Role of microRNAs in immunoregulatory functions of epithelial cells

Abstract

Epithelial cells (ECs) provide the first line of defense against microbial threats and environmental challenges. They participate in the host’s immune responses via the expression and secretion of various immune-related molecules such as cytokines and chemokines, as well as interaction with immune cells. A growing body of evidence suggests that the dysregulated function of ECs can be involved in the pathophysiology of a broad range of infectious, autoimmune, and inflammatory diseases, including inflammatory bowel disease (IBD), asthma, multiple sclerosis, and rheumatoid arthritis. To maintain a substantial immunoregulatory function of ECs, precise expression of different molecules and their regulatory effects are indispensable. MicroRNAs (miRNAs, miRs) are small non-coding RNAs that regulate gene expression commonly at post-transcriptional level through degradation of target messenger RNAs (mRNAs) or suppression of protein translation. MiRNAs implicate as critical regulators in many cellular processes, including apoptosis, growth, differentiation, and immune response. Due to the crucial roles of miRNAs in such a vast range of biological processes, they have become the spotlight of biological research for more than two decades, but we are still at the beginning stages of the use of miRNA-based therapies in the improvement of human health. Hence, in the present paper, attempts are made to provide a comprehensive overview with regard to the roles of miRNAs in the immunoregulatory functions of ECs. A better understanding of the molecular mechanisms through which immunoregulatory properties of ECs are manifested, could aid the development of efficient strategies to prevent and treat multiple human diseases.

Introduction

Epithelial cells (ECs) such as those in lining the skin, gastrointestinal tract, respiratory tract, and oral cavity provide the first line of host defense against foreign bodies and injury [1]. In addition to their role in creating a physical barrier, ECs are critical in the recruitment of immune cells to the affected site and contribute either independently or in collaboration with resident/ recruited immune cells to provide epithelial tissue immunity [2, 3]. To perform these functions, ECs express a wide range of biomolecules associated with the immune response, including cytokines, chemokines, co-stimulatory molecules, and major histocompatibility complex (MHC) class I and II. Moreover, ECs are equipped with pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) which enable them to recognize distinct pathogen-associated molecular patterns (PAMPs) and to participate in the initiation of appropriate immune responses against microbial pathogens [2, 3]. Different gene products regulate the EC functions. Addressing these molecules and their associated pathways will provide new perspectives to understanding malignant diseases related to the dysfunction of ECs.

MicroRNAs (miRNAs, miRs) are a class of small non-coding RNAs that regulate gene expression at the post-transcriptional level mainly through degradation or translational repression of target mRNAs. MiRNAs play important roles in various cellular processes including development, differentiation, apoptosis, and immune response [4,5,6]. Furthermore, there are growing evidence concerning the contribution of miRNAs in the regulation of almost all aspects of EC functions such as renewal and wound healing [7,8,9], epithelial/endothelial barrier maintenance [10, 11], response to oxidative stress [12, 13], autophagy [14] and epithelial immunity [15, 16]. Also, previous studies have explored that dysregulation of miRNAs in ECs is associated with several immune-related disorders such as inflammatory bowel disease (IBD) [11, 17], and asthma [18]. Therefore, in this review, our main focus is directed toward miRNA involvement in the regulation of immune response by ECs. As well, we summarize multiple extracellular roles of miRNA in mediating epithelial-immune cell communications. Of note, we provide an overview of the current knowledge about the miRNA regulatory effects in the modulation of EC function in confronting COVID-19 infection.

Better understanding of the immunoregulatory features of ECs and the mediators that play a fundamental role in which, will guide future research to design efficient therapeutic interventions against various infectious and inflammatory diseases.

Epithelial cell functions: from physical/biochemical barrier to immune protection

The ECs protect the host with the formation of a physical and biochemical barrier separating the host body from the external environment. In addition, the ECs can respond to danger signals such as microbial stimuli and contribute to the regulation of both tolerogenic and immunogenic responses [19]. Given the important role of ECs in the establishment of protective immunity, disruption of EC homeostasis creates the risk of infection and inflammatory disorders.

Tight junction proteins (TJPs), production of mucous layer, secretion of broadly targeted antimicrobial proteins (AMPs), and transcytosis of secretory immunoglobulin A (SIgA) are among the main mechanisms that contribute to the protective function of the epithelial barrier [19]. Also, the epithelium can respond to pathogens by secretion of various cytokines responsible for recruiting immune cells to infected or injured sites [20].

In the following paragraphs, we briefly discuss evidence about the protective mechanisms by which the epithelium improves host defense against invading pathogens.

