Molecular Signature and miRNA Pattern of CD4 + T Lymphocytes in Plaque Psoriasis

Abstract

Psoriasis is a global health problem affecting approximately 3% of the population. Because this autoimmune and autoinflammatory disease significantly reduces the quality of life and carries the risk of serious complications and comorbidities, all efforts have been directed towards developing effective therapeutic strategies that allow for a significant restoration of immunological homeostasis. To make this possible, basic research is being conducted, among others, to discover the molecular basis responsible for the pathogenic activation of autoreactive lymphocytes. At the same time, microRNAs seem to be very interesting candidates for epigenetic modulation of signaling pathways disturbed in psoriasis, which are themselves also affected in its pathogenesis. Therefore, our narrative review comprehensively summarizes the current knowledge on the molecular signature and miRNA profile that appear characteristic for psoriasis-related CD4 + T lymphocytes and hypothesizes the effects of microRNA dysregulation based on the results of a contextual functional analysis of selected candidates having the greatest impact on the pathogenesis of psoriasis. Detailed considerations allowed us to uncover the most important signaling pathways and microRNA molecules active in pathogenic CD4 + T lymphocytes that are involved in psoriasis progression, and also suggested other microRNAs that appear to have potential therapeutic activity, with the leading opposing roles of interferon regulatory factor 4, microRNA-155 and microRNA-150.

Introduction

Psoriasis is a chronic autoimmune skin disease characterized by a wide variety of clinical manifestations, with two extreme phenotypes, i.e. plaque and generalized pustular psoriasis. While the latter is primarily driven by neutrophils and IL-36, plaque psoriasis depends on dysregulation of cellular immunity, including CD4 + T lymphocytes [1]. All clinical forms of psoriasis, including plaque psoriasis, are strongly influenced not only by genetic predisposition [2], but also by environmental factors, including chronic dysbiosis within the gut-skin axis [3], which disrupt the fate of T cells.

After decades of intensive research, autoreactive CD4 + helper T (Th) cells releasing IL-17 (Th17 lymphocytes) emerged as central players in the pathogenesis of psoriasis. Their pathogenic activity results from severe dysregulation of molecular pathways and miRNA signaling that drive tissue inflammation and systemic complications. Supported by other cells, including Th populations, Th17 lymphocytes contribute not only to the complex pathogenesis of psoriasis but also to other related autoimmune diseases, becoming extremely interesting therapeutic targets.

Recent advances have revolutionized our understanding of the immunological pattern of psoriasis, including the single-cell transcriptomic identification of pathogenic immune cell subsets and memory T cell populations in affected tissues, particularly the skin. Furthermore, epigenetic dysregulation, including miRNA signaling abnormalities, accompanying immune imbalances, all contribute to the variable clinical course and response to biological therapies. Discussing these aspects is crucial today, because despite enormous scientific progress, it still has not translated into personalized management and therapies. This requires prior collection of individual knowledge elements and identifying missing ones for further research. This is precisely what review articles are designed to achieve. Therefore, we built our narration about the molecular and miRNA signatures of psoriatic CD4 + T lymphocytes around the hypothesis that their targeting is a promising strategy for new biomarkers and therapies that will help overcome unresolved clinical problems such as treatment resistance, disease relapse, and incomplete restoration of immune homeostasis in psoriasis, especially after integration with multi-omics technologies.

Knowledge in this field, as mentioned, is selective and consists of many small elements that need to be put together like scattered puzzles. Therefore, we provided a summary of the latest progress regarding molecular dysregulation and miRNA profile of CD4 + T lymphocytes in plaque psoriasis with a particular emphasis on Th17 cells, in order to provide a comprehensive picture of affected signaling pathways that may become targets for future precision therapies. To strengthen our considerations, we also performed a contextual functional analysis of selected miRNAs with the greatest impact on the pathogenesis of psoriasis.

The presented literature was searched primarily in the PubMed and Google Scholar databases, using a combination of various general keywords, including in particular “autoimmunity”, “psoriasis”, “CD4 + T cells/lymphocytes”, “helper T cells”, “Th1”, “Th17”, “regulatory T cells/lymphocytes”, “molecular pathways/paths/networks”, “signaling pathways/paths/networks/circuits”, “miR/miRNA/microRNA”, “immune regulation”. Experimental and review articles written in English, published in reputable journals, and clearly related to the topics discussed, i.e. molecular pathways regulated by miRNAs in CD4 + T lymphocytes in plaque psoriasis, were then selected, starting with the most recent ones. After determining the most important molecular pathways and miRNA molecules involved in the broadly understood regulation of individual CD4 + T lymphocyte populations in psoriasis, we then searched for articles that would describe in detail the relationships between these molecular pathways and miRNAs in the broad context of autoimmunity and inflammation in order to draw final conclusions from our considerations about plaque psoriasis.

A Brief Clinical Overview of Psoriasis

Due to the predominant cutaneous symptoms, psoriasis was long considered a skin-limited disorder. Nowadays, understanding of its pathogenesis is much broader and emphasizes the central role of autoinflammatory and autoimmune processes initiated by genetic predisposition and/or environmental trigger. All these factors influence the course of the disease and the dominant clinical symptoms in each person with the two extreme phenotypes described, i.e. plaque and pustular psoriasis. While the latter is considered to be mostly driven by autoinflammation [4], plaque psoriasis involves the pathogenic activation of self-reactive helper T (Th) lymphocytes [5].

Accordingly, plaque psoriasis (psoriasis vulgaris) is the most common clinical subtype developing in around 90% of patients, characterized by skin lesions formed by well-defined, dry, erythematous, thickened plaques with flaky silvery scales, most typically covering the scalp, elbows, knees, and lower back. Distinctive histological features include increased but simultaneously impaired keratinocyte proliferation with markedly reduced turnover time, parakeratosis, and acanthosis, accompanied by immune cell infiltration and angiogenesis. Development and maintenance of psoriatic plaques are orchestrated by a complex network of cellular and cytokine interactions with IL-23/IL-17 axis playing a central role by linking dysregulated innate and adaptive immune responses to the abnormal functioning of the epidermis. Consequently, autoreactive effector CD4 + T lymphocytes are largely present in psoriatic lesions, with Th1 and Th17 subpopulations attracting significant attention together with impaired regulatory T (Treg) cell responses. Moreover, pathogenic CD4 + T cells and their abnormal functioning can also be observed in circulating blood and unaffected skin of psoriatic individuals, and is closely related to numerous comorbidities, including psoriatic arthritis, metabolic syndrome and cardiovascular diseases, which further underlines the systemic nature of plaque psoriasis. As discussed below, the role of epigenetic regulation in psoriasis is currently being under intense investigation. Accumulating evidence points toward certain non-coding RNA molecules, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), which are involved in the disease modulation and might be a promising target in novel treatment options.

In Poland, over a million people suffer from psoriasis, constituting about 3% of citizens, which corresponds to a global percentage of the total world population. Unlike a healthy human, whose epidermis matures and peels off over a period of about 30 days, in a person with psoriasis this process takes 3–4 days, causing painful and itchy lesions that crack and bleed easily. This is a huge psychological, social and economic problem, making it difficult to perform professional duties by the patient. Moreover, constant damage to the skin’s barrier continuity predisposes to infections and disturbs immunological, regenerative and ionic/metabolic homeostasis in the largest tissue of the body. This, in turn, affects the functioning of the entire organism, making psoriasis a chronic, incurable disease of civilization [6, 7].

Currently available treatments for psoriasis include topical creams and ointments designed to inhibit excessive keratinocyte growth and remove scales, phototherapy, which modulates the function of skin-infiltrating immune cells, and systemic medications, which are intended to induce general immune suppression. However, none of these treatments can restore immune tolerance or selectively inhibit the immune cells involved in the pathogenesis of psoriasis.

Risk Factors, Genetic Susceptibility and Comorbidities

It is estimated that a genetic predisposition exists in up to 60% of people with psoriasis, and this percentage is even higher in children of both parents with the condition. However, people without a family history of psoriasis can also develop it, as the disorder can be triggered by environmental factors such as stress, streptococcal and other infections, trauma, burns, or certain medications. The latter include beta-blockers, lithium and tumor necrosis factor alpha (TNFα) antagonists that are well known to induce de novo or exacerbate the disease [8, 9].

Recently, the gut microbiome has emerged as one of the most important factors controlling the development and fate of Th17 lymphocytes. Namely, chronic dysbiosis has been found to promote pathogenicity of Th17 lymphocytes that may further drive psoriatic inflammation together with the oxidative stress [10–12]. Furthermore, the predominance of individual risk factors determines the clinical outcome, i.e., whether the disease progresses towards a plaque or pustular phenotype, and thus clinical symptoms may vary over the course of the disease in each patient [13].

