Recent research has shown that epigenetic abnormalities significantly contribute to the dysregulation of subsets of B cells in CVID patients [6]. It has recently been revealed that the regulation of gene expression by miRNAs plays a significant role in modulating the activation and function of B cells [8]. Even so, it is still unclear how particular miRNAs function in this process. In the present study, our goal was to investigate the expression of hsa-miR-142-3p and hsa-miR-155-5p, which are epigenetic modulators, in CVID patients, and we observed that the hsa-miR-142-3p and hsa-miR-155-5p expression levels in blood samples from CVID patients were decreased compared with those in blood samples from healthy controls.
MiR-142 is extensively expressed in hematopoietic tissues and is involved in lineage differentiation in these tissues. [33, 34]. Multiple studies have indicated the essential function of miR-142 in immunological and inflammatory responses; furthermore, the expression of miR-142-3p is increased in immune cells after the induction of inflammation [35, 36]. MiR-142 is known to be a crucial modulator of lymphopoiesis and is necessary for the normal function and development of dendritic cells (DCs), mast cells, and megakaryocytes in mice [37–39]. MiR-142 is also essential for the function of mature B and T cells in addition to normal lymphocyte development [40]. Studies on mouse models with miR-142 deficiency have shown that miR-142 is essential for lymphocyte homeostasis, and miR-142 deletion in mouse models leads to defects in Marginal Zone B (MZB) cells, B1 B cells, and peripheral T-cell development. [41]. Ablation of miR-142 in mouse models causes defects in the humoral immune response regardless of a large expansion of the B lymphocyte compartment, suggesting that miR-142 is essential for efficient B-cell function [41]. It has been observed that mouse models with miR-142 deficiency fail to mount specific antibody responses after exposure to antigen and exhibit defective formation of germinal center (GC) B cells and differentiation of plasma B cells [42]. Moreover, miR-142-3p acts as a critical gene controlling the expression profile of mature B cells, and miR-142-3p may control cell homeostasis through the B-cell activating factor receptor (BAFF-R) [41].
Another miRNA examined in this study is miR-155, whose key role has been identified in various studies as a critical modulator of the immune response [43, 44]. MiR-155 is a particular miRNA for the hematopoietic cell system and is mainly expressed in the spleen and thymus, especially within both B and T lymphocytes, and controls various biological functions in the immune system by regulating the transcriptome in lymphocytes [45, 46]. Deregulation of miR-155 has been identified in chronic inflammation, autoimmunity, neoplasms, and fibrosis. Additionally, miR-155 overexpression enhances the proliferation of pre-B cells and promotes B-cell lymphoma [43, 47]. MiR-155 is required for proper DC activation, functions as a positive modulator of inflammatory cytokine production and contributes to inhibiting the migration of neutrophils and enhancing degranulation [48]. MiR-155 is required for the maintenance, maturation, and effector response of NK cells and regulates the production of IFN-γ in response to various stimuli [49]. MiR-155 is required for the optimal activity of CD8 + T lymphocytes to eliminate tumor cells and pathogens and to develop a memory response [50]. The expression of miR-155 is required for the maintenance of normal B-cell function and differentiation into antibody-producing cells [51]. miR-155 is a positive modulator of the proliferation and differentiation of Tfh cells and promotes Th17 and Th1 differentiation [52]. Additionally, Th2 cells express miR-155, which is involved in the immune response in these cells [53].
miR-142-/- and miR-155-/- mouse knockout models have been shown to exhibit a CVID phenotype with hypogammaglobulinemia, immunodeficiency, lung disease, inflammation of the intestine and polyclonal lymphoproliferation, highlighting the critical contribution of miRNAs to the control of B-cell activity [41, 44]. Several findings have shown that deregulation of miR-142 and miR-155 is implicated in most comorbidities found in CVID patients, such as autoimmune and neoplastic clinical complications [12]. MiR-142 and miR-155 are evolutionarily conserved between mouse species and humans, which allows for the clinical use and transfer of studies in mouse models to humans [13, 54]. The CVID phenotypes in the miR-142-/- and miR-155-/- mouse knockout models are in accordance with our finding that the expression of these two miRNAs is decreased in CVID patients.