Role of TJPs in epithelial barrier function

The ECs are joined by tight junctions. TJPs, located at the tight junctions, comprise transmembrane (or integral membrane) proteins (such as junctional adhesion molecules (JAMs), tricellulin, claudins, and occludin) and peripherally associated scaffolding proteins (such as ZO (zonula occludens)-1, -2 and -3). These proteins determine the mucosal permeability and regulate the transport of solutes, ions, and water through the paracellular pathway of ECs [21,22,23]. Several lines of evidence demonstrate the importance of TJPs in the regulation of epithelium function and prevention of severe inflammatory responses. For instance, Yuki et al. reported that levels of ZO-1 and claudin-1 proteins were decreased in the skin of patients with atopic dermatitis [24]. In another study, Krug et al. reported that tricellulin, a protein that participates in organization of tricellular as well as bicellular tight junctions [21], was decreased in patients with ulcerative colitis, and its reduction increased the paracellular passage of macromolecule [25].

Expression of AMPs as a potent arm of the innate immune system in the epithelial barrier

AMPs are charged peptides that act as a protective part of the host ҆s innate immune system against a broad range of bacteria, fungi, and viruses. For example, cathelicidins― an important group of cationic AMPs― convert into their mature form (LL-37 in humans and mCRAMP in mice [26]) through extracellular cleavage by proteinase-3 [27]. The AMP LL-37 is produced by various human cell types such as neutrophils [26], mast cells [28], monocytes [29], and ECs from different organs including intestine [30], gastric [31], lung [32, 33] and mouth [34]. This AMP showed antimicrobial activity against a variety of pathogens such as Pseudomonas aeruginosa [27, 35], Helicobacter pylori [31], Staphylococcus aureus [36, 37], Candida albicans [34], and respiratory syncytial virus (RSV) [38]. Besides direct antimicrobial activity [38, 39], LL37 shows diverse immunoregulatory functions against infection. Wang et al., reported that LL37 enhances bacterial phagocytosis in human macrophages. Furthermore, the expression of Fcγ receptors (including CD32 and CD64), TLR4, and CD14 was increased on LL-37-treated macrophages [40]. Treatment with LL-37 significantly enhanced interleukin (IL)-6 and IL-8 release from human bronchial epithelial IB3-1 cells [27]. As such, Neumann et al., found a role for LL-37 in the formation of neutrophil extracellular traps [41]. In addition, another study reported that mouse and human cathelicidins released by neutrophils promoted differentiation and survival of Th17 cells, and directed subsequent adaptive immune responses through which [26]. As an example of the pathophysiological role of LL-37 in disease progression, Jiao et al. study provided evidence that elevated levels of LL-37 induced asthma exacerbation through the activation of eosinophils interacting with bronchial ECs in inflammatory airway [42]. This evidence and other similar findings indicate that AMPs such as LL37 mediate communications between the ECs and immune cells.

Other AMPs such as defensins, are also produced by ECs and protect the host against pathogens [43], however, providing a comprehensive overview with regard to different types of AMPs is beyond the scope of the present article.

Secretion of IgA and immunity in epithelial barrier

Secretory IgA (SIgA) is the predominant antibody isotype on mucosal surfaces of humans and many other mammals which plays important roles in protection against pathogens without leading to inflammation because of its inability to activate the complement pathway [44]. In addition, the production of SIgA regulates the commensal microbiota composition to maintain a healthy balance between the host and the microorganisms [44, 45].

IgA-producing plasma cells abundantly reside within the lamina propria of the gastrointestinal tract, but a significant number of these cells are also found in the other mucosal sites such as upper and lower airways [44] and genital tract [46]. The multimeric IgA produced by local plasma cells in the lamina propria is transported across the ECs ― which express poly-Ig receptor (pIgR) on their basolateral surfaces― into the mucosal lumen [44, 45]. Moreover, IgA-producing plasma cells are also present within non-mucosal sites such as in the mammary gland [47], bone marrow [48], and brain tissue [49], although data suggests that systematic and mucosal IgA producing plasma cells are of the same origin [47,48,49].

The major part of mucosal IgA-secreting plasma cells is derived from activated B-cells in mucosal-associated lymphoid tissues (MALT). The majority of MALT is localized along the gut, termed as gut-associated lymphoid tissues (GALT). The GALT includes several structures which the Peyer’s patches (PPs) are the main IgA-inductive sites among them [45]. Activated naïve IgM B cells in the gut differentiate into IgA-secreting plasma cells by class-switch recombination (CSR) from Cμ to Cα in the constant region of the Ig heavy chain. This process is dependent on priming by mucosal dendritic cells (DCs) carrying various antigens and live bacteria from the luminal surface into the PPs [19, 45, 50].