The high variability of clinical phenotypes makes it difficult to study genetic risk factors [14]. Nevertheless, genome-wide association studies (GWAS) have uncovered approximately 100 susceptibility loci for psoriasis in humans. Most of them are linked to immune system functioning and keratinocyte development. The greatest risk is attributed to a specific allele of a major histocompatibility complex (MHC) class I region situated at chromosome 6, namely HLA-Cw6 (C*06:02). This allele has long been strongly associated with early-onset psoriasis and its guttate subtype. Nowadays, its role in autoantigen presentation to CD8 + T lymphocytes is under intense investigation, as mentioned below, especially that it has been found to promote guttate psoriasis driven by streptococcal infection [15]. The second group of susceptibility loci includes the genes encoding cytokines, such as IL-17, IL-23, and IL-12, as well as cytokine receptor subunits. Finally, several genes affecting T cell function (e.g. RUNX3, STAT3, IRF4), and related molecular pathways (for instance c-Rel, CARD14, CARM1 from NF-κB path and TYK2 from JAK/STAT path) have been associated with the increased risk of psoriasis [16–20]. Translating this information into understanding immune cell dysfunction will thus be a huge contribution to precision medicine and therapy of psoriasis [21].

In addition, a consistent overactivation of pro-inflammatory mechanisms, after crossing a certain threshold, eventually result in the immunological tolerance breakdown, with chronic inflammation and autoantigen presentation driven by stimulation of Toll-like receptors (TLR) 7 and 8 [22]. Besides, while chronic inflammation has long been linked to aging, psoriatic patients demonstrate sign of premature immunosenescence, characterized by the presence of functionally senescent, terminally differentiated circulating CD8 + T cells [23, 24], which also seems to affect the disease course.

There is increasing evidence pointing to the significant systemic impact of psoriatic autoinflammatory and autoimmune processes on the body’s functioning. Circulating pro-inflammatory cytokines (e.g. IL-17 A, TNFα, IL-6), as a sign of systemic inflammation, were shown to correlate with disease severity. Moreover, skin biopsies from seemingly healthy skin of patients nevertheless preserves certain levels of inflammation markers typical for psoriatic lesions, altogether promoting serious systemic complications.

The most common complication of psoriasis, which sometimes may occur as a separate clinical disorder, is psoriatic arthritis [25, 26]. However, the concept of “psoriatic march” demonstrates that this disease is often complicated by a number of clinically serious comorbidities, such as Crohn’s, cardiovascular, liver and kidney diseases, type 2 diabetes and certain cancers [27]. A very recent review highlighted that IL-17 axis plays the central role in other psoriasis comorbidities, including metabolic syndrome [28]. Therefore, the development of innovative, precise therapies that allow for the permanent restoration of immunological homeostasis in people with psoriasis would be extremely helpful in regaining the quality and length of the patient’s life. For this reason, it is necessary to identify molecular pathways whose dysregulation leads to pathogenic activation of autoreactive effector T lymphocytes, causing the occurrence of clinical symptoms in plaque psoriasis.

Therefore, our narrative review comprehensively summarizes the current knowledge on the molecular signature and miRNA profile that appear characteristic for psoriasis-related CD4 + T lymphocytes.

General Overview of T Lymphocytes in Psoriasis

Various classifications are used to characterize T lymphocytes, and one of them, based on the type of T cell antigen receptor (TCR), distinguishes gamma/delta (γδ) and alpha/beta (αβ) T lymphocytes. Physiologically, a small subpopulation of γδ T cells acts independently of classical MHC molecules, enabling a rapid innate immune response to stress and infection, particularly in tissues such as the skin, gut, and lung. In contrast, the much more numerous αβ T cells provide a conventional, adaptive immune response against a variety of pathogenic peptide antigens. Moreover, T cells can be further divided into CD4 + and CD8 + subtypes, both of which play a significant role in psoriasis pathogenesis. Epidermal CD8 + T cells recognizing autoantigenic epitopes mediate a self-damaging cytotoxic effector reaction and deliver locally acting cytokines that enhance psoriatic inflammation [29]. Albeit, initiation, spread, and prolongation of autoimmune response depend on an imbalance between different CD4 + T cell subpopulations. Therefore, the latter are of increasing research interest when considering the possibility of lasting restoration of immunological tolerance in autoimmune diseases. For this purpose, detailed identification of the molecular signature and the possibility of epigenetic regulation of CD4 + T cell functioning is crucial for planning future personalized therapies.

One cannot deny the important pathogenic and regulatory role of γδ T cells in psoriasis, which has already been discussed elsewhere [30–32]. However, only few (less than 1%) γδ T cells are CD4+, while the majority is double negative [33]. Henceforth, this review focuses on conventional CD4 + αβ T lymphocytes, which can be functionally classified as helper or regulatory T cells, among which Th1, Th2, Th9, Th17, Th22, and Tfh as well as FoxP3 + Treg and FoxP3- Tr1 subpopulations, have been distinguished [34].

Type 1 regulatory T cells (Tr1) constitute a major subset of FoxP3- natural regulatory T cells that efficiently produce IL-10 to maintain immune tolerance and control inflammation in both humans and mice [35]. They play a key role in preventing autoimmunity, and their dysregulation is observed in psoriatic inflammation [36–38]. Importantly, Tr1 cells differentiate in response to IL-6 and IL-27, and the latter cytokine, a member of the IL-12 family [39], is responsible for blocking the Th17 phenotype shift despite of the activation of STAT1 and STAT3 [40]. Conversely, as mentioned below, IL-6 together with IL-23 induce the maturation of pathogenic Th17 cells, and this phenomenon clearly confirms that even a single cytokine switch may significantly change the direction of the cell differentiation process.

In psoriasis, the function of classical CD4 + FoxP3+ Treg cells is also severely impaired, and they may even develop an exhausted phenotype [38]. However, it should be noted that in inflammatory environments, Treg cells can acquire an intermediate phenotype associated with the expression of a cell lineage-specific transcription factor that is thought to support the inhibitory activity of Treg cells adapted to the current tissue conditions. In particular, in a response to IL-12 and IFNγ signaling, Treg cells can become T-bet + and thus express a Th1-like phenotype, which seems to inhibit Th1 cell activity [41]. However, under the influence of IL-6 and IL-23, they gain a Th17-like phenotype, which has been shown to make RORγt+ Tregs rather pro-inflammatory due to the ability to release IL-17 [42].

The development and physiological role of helper T lymphocytes, including Th1, Th2, Th9, Th17, Th22, and Tfh cells, have been discussed in detail in other reviews [34, 41, 43]. The cytokine milieu provided by helper T cell subpopulations physiologically creates a complex network of interregulated cascades that confer immune homeostasis. However, disruption of these cascades leads to the predominance of Th1 and Th17 lymphocytes in psoriatic lesions and a reduction in the number and/or function of Th2 and Treg lymphocytes. Nevertheless, in generalized pustular psoriasis, some observations imply that Th2 lymphocytes might play a supporting pathogenic role [44], and may potentially drive autoantibody formation in patients whose disease progresses to psoriatic arthritis [45]. But this requires further investigation, as is the case with the role of T follicular helper (Tfh) cells. Intriguingly, circulating and skin-infiltrating Tfh lymphocytes exhibit an activated phenotype in patients with psoriasis and positively correlate with symptom severity [46]. Nevertheless, their exact role in psoriatic inflammation remains unclear, but may be related to the secretion of IL-21, which in turn could drive the differentiation of Th17 cells. At the same time, skin-tropic Th9 and Th22 cells are important for cutaneous homeostasis, but their overactivation is observed in psoriatic inflammation [47], with IL-9 appearing to increase cytokine release by other infiltrating helper T lymphocytes, while IL-22 seems to cause pro-inflammatory activation of keratinocytes [48]. As a result, the latter cells release IL-17 C that strongly enhances IL-17 A-related pathogenicity of Th17 lymphocytes [49]. It is also worth noting that recently attention has been drawn to resident memory T cells, which may be responsible for the recurrence of skin lesions due to their persistent pro-inflammatory phenotype [50]. However, as mentioned above, among CD4 + cells, Th1 and Th17 lymphocytes play a key role in driving psoriatic inflammation and autoimmunity in plaque psoriasis [48], and STAT4 has been proposed to maintain the expression of IL-23 receptor by memory Th17 lymphocytes [51]. In turn, IL-23 seems to drive STAT4 activity in these cells [52].