Considering the activity of miR-155 and miR-142 in the immunity and function of B cells, it can be concluded that defects in miR-142 and miR-155 expression may cause disturbances in the function of B cells, and according to the results of previous studies on mouse knockout models and the decrease in the expression of these two miRNAs in CVID patients in this study, it can be suggested that defects in miR-155 and miR-142 may be involved in the pathogenesis of CVID.
The evaluation of the clinical effect of miRNAs in CVID disease is an initial step, and only limited studies have investigated the expression profile of miRNAs in CVID disease [9–11]. Although recent findings revealed the significance of the varied expression of miRNAs and epigenetic regulation in the defective differentiation of B cells, further study is required to clarify the association between the expression of miRNAs and the development of B cells and to evaluate whether these miRNAs might serve as novel potential biomarkers for CVID patient management and treatment.
Systems biology approaches provide valuable strategies for identifying the underlying pathogenic mechanism of diseases and developing new therapeutic targets for the prevention and treatment of diseases [55]. In this study, a PPI network was constructed for the common targets of hsa-miR-155 and hsa-miR-142, and the hub genes in the PPI network were identified. Interestingly, we identified 5 hub genes in which pathogenic mutations have been reported in PIDs, including PTEN, NFE2L2, TGFBR2, MSH6, and SOCS1. Additionally, it has been reported that the RAC1 and IL6 genes directly contribute to the pathogenesis of PID diseases, and the FOXO3, HIF1A, and SIRT1 genes contribute to immune and inflammatory responses. Previous findings verifying the significance of these hub genes in immunity and PID pathogenesis, which are mentioned below, concern each of these genes. PTEN is a tumor suppressor gene and is implicated in the homeostasis and development of B cells [56]. PTEN deficiency impairs CSR, and its pathogenic mutations have been identified in CVID patients [22]. NFE2L2 encodes a transcription factor that controls the expression of many genes involved in inflammatory and immune responses [57]. NFE2L2 is crucial for controlling antiviral and innate immune responses. NFE2L2 also inhibits the transcription of proinflammatory cytokines and the activation of IL6, which suppresses the inflammatory response of macrophages [58]. Autosomal dominant mutations of NFE2L2 were identified in patients who presented with immunodeficiency, hypogammaglobulinemia, recurrent infections, and developmental delay [23]. Another hub gene, TGFBR2, is a member of the TGF-β receptor subfamily [59]. TGFβ superfamily members act as crucial modulators of B-cell function at various stages of development in the bone marrow and in differentiation into plasma cells that secrete antibodies [60]. Mutations in the TGFBR2 gene have been reported in patients with PIDs. Gain-of-function mutations in TGFBR1/2 result in hyper-IgE syndrome (HIES) and combined immunodeficiency (CID) with associated or syndromic features named Loeys‒Dietz syndrome (LDS), and loss-of-function variants in TGFBR2 are a predisposing risk factor for generalized pustular psoriasis (GPP) and adult-onset immunodeficiency syndrome (AOID) [24, 25]. MSH6, a DNA repair protein, acts in somatic hypermutation (SHM) and class-switch recombination (CSR) by repairing double-strand breaks in DNA [61]. MSH6 deficiency is associated with defects in CSR and SHM processes and antibody production [61]. Genetic defects in the MSH6 gene contribute to PIDs, which are associated with defective production of switched isotypes immunoglobulin (IgG/IgA/IgE), such as CVID [26]. SOCS1 encodes a suppressor protein of cytokine