Briefly, in the presence of cognate CD4 + T cells, interaction between CD40 on the surface of B cells and its ligand (CD40L) on T cells as well as secretion of multiple cytokines lead to high-affinity antigen-specific IgA production to neutralize the pathogens [19, 45, 51]. In addition, in the absence of T cells, CSR to IgA could occur through the stimulation of B cells by APRIL (A proliferation-inducing ligand) and BAFF (B-cell activating factor of the TNF family) [19, 51], which their structure and function are related to CD40L [52]. A role is also known for APRIL and BAFF in support of survival of IgA + B cells and IgA-producing plasma cells [45, 50, 52]. In response to commensal bacteria, the production of APRIL and BAFF by ECs directly stimulates the B cells and triggers IgA CSR. Furthermore, ECs induce the production of APRIL and BAFF by mucosal DCs which intensify the effect on B cell stimulation [19, 52]. However, there are several other important factors influencing IgA CSR which were well discussed in previous studies [45, 50].

Expression of immunoregulatory molecules by ECs and interaction with immune cells

It is noteworthy that epithelial tissues contain a complex network of resident immune cells that play crucial roles in host defense and tissue homeostasis. Tissue-resident immune cells are both myeloid and lymphoid cell subsets mainly including mononuclear phagocytes, innate lymphoid cells, tissue-resident T cells, and IgA-secreting plasma cells [19, 53]. In response to a challenge, such as invading pathogens and tissue injury, ECs exert their influence on priming of immune responses via communications with immune cells resident in the tissue and those that are infiltrated from the periphery to resolve the challenge, hence, restore the tissue to its original condition [53].

The production and secretion of numerous immunoregulatory signals by ECs such as transforming growth factor-β (TGF-β) [54, 55], IL-8 [20, 55], thymic stromal lymphopoietin (TSLP) [55, 56], IL-25 [57, 58] and many other biomolecules provide possible tools for the translation of stimuli-derived signals from ECs to immune cells and promote cross-talk between them. We summarized several biomolecules secreted by ECs as well as their immune-related functions in Table 1. In the following, several regulatory interactions of ECs in the immune system are mentioned.

Table 1 Immune-regulatory mediators secreted by epithelial cells

Previous data showed that human corneal ECs can internalize Aspergillus flavus spores via actin-mediated endocytosis [94] and differentially express distinct sets of gene transcripts associated with tumor necrosis factor (TNF) signaling, Th17 differentiation, NF-κB signaling, chemokine signaling and B cell receptor signaling against fungal infection compared to control [95]. After stimulation with killed Aspergillus fumigatus, pro-inflammatory cytokines such as CXCL1, TNF-α, and IL-6 and activation of P38 MAPK were induced through LOX-1 (lectin-like oxidized low-density lipoprotein receptor 1) in rat corneal ECs. Also, the expression of CXCL1 and TNF-α was found to be elevated through LOX-1 in human corneal ECs [69]. Moreover, corneal ECs upregulated the expression of dectin-1 [96], TLR-2, TLR-4, IL-1β, and IL-10 upon stimulation with A. fumigatus antigens [97].

Pulmonary ECs infected with different strains of Mycobacterium tuberculosis at early stage can produce a wide range of cytokines, chemokines, growth factors and PRRs such as IL-6, IL-8, interferon (IFN)-γ, TNF-α, granulocyte colony-stimulating factor (G-CSF), granulocyte–macrophage colony-stimulating factor (GM-CSF), TLR3, TLR5, and TLR2 [20].

Keratinocytes are the main cell type of the epidermis― the outermost layer of skin ― which in addition to providing a physical barrier, can express different types of cytokine receptors and PRRs such as TLRs, nucleotide-binding oligomerization domain-like receptors (NLRs), and RIG-I–like receptors (RLRs). Furthermore, they produce a wide variety of cytokines, chemokines, growth factors as well as AMPs [98]. For example, human keratinocytes and mouse skin produce inflammatory mediators IL-6, IL-1β, IL-8, cyclooxygenase (COX)-2, and monocyte chemoattractant protein (MCP-1) mediated by NF-κB signaling in response to ultraviolet B (UVB) irradiation [59]. Under the mediation of IL-25, keratinocytes can produce pro-inflammatory cytokines and chemokines via activation of the STAT3 pathway in a murine psoriasis model― a chronic autoinflammatory skin disease― indicating that keratinocytes play a critical role in the pathogenesis of this disease [82].