Of crucial importance, Saravia et al. recently highlighted the remarkable plasticity of CD4 + αβ T cells as they adapt to the current microenvironment [41]. Interestingly, their ability to adopt an alternative transcriptional signature upon receiving specific signals is at least partly due to epigenetic modifications, including bivalent histone modifications maintained in CD4 + T cells near all major regulatory genes regardless of the differentiated cell phenotype. This feature further complicates research on CD4 + T cells, especially in the context of applying the acquired knowledge in clinical practice.

Naive T Cell Priming

A set of factors, not yet fully understood, triggers psoriatic inflammation through pro-inflammatory activation of keratinocytes and dermal antigen-presenting cells (APCs), including plasmacytoid and myeloid dendritic cells (DCs), Langerhans cells and macrophages [53]. The dependence of the production of cytokines from IL-12 family, including IL-12 and IL-23 that polarize the autoimmune reaction typical of psoriasis, on the activation of various TLRs indicates the involvement of microbial products (pathogen-associated molecular patterns, PAMPs) along with endogenous damage-associated molecular patterns (DAMPs) [54–56]. Both therefore link the onset of psoriasis to, respectively, a dysbiotic microbiota, as well as tissue injury and cell death. Of note, a recent comprehensive review clearly demonstrated that dysregulated helper T lymphocyte (Th) responses can be caused by bacterial and fungal dysbiosis not only in the skin but also throughout the entire gastrointestinal tract from the tonsils to the intestines and colon [3]. It can therefore be assumed that psoriatic inflammation begins with the recognition of immunogenic microbial peptides by naive CD4 + T cells, which differentiate towards Th1 and Th17 lymphocytes. Then the activated T lymphocytes migrate and accumulate in the epidermis, and release pro-inflammatory cytokines that induce local inflammation involving keratinocyte and APC stimulation along with neutrophil infiltration [3]. As a result, keratinocytes release antimicrobial peptides, including LL-37, as well as keratin-17 and phospholipase PLA2G4D, the latter is also derived by mast cells, while melanocytes secrete ADAMTSL5. Importantly, LL-37, keratin-17 and ADAMTSL5 together with lipid neoantigens generated in a reaction catalyzed by PLA2G4D are now recognized autoantigens that initiate an autoimmune response in psoriasis [57]. Mechanistically, lipid neoantigens are presented by CD1a+ Langerhans cells to γδ T cells, while ADAMTSL5, complexed with HLA-Cw*06:02, is recognized by intraepidermal CD8 + T lymphocytes exhibiting the unique TCR variant Vα3S1/Vβ13S1. Furthermore, HLA-Cw*06:02-restricted CD8 + T lymphocytes can also recognize keratin 17 due to its molecular mimicry with the surface M protein of group A β-hemolytic streptococci. Finally, LL-37 aggregates with self-DNA originating from neutrophil extracellular traps (NETs), and in this form is endocytosed by plasmacytoid DCs (pDCs). Owing to TLR7 and TLR9 ligation by LL-37-aggregated nucleic acids, pDC excessively produce IFNα to activate myeloid/conventional DCs and dermal macrophages. These APCs sustain autoimmune reaction by releasing pro-inflammatory cytokines, such as TNFα, IL-6, IL-12 and IL-23, with the latter two driving pathogenic Th1 and Th17 cell responses [57], as discussed below. Importantly, HLA-Cw*06:02 allows for the cross-presentation of extrinsic psoriatic autoantigens by DCs to naive CD8 + T cells, while cross-primed Th1 lymphocytes enable their efficient activation and proliferation in draining lymph nodes [58]. From the immunological point of view, this is the main reason why CD8 + T lymphocytes play a pathogenic role mainly in genetically susceptible individuals with the HLA-Cw*06:02 haplotype [59]. After migration to the epidermis, antigen-specific CD8 + T cells recognize autoantigenic peptide complexed with HLA-Cw*06:02 on keratinocytes, and release IFNγ to drive local inflammation. However, these cells appear to rapidly mature towards a resident memory T cell phenotype rather than causing direct cytotoxic damage to the epidermis [60]. In parallel, cytokines derived from Th1 and Th17 lymphocytes induce excessive proliferation and abnormal differentiation of keratinocytes. This, in turn, leads to epidermal thickening and the development of inflammatory skin lesions characteristic of plaque psoriasis [57, 58].

Th1 and Th17 Cell Differentiation

Physiologically, Th1 lymphocytes provide an acquired immunity against intracellular pathogens by enabling cytotoxic activation of macrophages, NK cells, and CD8 + T lymphocytes. Whereas, Th17 lymphocytes are essential for protecting mucosal barriers against extracellular pathogens, including bacteria and fungi. However, these important types of immune response can be skewed towards a harmful autoimmune reaction after the breakdown of immune tolerance mechanisms due to chronic inflammation, dysbiosis and tissue damage [61].

Analogously to physiological conditions, characteristic combination of psoriasis-activating stimuli forces the immune sentinel cells, macrophages and DCs especially, to initiate selective signaling that biases immune response towards type I [56, 62]. Such signaling includes the production of IFNα/β and IL-12 [63, 64], as well as IL-27, which is believed to act upstream of IL-12 at the early stage of Th1 cell commitment [65], whereas in the later phase of inflammation it is suggested to metabolically inhibit the functioning of Th1 lymphocytes and prevent the activation of Th17 cells. Therefore the role of IL-27 in psoriasis is considered divergent [66–68]. Simultaneously, NK cells, as first-line innate cytotoxic defenders, become activated and immediately begin to release IFNγ [69]. The latter cytokine activates STAT1 in naive CD4 + T lymphocytes, forming an immunological synapse with antigen-presenting DCs and macrophages. STAT1 dimerization and nuclear translocation enable the expression of T-bet, which in turn transcriptionally induces the synthesis of IFNγ and IL-12Rβ2 [70]. Given the postulated intracellular accumulation of IL-12Rβ1 transcripts [71], the assembly of both receptor β chains renders CD4 + T lymphocytes responsive to IL-12 signaling. Following the engagement of the p40 and p35 subunits of IL-12 with IL-12Rβ1 and IL-12Rβ2, respectively, Tyk2 and Jak2 kinases phosphorylate STAT4, which then forms homodimers and translocates to the nucleus to drive transcription of many genes associated with Th1 cell development and function. Of note, autocrine IFNγ signaling maintains T-bet transcriptional activity to perpetuate the Th1 phenotype, while its sustained release induces systemic inflammation [72], and has been proposed to program DCs and macrophages to secrete IL-23 [48]. Moreover, Th1 lymphocytes interact with macrophages via CD40-CD40L to augment IL-12 production [73], which recently has also been proposed to increase IL-23 secretion [74]. It can therefore be assumed that the prolonged activation of Th1 lymphocytes precedes and favors the differentiation of pathogenic Th17 cells in a positive feedback loop.

Along these lines, TGFβ and IL-6 were found to activate the differentiation of naive CD4 + T cells towards a Th17 phenotype by inducing STAT3 homodimerization, which enables autocrine IL-21 signaling [75], which is essential for inflammatory activation [76]. Nevertheless, the pathogenicity of Th17 cells is driven by IL-23 signaling [77], which is enabled by IL-23 receptor expression stimulated, among others, by IL-6 [78, 79]. Specifically, upon binding of the p40 and p19 subunits of IL-23 with IL-12Rβ1 and IL-23R, respectively, Tyk2 and Jak2 kinases phosphorylate STAT4 and STAT3 [80], allowing them to form homo- and heterodimers, which transcriptionally activate the expression of RORγt and sustained production of IL-17 [81], which causes pathological hyperplasia of keratinocytes [82]. As a consequence of psoriatic inflammation, numerous mediators are released, and some of them are considered the main self-antigens responsible for induction of autoimmune response [57]. It should be emphasized that the leading autoantigen derived from keratinocytes, i.e. cathelicidin LL-37, is recognized by both CD4 + and CD8 + T lymphocytes, and promotes the secretion of IL-17 by CD4 + cells [57, 83], which is an evidence that Th17 lymphocytes can also be autoreactive, as described below.

Although most of our current knowledge of T cell maturation comes from studies in mouse models, the basic principles of CD4 + T lymphocyte differentiation are well conserved across species, and thus mouse and human T cell subsets share similarities in key cytokines, master transcriptional regulators, and signaling pathways, as recently reviewed in detail elsewhere [41]. In this narrative review article, we therefore summarized the principles of molecular signature and miRNA pattern in different CD4 + T cell subpopulations that are crucial for psoriasis and have been discovered in mouse and/or human studies. Moreover, in order to enrich and validate our considerations and increase the significance of the conclusions drawn, we performed a contextual functional analysis of selected miRNA combinations to more precisely estimate their impact on the studied CD4 + T cells.