signaling pathways and is a regulator of adaptive and innate immunity [62]. SOCS1 haploinsufficiency has been identified in the pleiotropic form of PIDs with autosomal dominant inheritance and early-onset autoimmunity [27]. Additionally, a heterozygous de novo variant in the SOCS1 gene is known to be a pathogenic mutation in patients with CVID [63]. RAC1 and its homolog RAC2, members of the family of Rho GTPases, are known to be involved in immune responses such as the regulation of the homotypic adhesions of B lymphocytes, CSR, and the humoral-mediated response [64]. Pathogenic mutations in some genes of the Rho GTPase family, such as the Rac2, Cdc42, and RhoH genes, have been detected in PIDs [28]. RAC2 mutations have been detected in several patients with several forms of PID, such as CVID, severe combined immunodeficiency (SCID), and defects in phagocytes [65]. Abnormal activation of Rac1 has been reported in PID because of RhoGEF mutations that regulate the Rho GTPase family with guanine nucleotide exchange factor (GEF) activity [28]. According to the mutations found in members of the Rho GTPase family in PID, especially the close homolog of RAC1, i.e., RAC2, it seems necessary that pathogenic mutations in RAC1 also be evaluated in PID patients. IL6 encodes interleukin 6 (IL-6), which has immunoregulatory effects on different immune cells [66]. Studies have shown the contribution of IL-6 to CVID, humoral immunodeficiency, and autoimmune disorders [29]. FOXO3, a member of the FOXO protein family, was also identified as a significant hub gene in the PPI network of our study with a degree of 13. The FOXO proteins are a subgroup of the Forkhead transcription factor family that regulates various biological pathways and immune cell homeostasis, including B and T lymphocytes and other nonlymphoid lineages [30]. HIF1A is another hub gene that encodes hypoxia-inducible factor-1α (HIF-1α). The critical role of HIF-1α has been identified in the regulation of multiple aspects of the immune system, including the differentiation, development, and function of different immune cells, in dendritic cells, neutrophils, macrophages, and B and T cells [31]. The involvement of HIF in multiple inflammatory diseases and its important role in the immune system may also be implicated in PID pathogenesis. SIRT1 encodes a histone deacetylase that has anti-inflammatory activity and is known to be an important modulator of innate and adaptive immunity[32]. SIRT1 deficiency can impair the differentiation of B cells into plasma cells and enhance the secretion of proinflammatory cytokines by B cells and autoantibody generation, which can be a possible cause of autoimmune disorders [32, 67, 68]. The above information illustrates the important activity of hsa-miR-142 and hsa-miR-155 target genes in the immune system and in PIDs, which especially highlights the potential role of these two miRNAs in PID and CVID pathogenesis.
In this study, the expression of 17 hub genes from the PPI network of target genes of hsa-miR-142-3p and hsa-miR-155-5p was investigated in three GEO datasets. In two datasets, the expression of MSH6, FOXO3 was significantly differentially expressed and the expression of SOCS1 and SIRT1 was significantly differentially expressed in one of the datasets. According to the common perspective that miRNAs suppress the expression of their target genes, it is expected that with the downregulation of hsa-miR-142-3p and hsa-miR-155-5p, the expression of their target genes will increase. However, some studies have shown that with the dysregulation of miRNAs, the expression of their target genes can also be upregulated or downregulated [69, 70]. Therefore, dysregulation (upregulation or downregulation) of target genes of hsa-miR-142-3p and hsa-miR-155-5p can be expected.