The luminal surface of PPs is covered by the follicle-associated epithelium (FAE) which contains relatively limited numbers of goblet cells, enteroendocrine cells, and intraepithelial lymphocytes and is rich in specialized ECs known as microfold cells (M cells). M cells, which are phagocytic, constantly sample and transport luminal antigens to the underlying GALT. Then, M cells release their transcytosed material within intraepithelial pockets formed by their expanded basolateral side. Within these pockets, M cells interact directly with the immune cells residing in the subepithelial dome (SED) beneath the FAE. The antigens transported by M cells are then taken up by antigen-presenting cells (APCs) residing in the SED such as immature DCs. The antigen-primed DCs undergo a maturation process and migrate to the T-cell zone of GALT to present antigens to T cells, leading to the activation of antigen-specific B cells and ultimately the induction of mucosal immune responses including the production of IgA antibodies by lamina propria plasma cells [16, 99, 100].

ECs act as non-professional phagocytes

As mentioned in the above section, ECs are capable of phagocytosis and elimination of cell debris, dead cells, and invading pathogens [101, 102]. However, they use different phagocytosis mechanisms compared to professional phagocytes such as macrophages. Although ECs have a remarkably lower phagocytic efficiency compared to professional phagocytes, accumulating evidence indicates that their phagocytic activity has a significant contribution in maintaining tissue homeostasis as well as in eliciting an adequate innate immune response against pathogens [101].

Capasso et al. study showed that Pseudomonas aeruginosa was attached to apoptotic ECs or apoptotic bodies and internalized by surrounding ECs via efferocytosis― a mechanism in which phagocytes engulf and remove apoptotic cells. Finally, the bacteria were killed within the cells through lysosomal processes [103].

ECs act as non-professional antigen-presenting cells

In addition to acting as non-professional phagocytes, ECs can present different antigens by major histocompatibility complex (MHC) class I and MHC class II molecules to the intraepithelial lymphocytes― primarily a heterogeneous T cell population including conventional T cell, γδ T cell, NKT cell, CD4 + CD8αα + double-positive T cell [19, 104] ― and lamina propria lymphocytes [105, 106]. Thus, ECs have the potential to act as non-professional APCs and stimulate immune responses against numerous antigens [105, 106].

MHC- I molecules are expressed by most nucleated cells and mainly present endogenous antigens to cytotoxic CD8 + T lymphocytes. While, MHC- II molecules are predominantly expressed on the professional APCs (DCs, B cells, macrophages) and thymic epithelia, and primarily present antigens to CD4 + T cells [107]. However, evidence shows that MHC-II proteins and associated processing molecules are also expressed by non-hematopoietic cells, such as fibroblasts, myofibroblasts, lymphatic endothelial cells, and ECs [102, 106, 108,109,110,111] which provide an important prerequisite for them to function as non-conventional APCs [112]. Although numerous studies reported the role of IFN-γ as a critical inducer of MHC- II expression by ECs [102, 113, 114], limited evidence shows that there are potential IFN-γ independent mechanisms in the induction of MHC- II expression on ECs [115]. Despite the expression of MHC- II molecules on the surface of ECs being reported in both normal and inflammatory conditions, their expression level can be different between health and pathological conditions. For example, an elevated level of MHC-II expression was found in IBD and Epstein-Barr virus (EBV)-associated gastric cancer compared to the normal groups [113, 116].

In the context of antigen presentation through MHC-II by ECs, either immune-enhancing or immunosuppressive responses have been suggested. Several studies reported the upregulated expression of MHC-II by ECs under inflammatory conditions which activated effector CD4 + T cell responses [117, 118]. While, other studies reported conflicting findings and suggested a tolerogenic role of antigen presentation by ECs through regulatory T (Treg) cell expansion [119, 120]. These contradictory observations highlight the need for further investigations to illustrate the exact outcome of antigen presentation by ECs to effector or regulatory CD4 + T cells. The findings mentioned below support the ability of ECs for activation of T cells through antigen presentation.

Shenoy et al. study showed that antigen presentation by lung ECs critically regulated CD4 + resident memory T (T_RM) cell function and reported an important role of epithelial CD4 + T_RM cell immune interactions in establishing barrier immunity [106]. Koyama et al. found that MHC-II-expressing intestinal ECs have a pivotal role in alloantigen presentation to donor CD4 + T cells in vivo and thereby in the initiation of acute lethal graft-versus-host disease (GVHD)― an immunopathology mediated by mature donor T cells which recognize host alloantigens and leads to severe inflammation ― following allogeneic bone marrow transplantation. They also reported that intestinal EC-specific deletion of MHC-II abrogated lethal GVHD in the gastrointestinal tract [118].