Molecular Signaling Pathways that Determine CD4 + T Cell Fate in Psoriatic Inflammation

Although all Th cell populations are involved in psoriatic inflammation in different aspects [84], the main pathogenic role is attributed to the self-reactive Th17 lymphocytes. As mentioned above, their initial activation is supported by Th1 lymphocytes, differentiating in a response to IL-12 secreted by dermal APCs [61]. In turn, Th1 lymphocytes release IFNγ and TNFα that exacerbate epidermal injury and orchestrate Th17-promoting tissue microenvironment. Because CD4 + T cells constitutively express moderate levels of IFNγ receptor (IFNGR) [85], upon cell activation this cytokine can rapidly enhance expansion and survival independently of STAT1 [86]. Therefore, neutralization of IFNγ at the early stages of the development of the autoimmune response prevents Th17 cell polarization, but surprisingly, anti-IFNγ treatment at a later stage induces the opposite effect [87]. One can thus speculate that Th1 lymphocytes play a bidirectional role in controlling Th17 cell maturation and functioning. Namely, Th1 lymphocytes foster their Th17 counterparts in the acute phase of autoimmune response, while at a later stage they attempt to reduce Th17 pathogenicity in an IFNγ and retinoic acid-dependent manner [88, 89], likely after developing a Treg-like phenotype under the influence of retinoic acid [90, 91].

However, when control mechanisms fail, IFNγ favors pathogenic differentiation of self-reactive Th17 lymphocytes, and the main underlying molecular pathways are depicted in detail in the Fig. 1. Specifically, after tissue injury, keratinocytes release antimicrobial peptides, among which cathelicidin LL-37 bind to self-DNA and RNA originating from damaged cells. This induces the early stage of psoriatic dermatitis, characterized by the skin infiltration by plasmacytoid dendritic cells that release large amounts of IFNα and IL-12 following their activation by TLR7 and TLR9 agonists from LL-37-DNA/RNA complexes [92–94]. In turn, IFNα increases the expression of IL-15R in CD4 + T cells [95]. Simultaneously, upon IL-12 exposure, macrophages rapidly secrete IL-15 [96], which augments their expression of MHC class II enabling macrophages to act as professional APCs [97]. Similarly, IL-15 promotes myeloid dendritic cell maturation after stimulation with TLR7/8 ligands [98]. Finally, IL-15 up-regulates the expression of IL-1R by naive CD4 + T cells [99], which constitutively express the IL-6R, under the control of TNF receptor-associated factor 2 (TRAF2) and TRAF5 signal transduction molecules [100]. In parallel, macrophages attempt to heal the injured tissue, among others, by secreting TGFβ [101], but the exposure to TGFβ is an obligatory prerequisite for priming naive CD4 + T cells to the Th17 phenotype, as summarized in detail elsewhere [102]. It should be noted, however, that most of the available research evidence suggests that TGFβ1 promotes non-pathogenic maturation of Th17 lymphocytes, while other, yet not fully defined factors tilt self-reactive Th17 cells towards pathogenic effectors [102]. These factors seem to involve the sustained exposure to pro-inflammatory cytokines, as depicted in the Fig. 1, together with dysregulated miRNA signaling, as discussed below (please also refer to the Fig. 3), both beginning at the immunological synapse.

Fig. 1.

Postulated molecular pathways that guide pathogenic maturation of self-reactive Th17 lymphocytes at the immunological synapse. Cytokine signaling at the site of keratinocyte injury primes naive CD4 + T cell to the Th17 phenotype, while LL-37-DNA/RNA complexes are endocytosed by APCs (dendritic cells and macrophages). After migration to the draining lymph nodes, they form an immunological synapse with self-reactive CD4 + T cell. Recognition of autoantigenic determinant by TCR accompanied by costimulatory and IL-2 signaling promotes T cell proliferation, while released cytokines determine the differentiation shift towards pathogenic Th17 lymphocytes. IL-6R stimulation drives STAT3 phosphorylation, which in turn induces the expression of IL-23R enabling IL-23 to enhance STAT3 transcriptional activity. Synergistically with IL-23, IL-1β induces p38 signaling pathway, which triggers IRF4 to induce the transcriptional activity of RORγt, a lineage-specific transcription factor finally responsible for the development of Th17 effector phenotype, additionally augmented by autocrine IL-21 signaling. Note that well-established molecular pathways in Th17 cells are indicated by green arrows, and postulated pathways are indicated by blue arrows. Created with BioRender.com under a license for the Faculty of Medicine, Jagiellonian University Medical College

Fig. 3.

Extracellular vesicle (EV)-enclosed miRNAs as critical regulators transferred at the immunological synapse. A successful presentation of an extrinsic antigen encompasses three main conditions, i.e. epitope recognition by TCR accompanied by interaction of CD4 molecule with MHC class II, co-stimulation as well as paracrine signaling via cytokines. Currently, the latter condition is considered to involve the transmission of miRNA-enriched EVs. a Pro-inflammatory activation of dendritic cells causes the release of EVs containing miR-148a and miR-155, which promote Th1 and Th17 lymphocyte differentiation and the development of autoimmune responses, including those underlying the pathogenesis of psoriasis. b Both CD8 + Ts lymphocytes and CD4 + Treg lymphocytes secrete miR-150 in EVs, which induces a tolerogenic phenotype in dendritic cells, which in turn amplify the regulatory signal by releasing secondary EVs containing miR-150, promoting Treg cell maturation and ensuring immune tolerance. Created with BioRender.com under a license for the Faculty of Medicine, Jagiellonian University Medical College

Accordingly, primed CD4 + T cells circulate to a nearby lymph node in search of the self-antigen for which they are specific. Whereas, the LL-37-DNA/RNA complexes are endocytosed by professional APCs (myeloid dendritic cells and macrophages), which stimulates their TLR7/8 and TLR9 receptors [57, 103, 104]. After migration to the draining lymph nodes, APCs trigger a pro-inflammatory signaling cascade that results in the release of specific cytokines, namely IL-1β (in an NLRP3 inflammasome-dependent manner [105, 106]), p40, p35 and p19 subunits of IL-12 and IL-23 as well as IL-6 [107, 108], both in interferon regulatory factor (IRF) and NF-κB-dependent manner [109]. In parallel, processed epitopes derived from autoantigens are presented in a complex with MHC class II, while TLR7/8 agonism drives CD80 and CD86 expression [110]. Antigen recognition by TCR activates primed CD4 + T lymphocyte, which begin to synthesize the IL-2R receptor [111]. Simultaneously, CD28 ligation initiates p38 mitogen-activated kinase (MAPK) signaling pathway [112], and TCR drives the action of zeta-chain-associated protein kinase 70 (ZAP70), a tyrosine kinase that may alternatively phosphorylate p38 on tyrosine-323 (p38-Y323) [113]. In turn, p38-Y323 induces the transcriptional activity of IRF4 (interferon regulatory factor 4) in an NFAT (nuclear factor of activated T cells) dependent manner [114, 115], and IRF4 drives the metabolic reprogramming of differentiating CD4 + T lymphocyte [116]. Simultaneously, NFAT transcription factors promote IL-2 release that enables T cell to proliferate and launch the transcription of genes for effector functioning [117], including priming of the RORC locus for RORγt (retinoid-related orphan receptor gamma t) production [118]. Additionally, RORγt expression is induced by IL-1β, which, in addition to mTOR signaling [119], stimulates the p38 pathway leading to IRF4 activation [120]. This appears to promote the pathogenic shift of Th17 lymphocyte [121]. Simultaneously, IL-6 initiates the transcriptional activity of STAT3 [100], which is ultimately perpetuated by IL-23 signaling that induces STAT3 phosphorylation at tyrosine-705 (STAT3-Y705) [122]. Of note, IL-23 and IL-1β signaling synergizes to greatly increase STAT3 activity in a RelA-dependent manner, and, additionally, IL-1β blocks SOCS3 downregulatory action promoted by IL-23 [122]. Based on the preliminary data, one can speculate that p38-Y323 phosphorylates MSK1 (mitogen- and stress-activated protein kinase 1) [112], which in turn phosphorylates STAT3 at serine-727 (STAT3-S727) to drive pathogenic differentiation of Th17 lymphocytes [123, 124]. As a consequence, STAT3 activates MSK1 to sustain NFAT signaling [125], while IL-6 additionally promotes STAT3 phosphorylation at serine-727 [123]. Besides, NFAT stimulates the release of IL-21, which acts on differentiating Th17 cell in an autocrine manner to induce IRF4 transcriptional activity via STAT3 [126]. Simultaneously, IL-6 seems to maintain this pathway by promoting BATF (Basic leucine zipper ATF-like transcription factor), JUN and AP-1 (activating protein-1) interactions with IRF4, which greatly increases its DNA binding capacity [127–129]. As a result, Th17 lymphocyte acquires a fully developed effector phenotype and drives pathogenic inflammation in psoriasis due to the sustained activation of pro-inflammatory signaling pathways, discussed in detail below.