As mentioned in the Results section, the significant enriched Reactome pathways for the common targets of hsa-miR-142-3p and hsa-miR-155-5p suggest the involvement of the FoxO-mediated signaling pathway, the TGF-β receptor complex, VEGFR2-mediated vascular permeability, interleukin-4 signaling and interleukin-13 signaling. According to several studies mentioned below, it seems that the enriched pathways of common targets of the two miRNAs may have more important effects on the immune response and PID pathogenesis. Considering that miR-142-3p and miR-155-5p have different biological activities in addition to regulating immune responses, it is possible that the common gene targets of these two miRNAs play relatively important roles in immune system activities. The FoxO signaling pathway was significantly and repeatedly observed in the enrichment analysis. As mentioned earlier, FOXO3, a member of the FOXO protein family, was also identified as a significant hub gene in the PPI network of our study with a degree of 13. Other critical members of the FOXO protein family involved in the immune system include FoxO1, which is particularly critical for the differentiation, isotype switching, survival, and proliferation of B cells, and it also has a critical function in germinal center development, and the depletion of FoxO1 results in reduced somatic hypermutation and isotype switching, leading to defective antibody production [71]. Foxp3 is another member of the FOXO family, and its mutation has been linked to immune dysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) [25]. Recent findings have demonstrated that Foxp3 contributes to the development of CVID [72]. The expression of Foxp3 has been shown to exert a suppressive impact on Treg cells and hinder B and T-cell proliferation and activation. Additionally, a decrease in the frequency of CD4 + CD25highFoxp3 + regulatory T cells and the level of FOXP3 expression has been reported in the peripheral blood of CVID patients with an autoimmune disorder, which may indicate the involvement of Treg lymphocytes in the pathogenesis of CVID [72]. Additional studies are needed to fully elucidate the role of Treg lymphocytes and Foxp3 in the pathogenesis of CVID, but it appears that a reduction in Treg cell number and Foxp3 expression may indirectly promote CVID development.
Our enrichment analysis revealed that the TGF-β receptor complex was a significant pathway, and TGFBR2 was identified as a significant hub gene in the PPI network of our study. TGF-β signaling is necessary for regulating different biological functions in immune responses [59]. TGF-β is an immunosuppressive mediator, and defects in its receptor (TGFBR1/2) are associated with inflammatory and immunodeficiency diseases, as previously mentioned [24, 25]. Differential gene expression analysis has shown that the TGFB1 gene is dysregulated in CVID patients [6]. According to the reported evidence of the activity of the TGF-β superfamily in immunity and its involvement in inflammatory and immunodeficiency diseases, the involvement of TGF-β signaling in the pathogenic process of CVID is hypothesized, and it seems that the examination of pathogenic mutations in the genes implicated in the TGF-β signaling pathway in CVID diseases is necessary. Our enrichment analysis revealed that VEGF/VEGFR signaling is another biological pathway that can be considered for further investigation as a potential mechanism in CVID pathogenesis. VEGF/VEGFR signaling modulates innate and adaptive immunity, and recent research in cancer has shown that VEGF has immunosuppressive activities in addition to proangiogenic properties [73, 74]. Different types of VEGF receptors are expressed in immune cells and regulate the activity of these cells [75]. VEGF inhibits CD3 + T-cell proliferation and reduces CD4 + and CD8 + T-cell numbers by decreasing hematopoietic progenitor cells [74]. VEGF suppresses inflammation in bacterial infection by affecting macrophages and mediates the migration and infiltration of macrophages [75]. Other evidence has shown that VEGF inhibits the proangiogenic and immunologic functions of macrophages in a rodent model of glioblastoma [76]. VEGF can also affect the function and maturation of DCs and can suppress DC differentiation [77]. Studies have reported that blockades of VEGF-C and VEGF-A can induce the activation of CD8 + and CD4 + cells, enhance the antigen-presenting properties of DCs, augment macrophage-mediated cytotoxicity, and activate the complement cascade [78]. The reported evidence indicates the effect of VEGF/VEGFR signaling on the immune system, but more studies are required to completely identify the mechanisms of its interaction and its possible involvement in the development of immunodeficiency diseases.
One of the limitations of this study was that we could not examine a larger number of patients. Although this problem has been observed in other miRNA studies in CVID patients and fewer than 12 patients were evaluated in these studies [9–11], the small sample size is one of the limitations of studying rare diseases. On the other hand, we examined the expression of hub genes only in the datasets, and for further studies, we suggest that the expression of hub genes be considered in CVID.