Hatano et al. reported that antigen presentation by IFN-γ- pretreated murine small intestinal ECs induced antigen-specific proliferation in CD4 + intestinal intraepithelial lymphocytes (IILs) and enhanced IFN-γ secretion by these cells [105]. As another example, Dotan et al. reported that co-culture of intestinal ECs isolated from IBD patients with autologous or allogeneic healthy peripheral blood T cells stimulated the proliferation and IFN-γ secretion in CD4 + T cells which were significantly greater degree than those in T cells stimulated with normal intestinal ECs. Moreover, blockade of MHC-II (DR) harnessed CD4 + T cell proliferation and the IFN-γ secretion in IBD intestinal EC- CD4 + T cell co-cultures, with a lesser effect in the normal intestinal EC- CD4 + T cell co-cultures [117].

About the extensive capabilities of ECs, in the above section, we attempted to provide a short overview of the manifold functions of these cells in immune defense which should be given more attention in future studies.

MiRNAs and epithelial immune responses

Accumulating data indicates that miRNAs play key roles in determining the fate and modulation of functions of ECs, such as proliferation [121], differentiation [16, 79], apoptosis, and autophagy [122] through targeting different genes and signaling pathways. Nakato et al., with the generation of mice harboring intestinal EC- specific deletion of Dicer1, found that intestinal epithelial miRNAs (miRNAs in FAE) play a significant role in the differentiation and function of M cells and contribute to mucosal immune homeostasis [16].

MiRNAs affect the epithelial and endothelial permeability through the regulation of TJP expression. For example, miR-122a, miR-144, and miR-200C-3p can increase intestinal tight junction permeability by directly targeting and degradation of the occludin mRNA [123,124,125]. MiR-29 can increase intestinal epithelial permeability by directly targeting and reduction of the claudin-1 mRNA [126]. MiR-144 promotes intestinal permeability by directly targeting ZO1 mRNA [123] (Fig. 1). Also, miR-21-5p increases intestinal epithelial permeability via induction of ARF4 (ADP ribosylation factor 4) expression (ARF4 is not a direct target of this miRNA) [127]. Dysregulation of epithelial barrier function contributes to a broad range of autoimmune and inflammatory diseases [11, 124].

Fig. 1

Schematic drawing that briefly illustrates (A) the microRNA involvement in the modulation of immune response by epithelial cells; and (B) the effect of microRNAs in epithelial permeability through the regulation of tight junction protein expression

Moreover, epithelium-expressed miRNAs act as mediators for crosstalk between ECs and the immune system (Fig. 1). Biotin et al. study, using a mouse model of inactivated Dicer1 in the gut, showed that epithelial miRNAs play a fundamental role in the induction of the anti-parasitic Th2 (T helper type 2) responses and modulation of gut mucosal immunity. Particularly, they showed that miR-375 expression in mouse colonic epithelium induced higher expression of RELMβ and TSLP― two epithelium-derived cytokines that regulate mucosal anti-parasitic Th2 response [79].

Kawasaki et al. found that miR‑429 exerts anti‑inflammatory function through the suppression of inflammatory cytokines such as IL‑8 by inhibiting the NF‑κB pathway in gingival EC line (squamous cell carcinoma Ca9‑22 cells) [15]. In Chen et al. study, stable knockdown (KD) gingival EC lines for several epithelium-expressed miRNAs were constructed and their inflammatory response to infection with periodontal pathogens was assessed. They reported that pathogen-stimulated miR-126 KD cells produced lower IL-8 and CXCL1 levels than wild-type cells. In contrast, infection of miR-155 KD and miR-210 KD cells showed higher IL-8 and CXCL1 expression than wild-type cells [60].

In the irradiated mouse model, oral gavage with hydrogen-water increased the miR-1968-5p level in the small intestine. MiR-1968-5p directly targeted and downregulated the MyD88 (myeloid differentiation factor 88) expression and alleviated the intestinal injury induced by irradiation [128]. It is worth noting that MyD88 was known as a key player in inflammatory signaling pathways downstream of IL-1 receptor (IL-1R) families and mammalian TLRs [129]. A study on the function of miR-146a in keratinocytes identified this miRNA as a regulatory agent in keratinocyte innate immunity in which TLR2- induced miR-146a acted as a negative feedback regulator via suppression of the inflammatory mediators such as IL-8, CCL20, and TNF-α. In addition, the study showed that miR-146a repressed the chemotactic attraction of neutrophils by keratinocytes [61]. As well, the Li et al. study reported that miRNA-23a-enriched exosomes from hypoxic tubular ECs mediated the cross-talk between these cells and macrophages to promote renal tubulointerstitial inflammation [130]. Thus, the blockade of miRNA transfer between ECs and immune cells may act as a potential therapeutic approach to ameliorate an immune-related disorder. Further findings concerning the role of miRNAs in the regulation of immune-related target genes expressed in ECs were presented in Table 2.