It is also worth noting that the effector pathogenicity of Th17 lymphocytes is attributed to their ability to produce IFNγ, as found in patients with Crohn disease resistant to anti-IL-17 therapy [130], and more recently in psoriatic patients treated with anti-IL-23 monoclonal antibody [131]. Of note, Th17 cells release IFNγ independently of T-bet in response to IL-23 [132], which explains the selective impact of risankizumab on various subtypes of human IL-17-expressing T lymphocytes [131]. Besides, pathogenic Th17 cell-derived IFNγ has recently been proposed to augment IL-23 release by human monocytes [133], creating a positive feedback loop to drive pathogenic autoimmune response.

Moreover, some researchers speculate that self-reactive Th17 lymphocytes may develop from Treg cells under certain conditions [134, 135], while others suggest that T cell differentiation fate is governed by extrinsic factors, including cytokines, as well as by intrinsic factors, such as TCR avidity for autoantigens, whereby the higher the avidity, the greater the chance of differentiation into Treg lymphocytes [136]. However, one can speculate that, analogously to CD8 + T cells [137], higher self-antigen binding capacity may enhance the response of CD4 + T lymphocytes to IL-15, thus priming them for the Th17 phenotype when other conditions defined by cytokines and miRNAs are met, as summarized in this review.

It can also be speculated that some chemokines, including CXCL9-11, which are known to attract effector T cells to the site of psoriatic inflammation may shape their differentiation. In some preliminary experiments, CD4 + T cell treatment with CXCL9 and CXCL10 has been shown to stimulate STAT1, STAT4 and STAT5 phosphorylation and thus promote Th1/Th17 phenotype, while alternatively, IL-10-producing Tr1 cells were induced by CXCL11and CXCL12 exposure through mTOR, STAT3 and STAT6 pathways [138–140]. However, the significance of such mechanism is likely limited, since relatively small fraction of effector CD4 + T lymphocytes in the skin express CXCR3 or CXCR4, especially when compared to CCR6 [141, 142], even though the expression of CXC10/11/12 and their corresponding receptors in psoriatic skin biopsies is higher compared to healthy skin [143–147].

Nevertheless, the effector function of autoreactive Th17 lymphocytes accompanied by other CD4 + T cell subpopulations is associated with the hyperactivation of several important molecular pathways, and their central factors are discussed below.

STAT3

IL-6 was the first cytokine identified to activate STAT3 [148], which has now been confirmed to act downstream of other cytokines (including IL-23 and IL-21), growth factors, and signaling molecules. Moreover, de novo germline STAT3 activating mutations have been included in the list of monogenic causes of genetic autoimmunity [149]. Thus, its unrestrained transcriptional activity plays a central role in autoimmunity but due to its ability to cooperate with various transcription (co)factors, the actual outcome of STAT3 activation is highly dependent on the cell type and the specific signaling status of the microenvironment. Nevertheless, STAT3 is an essential transcription factor for both pathogenic and non-pathogenic Th17 cell differentiation, as it enables the expression of RORγt, a lineage-specific transcription factor for IL-17-secreting T cells, as well as a majority of molecules involved in their effector functions [150], including those involved in the pathogenesis of comorbidities, such as psoriatic arthritis [151]. Accordingly, when comparing patients with active and inactive psoriatic arthritis, STAT3 phosphorylation was detected in all blood CD4 + T cell subsets previously defined by single-cell mass cytometry, with the most dominant increase found in Th1 and Tfh lymphocytes [152]. These observations confirm the significant role of STAT3 activation in psoriasis complications and point again to the still unclear involvement of Tfh lymphocytes in the pathogenesis of Th17-related autoimmunity [153, 154]. Of note, elegant studies by Alshekaili and coauthors in people with either gain or loss-of-function mutations have shown that STAT3 regulates the cytotoxicity of Tfh lymphocytes, while their exhausted counterparts are resistant to this effect [155]. Taking into account that chronic self-antigen stimulation causes T cell exhaustion, one can thus speculate that STAT3 unresponsiveness is closely related to the self-tolerance state. Along these lines, IL-6, IL-21 and IL-23-triggered STAT3 phosphorylation has been proposed to skew Treg cells towards pathogenic functions in patients with psoriasis [156], altogether strongly suggesting that STAT3 activation in CD4 + T cells interrupts their tolerant phenotype.

This seems very likely in the case of psoriasis, as this autoimmune skin disease often occurs in patients with STAT3 Gain-Of-Function Syndrome [157]. Among others, the resulting STAT3 overactivation may also disturb skin homeostasis by reducing the release of IL-9 by Th9 lymphocytes [158], especially that their homeostatic function relies on STAT3 phosphorylation at physiological levels [159]. On the other hand, however, some studies suggest that efficient STAT3 activity in APCs is required to induce antigen-specific CD4 + T cell tolerance, while its disruption breaks their tolerant phenotype [160]. Besides, STAT3 overactivation in T cell lymphoma promotes the development and regulatory function of Tr1 lymphocytes [161]. These findings again confirm that STAT3 effects are highly dependent on cell type and signaling milieu, as mentioned above, and that the clinical outcome is determined by which effects dominate over others.

Thus, from a therapeutic perspective, selective STAT3 inactivation in self-reactive CD4 + T cells could become an effective approach to restoring immune tolerance. One can assume that this may be achieved by targeted delivery of miRNA-based therapeutics, because the lack of response to STAT3 may be due to either epigenetic regulation by miRNAs or loss-of-function genetic mutations [162]. It should be stressed that the latter rare genetic abnormality leads to the development of an Autosomal-Dominant Hyper-IgE Syndrome [163]. This can be avoided by using selective miRNA-based therapies, which would constitute their advantage over other genetic methods.

Furthermore, human Th22 lymphocytes also express high STAT3 activity, which, interestingly, can be antagonized by STAT1 in order to limit the secretion of IL-22 involved in psoriasis pathogenesis [164], as described below. However, in certain circumstances, this regulatory loop may induce a deleterious effect, because STAT3-triggered IL-22 secretion has been shown to control the barrier function of epithelial cells during colitis [165]. These observations provide another example of the complexity of STAT3 signaling translating into various cell effector functions and clinical outcomes.

It should also be stressed that suppressor of cytokine signaling 1 (SOCS1) and SOCS3, negative regulators of STAT1 and STAT3 phosphorylation by Jak kinases, were shown to regulate naive CD4 + T cell fate between Th1 and Th17 program. In detail, SOCS1 deficiency supports Th17 differentiation, while SOCS3 deficiency hampers IL-6 and IL-23 signaling and pushes CD4 + T cells towards Th1 phenotype under certain microenvironmental conditions [166, 167]. Although their role is yet not well-defined for Th1/Th17 cell functioning under psoriatic conditions, SOCS1 and SOCS3 levels are decreased in skin biopsies of psoriatic mice, and etanercept led to the increase of their expression with lower Th1/Th17 infiltration. Besides, SOCS3 deletion was demonstrated to support Th17 development in experimental autoimmune encephalomyelitis (EAE) [168].

Retinoic Acid-Related Orphan Receptors (RORs)

Until recently, the effector functions of Th17 lymphocytes downstream of STAT3 were thought to be controlled solely by RORγt, which, among others, activates the transcription of genes encoding cytokines from the IL-17 family and the IL-23 receptor. As a consequence, RORγt was established as a Th17 lineage-specific transcription factor [169], which activity is greatly enhanced by post-translational sumoylation [170]. Afterwards, however, it was discovered that a very similar set of genes can also be compensatorily regulated by RORα in human Th17 cells [171]. Furthermore, RORα expression is also induced by TGFβ and IL-21 in a STAT3-dependent manner, and its gene polymorphisms correlate with psoriasis severity [172, 173]. Thus, RORα began to be considered an important transcription factor that enables cell polarization towards the Th17 phenotype [174], while RORγt was proposed to preserve this phenotype and thus induce Th17 cell pathogenicity [172, 175]. This hypothesis seems to be supported by the observation that RORγt deficiency significantly reduces the activity of RORα and STAT3 [176], while depletion of RORα weakly reduces Th17 cell pathogenicity [177]. On the other hand, RORα sustains RORγt expression by promoting its transcription, thereby potentiating Th17 cell pathogenicity [177]. Therefore, this effect may be responsible for the chronic inflammatory activation of Th17 lymphocytes, which has recently been assigned to RORα [178].

