (IFN-γ) therapy [8]. Studies demonstrated that IFN-γ therapy in CGD patients, increased superoxide production by phagocytes, but it has some limitations; for example, it is very expensive and requires three injections per week. It has also some side effects such as fever, headaches, fatigue, irritability, vomiting, neurotoxicity, leukopenia and flulike syndrome [9], [10], [11]. So, it is useful to find alternative strategies for saving these individuals from infections. Various reports have indicated that mesenchymal stem cells (MSCs) have protective effects on the function and viability of neutrophils [12], [13], [14], [15], [16], [17], [18], [19], [20], [21], [22], [23].

MSCs are multipotent progenitor cells that have some special characteristics such as self-renewing, multilineage differentiation and immunomodulatory functions [24], [25]. Recently, it was suggested that, one of the principal ways that MSCs influence other cells is releasing some mediators such as exosomes that are a subset of extracellular vesicles (EVs) [26]. Previously the effects of the MSC-exosomes and conditioned media (CM) on the function and viability of neutrophils that were isolated from healthy individuals were investigated [21], but these effects on the neutrophils isolated from CGD patients are not investigated.

Therefore, we evaluated the effects of two treatments including MSC-exosomes and MSC-CM on neutrophil function and apoptosis in CGD patients and healthy individuals and then compared them with each other (Fig. 1). This would be used for saving CGD individuals from vigorous infections by improving neutrophil function.