Table 2 Immune-related miRNAs and their direct target genes expressed in epithelial cells (ECs). MiRNAs directly target the mentioned genes and downregulate their expression and functions

Role of EC miRNAs in the control of microbial infections

The role of miRNAs in the interactions of the epithelium with the microbial pathogen has been widely investigated [151,152,153]. In this context, accumulating data reported that miRNA-mediated immune responses are involved in either pathogen survival or pathogen elimination. Several examples are mentioned as follows.

Upon influenza A virus infection, miR-136 is up-regulated in A549 human lung ECs. Subsequently, this miRNA mediates the up-regulation of several cytokines including IL-6 and IFN-β, and stimulates innate immunity by acting as a ligand for RIG-I (retinoic acid-inducible gene 1) leading to suppression of virus replication [70]. On the other hand, influenza A virus downregulates miR-17-3p and miR-221 in human lung ECs during the early-stage infection which this causes enhanced viral replication possibly through GALNT3 (GalNAc transferase 3) upregulation [154].

Aguilar et al. indicated that Salmonella typhimurium infection induced changes in the miRnome expression via downregulation of transcription factor E2F1. These changes promoted Salmonella replication in both infected epithelial and bystander cells [151]. Yang K et al. demonstrated that after Pseudomonas aeruginosa infection, miR-155 expression was upregulated in human and mouse corneas and was predominantly expressed in macrophages. Moreover, they found that miR-155 reduced the macrophage-mediated elimination of P. aeruginosa by targeting Rheb (Ras homolog enriched in the brain), and therefore, involved in corneal susceptibility to P. aeruginosa keratitis [155]. Another study indicated that Salmonella enterica infection increased miR-128 expression in intestinal ECs which, in turn, decreased the levels of EC-secreted M-CSF (macrophage colony-stimulating factor), leading to impaired M-CSF–mediated macrophage recruitment. It is noteworthy that M-CSF was confirmed as a direct target of miR-128 [74].

Recently, several studies have reported the possible roles of host miRNAs to serve as anti- or pro-viral effectors among COVID-19 patients and provided new perspectives to develop preventive and treatment strategies based on miRNAs. For example, Lu D et al. reported that miR-200c can directly target and inhibit the expression of angiotensin-converting enzyme 2 (ACE2) ― known as a receptor for the spike protein of SARS-CoV-2 which plays fundamental roles during the COVID-19 infection― in cardiomyocytes [156]. Given that ACE2 is remarkably expressed in different tissues including the lung, heart, kidney, intestine, liver, testis, and central nervous system [156, 157], miR-200c could be an interesting topic for future research to design a potential strategy for prevention and treatment of complications during the COVID-19 infection.

According to a few studies, several viruses use the "miRNA sponge effect" to disrupt the pathways regulated by host miRNAs. Through this mechanism, the viral genome acts as miRNA sponges that competitively interact with host miRNAs to deplete specific miRNAs and cause the disruption of miRNA/natural target interactions [158, 159]. For example, a recent study reported that hsa-miR-302c-5p― a key regulator of ACE2― can be sponged by the SARS-CoV-2 genome. This effect potentially led to an elevated expression of ACE2 [158] which was found to be associated with severe COVID-19 disease [160]. Therefore, focusing attention on such studies could be helpful to explore the exact role of miRNAs in the regulation of EC immune responses to microbial infection and may provide a promising target for clinical treatment of infectious diseases.

A brief overview of several studies reporting miRNAs expressed in EC and their respective function in the immune system and immune disorders is presented in Table 3.