Besides, the influence of RORγt transcriptional activity on Th17 cell effector functions is determined by cooperating factors. Specifically, RORγt-driven Runt-related transcription factor 1 (Runx1) stimulates the release of galectin-3 by Th17 lymphocytes, which in turn increases CCR6 expression via IL-1β delivered by galectin-recruited macrophages. As a result, Th17 lymphocytes develop a pathogenic phenotype, and CCR6 expression enables them to migrate to inflamed tissues, where they induce autoimmune responses. In contrast, this pathway is dispensable for Th17-dependent antimicrobial defense [179]. It is also worth mentioning that CCR6 expression distinguishes Th17 lymphocytes from other helper T cell populations [180]. Moreover, in certain circumstances, Runx1 and Runx3 interchangeably cooperate with T-bet to stimulate the release of IFNγ by pathogenic Th17 cells [181], while other psoriasis-related cytokine production is additionally driven by Runx3 itself [182]. From another perspective, Runx1 have been suggested to trigger RORγt activity to enhance IL-22 production by differentiating CD4 + T cells [183], which implies the importance of RORγt in maturation of Th22 lymphocytes that may potentially exacerbate psoriatic inflammation [184].

Interestingly, microbiota-driven RORγt expression by peripherally-induced Treg lymphocytes has been implicated to sustain their suppressive activity [185], suggesting that this transcription factor may also be involved in immune tolerance. Along these lines, in inflammatory conditions, it has been demonstrated that self-antigen-specific RORγt+ Treg cells develop from thymic precursors to downregulate autoimmune responses [186, 187]. Moreover, ERK2-mediated phosphorylation of RORγt at serine 182 skews CD4 + T lymphocytes towards Treg phenotype to reduce Th17-driven inflammation and maintain tissue homeostasis [188]. Finally, some preliminary observations suggest that the instability of RORγt+ Treg cell function may promote autoantibody formation in psoriatic patients [189]. Conversely, γδ T and NKT cells expressing RORγt may contribute to Th17-dependent autoimmunity by releasing large amounts of IL-17, and therefore inhibiting RORγt transcriptional activity in these cells has been considered a promising therapeutic approach [190–193]. However, the effectiveness of systemic RORγt inhibition in psoriatic patients enrolled to the Phase II clinical trial was below expectations [194], likely due to the simultaneous abolition of the protective role of RORγt+ Treg cells. Altogether, these findings reaffirm that signaling pathways have different effects on cell function, depending on the cell type and microenvironmental conditions, and their eventual therapeutic modulation should be selectively targeted to a specific cell population rather than systemically induced.

IRF4

In psoriasis, IRF4 has attracted researchers’ attention because it acts downstream of the cytokine receptor pathway, activating RORγt and RORα upon exposure to TGFβ in combination with IL-6 or IL-21, respectively [172]. This in turn enables CD4 + T cells to produce IL-17 family of cytokines, IL-17 A especially [195]. Mechanistically, IRF4 binds gene sequences containing AP-1-IRF composite elements that are then targeted by BATF to promote the transcription of Th17-programing genes [196]. Besides, IL-21 stabilizes Th17 phenotype via maintenance of IRF4 activity [197]. In turn, IRF4 phosphorylation by Rho-associated kinase 2 (ROCK2) drives IL-21 production and amplifies Th17-related autoimmunity [198]. This seems to explain the increased level of IRF4 detected in the skin biopsies of psoriatic patients [199], and capability of ROCK2 inhibitor to alleviate psoriatic inflammation [200, 201]. As mentioned above, IRF4 transcriptional activity has also recently been shown to be activated by IL-1β and costimulatory signaling [120] (Fig. 1). Moreover, IRF4 responds to the strength of the TCR signal and determines the fate of CD4 + T cells by cooperating with Blimp-1 for effector functions or with Bcl6 for Tfh maturation [202]. Taking into account that Blimp-1 activation in psoriatic keratinocytes drives the pathogenic programing of Th17 lymphocytes [203], these findings strongly imply the central role of IRF4 in psoriasis and other Th17-dependent autoimmune diseases. Indeed, its pharmacological inhibition alleviates psoriasis, EAE and experimental autoimmune uveitis in laboratory animals [204–206]. Similar effect can be achieved by stimulating the action of IRF4-binding protein [207]. This makes IRF4 an extremely important transcription factor in the pathogenesis of psoriasis and thus important target for future therapies.

Furthermore, IRF4 has been proposed to promote the maturation and pathogenic functionality of Th9 lymphocytes by dose-dependently enhance the transcription of IL-9-encoding gene [208, 209]. In addition, CD4 + T cells of IRF4-deficient mice poorly differentiate towards a Th1 phenotype [210], which is associated not only with reduced T-bet and IFNγ expression but also with diminished aerobic glycolysis [211]. This is further accompanied by decreased mTOR activity and nutrient uptake, which greatly alters the Th1 cell fate [212]. Besides, IRF4 depletion protects mice from experimental colitis [195]. Ultimately, recent comprehensive studies have confirmed that in IRF4-deficient mice, Th17 cell differentiation is completely abolished, while FLI1 has been identified as a key IRF4 cofactor for Th17 cell functionality. Contrary, the same studies have shown that IRF deficiency does not affect the development of Treg cells, but even indirectly increases the expression of FoxP3 [213]. These observations are in contrast to others made in the context of tumor microenvironment, which have found an important role for IRF4 in the development of Treg cells responsible for the exhaustion of effector T lymphocytes [214]. However, some opposing data suggest that IRF4 diminishes Helios activity and PD-1 expression by CD4 + T lymphocytes, thereby sustaining their effector function, including, among others, IFNγ production [205]. Thus, one can assume that IRF4 depletion would restore immune check point pathway for proper control of self-tolerance. However, IRF4 inhibition should be selective for CD4 + T cells, as its downregulation in DCs may reprogram them towards a Th17-promoting phenotype [215].

NF-κB

As reviewed elsewhere, several genes associated with NF-κB pathway are considered to drive human susceptibility to psoriasis and its complications [216]. A recent article showed that activation of this pathway in tissue-resident memory T cells is responsible for recurrent psoriasis, but this effect has been attributed to CD8 + rather than CD4 + T lymphocytes [217]. Conversely, RNA-seq of CD4 + T cells from psoriatic patients revealed the dysregulation of NF-κB pathway associated with TNFα signaling [218]. From another perspective, enhanced NF-κB signaling in APCs, including DCs, positively correlates with the frequency of Th17 lymphocytes and preludes the lack of response to biological therapy with adalimumab [219]. This is likely due to the fact that c-Rel is required for the production of IL-1β and IL-6 by DCs after TLR7 stimulation [220], and these cytokines are necessary for the activation of pathogenic Th17 lymphocytes (Fig. 1). However, experiments carried out in NF-κB1-deficient mice uncovered that this pathway is pathogenic in γδ T cells rather than in conventional CD4 + T cells [221]. On the other hand, in CD4 + T cells, TCR stimulation promotes NF-κB signaling involving a molecular adapter CARD-containing MAGUK protein 1 (CARMA1), which allows for completion of Th17 differentiation by increasing chromatin accessibility of effector cytokine loci [222]. Besides, as mentioned above, RelA (a p65 subunit) enhances cytokine signaling to induce STAT3 activity in differentiating CD4 + T cells [122]. Therefore, it can be assumed that the NF-κB pathway plays an important supporting role in the differentiation of Th17 cells at various stages [223], but its inhibition is not sufficient to fully alleviate psoriatic inflammation.

Dysregulation and overactivation of molecular pathways leading to psoriatic inflammation are often associated with abnormal miRNA signaling. It should be emphasized that epigenetics explores the influence of environmental factors on gene expression patterns, leading to stable and durable, yet reversible, modifications of DNA, histones, and RNA that dynamically regulate the functions of individual cells in the body. These alterations are induced, among others, by non-coding RNAs, such as miRNAs, circular RNAs (circRNAs), and lncRNAs. However, one should remember that non-coding RNAs, miRNAs especially, modulate gene expression in various manners, which leads to different biological outcomes, depending on the disease, tissue and cell-specific context [224]. With this knowledge, we propose the central concept that miRNA networks act as molecular regulators of CD4 + T cell plasticity and determine the balance between pathogenic inflammatory responses and immune homeostasis in psoriasis to constitute the foundation for next-generation precision therapies.

miRNA Regulation of CD4 + T Lymphocytes in Psoriasis

Firstly, it should be noted that dysregulated epigenetic modifications are recognized as an essential trigger of various pathological processes, including the breakdown of immune tolerance and the induction of autoimmunity [225]. In this regard, non-coding RNAs should be interpreted as targets for epigenetic-based therapies. Along these lines, recent narrative and systematic reviews have highlighted the most important non-coding RNAs associated with aberrant molecular signaling in keratinocytes and immune cells that drive psoriatic inflammation [224, 226–234], among which miRNAs dysregulated in immune cells, CD4 + T lymphocytes especially, are the main focus of the following considerations. Of note, T lymphocytes exhibit a similar pattern of miRNA dysregulation in autoimmune diseases with miR-21-5p, miR-148a-5p, and miR-155-5p being the most frequently upregulated non-coding RNAs [231]. Thus, we hypothesized that these upregulated miRNAs may determine the pathogenic activity of CD4 + effector T cells and repress immune tolerance.

The Upregulation of Selected miRNAs Corresponds to Pathogenic CD4 + T Cell Activation

Accordingly, T-bet transcription factor has been found to induce miR-148a expression in Th1 lymphocytes [235], while RORγt, STAT3 and IRF4 facilitate miR-155 production in Th17 cells [236], similarly to NF-κB pathway activation [224], which, together with STAT3, seems to control miR-21 transcription after TCR stimulation [237], as found in humans and rodents. These correlations strongly indicate that increased production of selected miRNAs results from activation of lineage-specific transcription factors that shape CD4 + T cell fate. However, it has still not been entirely clear why their activity induces CD4 + T lymphocytes to exhibit pathogenic autoimmune behavior.

In an attempt to answer this extremely important question, we conducted the contextual functional analysis with the use of DIANA-miRPath v4.0 [238]. It revealed “FOXO signaling pathway” as the second most enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway modulated by miR-21-5p, miR-148a-5p, and miR-155-5p in humans (Fig. 2a). This path caught our attention because recent transcriptomic meta-analysis identified “FOXO signaling” as one of the core molecular pathways affected in plaque psoriasis [239]. Moreover, FOXO transcription factors are involved in maintaining immune cell homeostasis by regulating their survival, apoptosis, autophagy, oxidative stress, and functional maturation [240], and all these processes are affected in psoriasis-associated CD4 + T cells (Fig. 2b, c). Accordingly, the loss of Forkhead box protein O1 (FOXO1) transcriptional activity in Treg lymphocytes greatly impairs their suppressive function in psoriatic patients [241] and in imiquimod (IMQ)-exposed mice [242]. Similarly, psoriasis is characterized by reduced expression of sirtuin 1 (SIRT1) [243], which drives the STAT3 and NF-κB pathways, leading to excessive activation of Th17 lymphocytes and their secretory activity [244]. According to DIANA-TarBase v8.0 [245], FOXO1 and SIRT1 are targeted by miR-155 and miR-148a, respectively, which seems to explain their diminished functionality in psoriatic inflammation (Fig. 2c).

Fig. 2.

miR-21, miR-148a, and miR-155, which are overexpressed in psoriasis, differently modulate the FOXO signaling pathway in CD4 + T cells. a Top 20 enriched KEGG pathways collectively modulated by hsa-miR-21-5p, hsa-miR-148a-5p, and hsa-miR-155-5p that are overexpressed in psoriasis-associated T lymphocytes, analyzed and visualized using DIANA-miRPath v4.0 (direct target prediction based on TarBase v8.0, miRNAs annotation from miRBase-v22.1, classic analysis with P-value threshold 0.05 and FDR correction). b Homeostatic role of important orchestrators of the FOXO signaling pathway, namely SIRT1 and FOXO1, in regulating CD4 + T cell differentiation and activation in order to prevent autoimmunity development. SIRT1-mediated deacetylation enables FOXO1 transcriptional activity while blocking the DNA binding capacity of STAT3 and p65 from NF-κB complex. c Pathogenic effect of downregulation of SIRT1 and FOXO1 expression together with overexpression of miR-148a and miR-155, characteristic of psoriasis, on the maturation and functioning of Treg and Th17 lymphocytes. According to the literature and the results of our contextual functional analysis, SIRT1 and FOXO1 are key targets of miR-148a and miR-155, respectively, which seems to explain their reduced expression in psoriasis-associated CD4 + T cells. d Complex effects of miR-21 on the FOXO signaling pathway, involving STAT3 and TGFβ1-driven processes, which together appear to diminish Th17 cell pathogenic activity. b-d Created with BioRender.com under a license for the Faculty of Medicine, Jagiellonian University Medical College

At the same time, STAT3 pathway activation in human keratinocytes has been proposed to increase miR-21 expression [246], similarly to activated human dermal T cells, in which miR-21 prevents apoptosis and thus may directly contribute to their expansion during psoriatic autoimmune reaction [247]. However, the impact of miR-21 on FOXO signaling pathway in CD4 + T cells appears to be much more ambiguous (Fig. 2d). Currently, STAT3 is considered to induce miR-21 transcription [248], which seems to explain its overexpression in psoriasis-related T lymphocytes. However, miR-21 has also been shown to downregulate STAT3 expression in mouse Treg cells, preventing their polarization towards a Th17-like phenotype [249], and to be released in extracellular vesicles (EVs) by human Treg lymphocytes [250]. Moreover, TGFβ1 signaling is thought to induce a non-pathogenic phenotype in mouse Th17 lymphocytes [251] by enhancing FOXO1 expression [252], and miR-21 has been found to promote TGFβ1 pathway by targeting its negative regulator, SMAD family member 7 (SMAD7) in cell culture-based experiments [253]. But some studies suggest that TGFβ1 inhibits miR-21 expression, which unlocks the SMAD7-dependent negative feedback loop [254], while other findings show that TGFβ1 increases miR-21 generation and thus limits SMAD7 activity [253]. Nevertheless, previous research has demonstrated that overactivation of SMAD7 that blocks TGFβ1 signaling in mouse T cells causes severe intestinal inflammation [255]. Altogether, it could therefore be assumed that miR-21 favors non-pathogenic Th17 and Treg lymphocyte polarization, which appears to confirm the immune regulatory function attributed to this miRNA [256]. However, there are conflicting data regarding the effects of miR-21 on the inflammatory cascade in Th17-mediated autoimmunity. Exactly, inhibition of miR-21 has been demonstrated to alleviate psoriatic skin inflammation [246], and EAE [257], making it a promising therapeutic approach in autoimmune diseases [258]. Moreover, in cancer patients miR-21 has been suggested to induce maturation of pathogenic Th17 cells in a TGFβ1-dependent manner [259], which seems to contradict previous conclusions. However, the role of TGFβ1 signaling in Th17 cell differentiation is extremely diverse, reflecting the complexity of biological processes regulated by miRNAs at different stages and with distinct biological effects.

Although the closely related FOXO and TGFβ1 signaling pathways control Th17 lymphocyte maturation at multiple levels [102, 260], other psoriasis-related processes active in human and rodent CD4 + T cells are also affected by miR-155 and miR-148a. For instance, miR-148a promotes Th1 cell survival by abolishing Bim activity [235], while miR-155 blocks immune checkpoint regulatory pathway by influencing CTLA-4 transcription as well as sustains the Th17 phenotype by targeting SOCS1 [224]. Thus, the divergent effects of miR-21, miR-148a, and miR-155 overexpression on psoriasis-associated CD4 + T cells strongly imply the involvement of other, yet undiscovered mechanisms of their action in psoriatic inflammation, which are all context-dependent. It is also worth adding here that miR-155 may promote the pathogenic role of Tfh lymphocytes in psoriasis, while miR-146a seems to prevent it [261]. Such a contradictory function of miR-155 and miR-146a is widely observed in psoriasis-associated CD4 + T cells, as described in our review.

miR-210 is another miRNA whose expression is increased in CD4 + T cells of psoriatic individuals [262]. Mechanistically, IL-23 and TGFβ-induced HIF-1α epigenetically enhances the transcription of miR-210, which then targets STAT6 and LYN, thereby promoting Th1 and Th17 lymphocyte differentiation followed by IFNγ and IL-17 overproduction [263]. In addition, miR-210 has been linked to the impairment of Treg cell development in psoriasis due to its postulated ability to disturb FoxP3 expression [264]. A similar detrimental effect on the Th17/Treg balance has also been attributed to the overexpression of miR-200a in human CD4 + T lymphocytes [265]. Besides, some preliminary observations suggest that miR-210 can modulate FOXO signaling pathway [266], analogously to miR-155 and miR-148a (Fig. 2), while the impact of miR-200a is less understood. However, it is worth noting that inhibition of miR-200a-3p was found to increase SIRT1 activity [267], suggesting that miR-200a overexpression also affects FOXO signaling in psoriatic CD4 + T lymphocytes.