Table 3 MiRNAs expressed in epithelial cells (ECs) and their respective function in the immune system and immune disorders

Xeno-miRNAs and effects on immune system

Growing evidence points to certain subtypes of miRNAs which are codified by non-host genomes but are present in body fluids and tissues of different species of animals, including humans. They have been termed xeno-miRNA (xeno-miRs) which can modulate gene expression among various species and kingdoms. Xeno-miRs in humans have been reported from numerous exogenous sources, which among them plant miRNAs are the main source of these exogenous RNAs. Upon dietary intake, xeno-miRs from different sources such as plant [161,162,163,164] and milk [165, 166] are absorbed by gastrointestinal ECs, packaged into exosomes, subsequently secreted into the blood circulation and then delivered into recipient tissues/ cells [161, 163, 164], including the lung, liver, spleen, kidney, heart, DCs, adipocytes and macrophages [161, 163, 164, 167,168,169,170,171], where they regulate host- gene expression [163, 167, 172].

Numerous studies have confirmed the immunomodulatory effects of xeno-miRs on the mammalian immune system. Cavalieri et al. demonstrated that a wide range of miRNAs obtained from diverse plant species could act as TLR3 ligands in DCs. Also, they found that plant xenomiRs (for instance, Fragaria vesca miR168), via impairment of TRIF signaling, were able to reduce inflammation and the pathology development of autoimmune encephalomyelitis in the mouse model [168].

Plant miR159a and miR156c in nut exosome-like nanovesicles were found to have anti-inflammatory effects in vitro and in mouse models of adipose tissue inflammation via downregulation of TNF receptor superfamily member 1a (Tnfrsf1a) expression in macrophages and adipocytes, which in turn negatively regulate TNF-α signaling pathway [167]. Zhou et al. study suggested that absorbed plant miR2911 from honeysuckle decoction was transferred into the lung by exosomes through circulation, where it inhibited SARS-CoV-2 replication and accelerated the recovery process in COVID-19 patients [171].

Another study reported that plant miR2911, encoded by honeysuckle, directly targeted various influenza A viruses and inhibited viral replication [164]. Ginger exosome-like nanoparticle miRNAs (aly-miR396a-5p and rlcv-miR rL1-28-3p) reduced SARS-CoV-2-induced lung inflammation and apoptosis via inhibition of expression of viral RNA polymerase Nsp12 and spike genes [170].

Moreover, diet-derived exosome-like nanoparticles containing miRNAs can be taken up by the gut microbiota and are able to modulate their composition and function in mammals. In this regard, Teng et al. reported that mdo-miR7267-3p, one of the miRNAs present in ginger exosome-like nanoparticles, repressed monooxygenase ycnE expression in Lactobacillus rhamnosus, which increased the production of indole-3-carboxaldehyde (I3A)― a ligand for aryl hydrocarbon receptor (AHR)― leading to the induction of IL-22 production via activation of AHR pathway in gut lymphocytes. These actions improved gut barrier function and ameliorated colitis in mice [173]. Another study reported that bovine milk-derived extracellular vesicles through immune-related miRNAs changed gut microbiota composition, modulated their metabolites, and strengthened intestinal immunity in mice [174].

Interestingly, Li et al. study provided evidence that plant miRNAs (for instance, miR2911 derived from honeysuckle) in the maternal diet can be delivered to the fetus through the placenta and regulate fetal gene expression [162].

However, the direct effects of xeno-miRs on the immunomodulatory functions of ECs as well as xeno-miR-mediated cross-talk between ECs and neighboring immune cells have not been deeply explored yet. Future research in this field opens promising avenues for miRNA-based treatment of immune malignancies through diet.

It is noteworthy to underline that despite the above-mentioned evidence, several researchers have reported negative/negligible expression of xeno-miRs in body fluids or tissues of recipients and rejected the xeno-miR hypothesis [175,176,177]. It seems that technical issues such as experimental artifacts and cross-contaminations [176], xeno-miR degradation during the digestive process [175], and being selective of diet-derived xenomiR absorption (dependent on miR sequence) by animals [178] are the possible causes for studies where xeno-miRs were not detected in animal bodies. However, further studies are needed to resolve these contradictions.

Therapeutic potential of miRNAs to treat conditions involving EC disorders

In the context of miRNA roles in immunoregulatory functions of ECs, promising therapeutic applications of miRNAs are to use their immunomodulatory capacities to induce antimicrobial pathways during infection as well as to control the deregulated inflammatory responses in immune-related disorders such as IBD and asthma (as noted in Table 3). For instance, miR-128 level in mouse intestinal and colon tissues was upregulated during Salmonella enterica infection. The elevation in miR-128 level decreased the secretion of M-CSF by host ECs and the M-CSF–mediated macrophage recruitment, leading to the escape of Salmonella from macrophages (Fig. 1). On the other hand, intragastric delivery of anti-miR-128 promoted M-CSF–induced macrophage recruitment and suppressed S. enterica infection in mice [74]. However, despite extensive studies confirming potential therapeutic applications of miRNAs, few studies have been conducted as clinical trials and none of those have reached phase III [179] or led to Food and Drug Administration (FDA)– approved drug. Thus, it seems that the translation of these research findings into clinical treatments faces significant challenges.

As an example of miRNA-based therapy targeting ECs, we refer to the RG-101 designed for use in patients with chronic hepatitis C virus (HCV) infection. In which anti-miR-122 oligonucleotide was conjugated to N-acetylgalactosamine‒ a high-affinity ligand for the asialoglycoprotein receptor that is widely expressed on hepatocytes [180]. It is interesting to note that miR-122 was known as a crucial host factor for HCV replication. It binds to 5´ UTR of the HCV RNA and enhances genome stability and translation [181]. To evaluate the safety and efficacy of RG-101 in human subjects, 32 patients were enrolled in phase 1B randomized controlled trial study. The results showed that a single subcutaneous injection of RG-101 significantly reduced viral load in patients at week 4 of treatment. In addition, HCV RNA levels substantially decreased in all treated patients and were not detectable for at least 76 weeks (end of follow-up) in 3 patients with sustained virological response. Nonetheless, viral rebound‒ which is associated with mutations in miR-122 binding regions in the HCV 5´ UTR‒ was observed in most patients. Some severe adverse events, including intrahepatic cholestasis and hyperbilirubinemia, were reported in some patients [180]. Anti-viral immunity analysis showed that NK-cell frequency increased and NK-cell activating receptors (such as NKp30 and NKp46), NK-cell IFN-γ production, and IFN-γ-induced protein 10 (IP-10) level in plasma decreased after RG-101 administration. Moreover, HCV-specific T-cell responses did not significantly change in patients. Overall, the data suggested that the NK cells, and not adaptive immunity, may have involved in the control of HCV infection [182]. Given that miR-122 acts as a tumor suppressor in hepatocellular carcinoma [183], the possibility of long-term risk of hepatocellular carcinoma development in patients with HCV infection following RG-101 administration should be noticed.

In another phase 1 clinical trial study, the safety, optimal dosing, and efficacy of TargomiRs were tested in patients with malignant pleural mesothelioma (MPM). TargomiRs were developed as minicells loaded with miR-16 mimic with an anti-EGFR bispecific antibody to target EGFR-expressing tumor cells [184]. Mesothelial cells have characteristics of both mesenchymal and epithelial cells which line the serosal cavities (peritoneal, pericardial, and pleural) and internal organs [185, 186]. MiR-16 was reported to have tumor suppressor activity in MPM [187].

In the above-mentioned study, 26 patients received at least one dose of TargomiR. During the response evaluation, the following results were observed in patients: 5% with a partial response, 68% with stable disease, and 27% with progressive disease. Moreover, toxicity effects, such as inflammation symptoms, anaphylaxis, and cardiac events, which were dependent on the dose of TargomiR administration, were recorded [184].

In sum, although it is now clarified that miRNAs are key regulators of gene expression and their dysfunction is involved in many diseases, attempts to produce miRNA-based therapies did not end with a practical outcome. This issue is partly related to the inherent characteristics of miRNAs, including a large number of endogenous targets, low binding affinity with its target which leads to nonspecific actions, and degradation of miRNA mimics/ anti-miRNAs by circulating RNase enzymes [179]. In addition, severe immune-mediated adverse reactions, such as those were observed in MRX34 administration in several patients with advanced solid tumors [188], are other obstacles that remain to be overcome. Nevertheless, the development of the targeted delivery system in which miR mimics/anti-miRs were transported to the specific tissue, can improve the efficacy and safety of a miR-based therapy [179].

In total, considering the above-mentioned points, we believe that a safe therapeutic compound that restores disordered host cells to compensate deregulated miRNA at its physiological level rather than exogenously transferred miRNA mimics /anti-miRNAs could be beneficial to resolve the challenges related to the miR-based therapeutics. Further research is needed to be directed to identifying these compounds and their molecular mechanisms of action.

Conclusion

Taken together, the studies summarized in this review illustrate the various and multifaceted roles of miRNAs in the immunoregulatory functions of ECs. Although we attempted to provide a comprehensive review, however, an in-depth overview of all aspects related to this issue was not possible in the current paper due to space limitations. For example, with regard to this issue, one of the valuable aspects can be a deep understanding of the role of miRNAs in cross-talk between microbiota, ECs, and the immune system. However, it is very beneficial and practical that reliable knowledge provided from a comprehensive review be translated into the development of novel therapeutics supporting human health.