Interestingly, miR-20b levels in serum and lesional skin obtained from patients with psoriasis showed a similar upregulation pattern as miR-155 and miR-210, which implied that miR-20b may also exert a pro-inflammatory effect on CD4 + T cells [268]. However, miR-20b has been demonstrated to target RORγt and STAT3 and thus significantly impair Th17 lymphocyte development and pathogenic function in EAE [269]. Analogously, miR-142-3p belongs to a group of miRNAs whose expression is increased in psoriatic skin, but is also known for its preferential expression in hematopoietic cells [270]. Although considered to promote hyperproliferation of keratinocytes [271], miR-142-3p is found in mouse Treg cell-derived EVs, which are capable of tolerizing DCs [272]. Additionally, this miRNA has recently been proposed to block IFNγ production by CD4 + T lymphocytes by targeting RICTOR, a main component of mTORC2 that promotes Th1 cell differentiation [273]. Again, these findings suggest that miR-20b and miR-142-3p exert cell-specific effects, with a rather inhibitory action on Th1 and Th17 cells. Therefore, from a therapeutic perspective, this highlights the need for a detailed understanding of the complex role of individual miRNAs in psoriatic inflammation and for their cell-directed delivery in future therapeutic strategies.

From another point of view, dermal macrophages modulate the pathogenesis of psoriasis in a context-specific manner [1]. For instance, when macrophages express reduced activity of serum- and glucocorticoid-regulated protein kinase 1 (SGK1), another target of miR-148a, they promote Th17 cell infiltration into mouse skin [274]. Furthermore, autoinflammatory differentiation of monocyte-derived DCs is driven by the transcription factor PU.1, which simultaneously increases the expression of miR-148a [275], whereas TLR7 stimulation in plasmocytoid DCs and TNFα-triggered inflammatory activation of macrophages upregulate miR-155 expression [276, 277]. Since both DC subpopulations together with macrophages play a key role in helper T cell polarization during psoriatic inflammation, it can be assumed that miR-148a and miR-155 may be transferred to antigen-recognizing CD4 + T cells at the immunological synapse in order to promote their differentiation bias towards Th1 and Th17 phenotypes (Fig. 3a). Moreover, miR-155 targets Ets-1, a negative regulator of Th17 cell maturation [278], thereby enabling efficient IL-23 signaling in order to induce T cell polarization and IL-17 production [276]. Finally, CD4 + T cell activation further increases the intrinsic transcription of miR-148a and miR-155 [224, 235], which appears to epigenetically stabilize their pathogenic functionality initiated by antigen recognition.

Of note, the abovementioned transcription factor PU.1 in CD4 + T cells drives Th9 polarization in cooperation with IRF4. Although the role of Th9 lymphocytes in psoriasis has not yet been clearly understood, PU.1 appears to promote pro-inflammatory activation of these cells, while, among others, IL-9 promotes TNFα, IFNγ and IL-17 production [279, 280]. On the other hand, FOXO1 drives Th9 cell differentiation, while its pharmacological inhibition in CD4 + T cells promotes Th1 and Th17 polarization [281, 282]. Since miR-155 has been shown to target FOXO1 (Fig. 2), one could speculate that this miRNA may prevent Th9 cell maturation. However, in the case of staphylococcal infection in children, miR-155 was found to promote Th9 cell activity by affecting SIRT1 expression [283]. Simultaneously, PU.1 has been demonstrated to activate miR-148a transcription in human DCs [275], suggesting its possible positive role in Th9 maturation, while overexpression of miR-148a-3p downregulated IRF4, thereby impairing Th9 cell development, and its sponging by lncRNA reversed these effects [284]. Altogether, these findings provide further evidence that miRNA-induced effects are context and cell type dependent.

Th22 lymphocytes also contribute to the pathogenesis of psoriasis by secreting IL-22, which, among others, may cause the release of autoantigens, which in turn triggers autoimmune responses [184]. Their maturation depends on miR-31 [285], the expression of which is also increased in patients with psoriasis [286]. This miRNA promotes cytokine and chemokine release by keratinocytes [286], and also influences cell movement and stability [287], but its exact role in CD4 + T cells remains to be elucidated.

All these observations imply additional indirect effects of miRNAs primarily upregulated in T lymphocytes from psoriatic patients. Therefore, from a therapeutic perspective, multidirectional but selective regulation of overactivated miRNAs is of great importance for the epigenetic control of autoimmune diseases [234, 246, 288, 289], including psoriasis [290]. Among other means [291], this may be achieved by introducing therapeutic lncRNAs to sponge pathogenic miRNA as well as by antagonizing miRNA with complementary miRNA inhibitors (anti-miRs). However, due to numerous challenges, including cell-specific targeting, there is still much work to be done before these technologies can be widely used in clinical practice [233, 234, 292, 293].

Certain miRNAs Orchestrate CD4 + T Cell Fate in Immune Tolerance

From another perspective, similar beneficial therapeutic effects can be achieved by restoring the appropriate profile of miRNAs specifically downregulated in psoriasis [294]. Accordingly, miR-138 shows reduced expression in CD4 + T cells from psoriatic patients, promoting transcription of Runx3, which drives Th1 lymphocyte differentiation, while miR-138 overexpression abolishes these effects [295]. This indicates that restoring miR-138 signaling can at least partially counteract the pathological effects of miR-210 in CD4 + T cells [262]. Furthermore, T cell autoimmune tendencies correlate with decreased expression of miR-146a [231], believed to prevent NF-κB pathway activation [233]. Besides, miR-146a transmitted by Treg cell-derived EVs has been observed to impair T cell proliferation by targeting STAT1 and IRAK2, and its serum level has increased after successful treatment of psoriatic patients with etanercept [250]. This implies that miR-146a may prevent the pathogenicity of Th1 lymphocytes [296], as continued below. However, miR-146a can also stimulate pro-inflammatory activation of keratinocytes [262], and recently has been positively correlated with psoriasis severity in patients [297]. On the other hand, miR-146a is well known to reduce TLR signaling, and thus its deficiency may heighten the activity of macrophages and DCs [298], initiating psoriatic inflammation. As signaled above, these discrepancies confirm that miRNA-mediated epigenetic regulation is cell-, tissue-, and disease-specific, and likely also patient-specific, for instance due to single nucleotide polymorphisms (SNPs) [299–302]. On the other hand, some keratinocyte-directed observations suggest that miR-125b and miR-99a target STAT3, and both are silenced in psoriatic lesions [233]. It can therefore be assumed that these miRNAs may also be dysregulated in T lymphocytes, which would further contribute to the pathogenesis of psoriasis. This is likely because both of them have already been proposed to inhibit Th17 cell differentiation in other autoimmune conditions [303, 304]. Moreover, topical application of miR-125b to lesional skin of IMQ-treated mice efficiently alleviated the inflammatory symptoms [294].

Our functional context analysis [238] to predict the possible molecular outcomes of the collective impact of miR-99a-5p, miR-125b-5p, miR-138-5p, and miR-146a-5p on CD4 + T lymphocytes revealed that the “AMPK signaling pathway” is the fifth most enriched KEGG pathway affected by these miRNAs, with AMPKα1 being directly targeted by miR-146a (Table 1). Strikingly, an elegant study by Blagih et al. demonstrated that AMPKα1 is required for the functional maturation of Th1 and Th17 cells in mice with colitis [305]. Moreover, Th1 and Th17 cell differentiation is positively regulated by the nutrient-sensing PI3K/Akt/mTOR pathway [306–308], while their pathogenicity is driven by the HIF-1α axis [309, 310], and these paths are also co-repressed by all four analyzed miRNAs (Table 1). Hence, these findings clearly indicate that psoriasis-associated downregulation of miR-99a, miR-125b, miR-138, and miR-146a in CD4 + T lymphocytes enables signaling cascades essential for effector responses, and links metabolic dysregulation with autoimmunity [311–313]. In this regard, restoring physiological miRNA signaling in CD4 + T lymphocytes would lead to stable re-induction of immune tolerance along with balanced Treg and helper T cell reactivity.

Table 1.

The most important KEGG pathways collectively modulated by hsa-miR-99a-5p, hsa-miR-125b-5p, hsa-miR-138-5p, and hsa-miR-146a-5p, that are downregulated in psoriasis-related CD4 + T lymphocytes, analyzed using DIANA-miRPath v4.0 (direct target prediction based on TarBase v8.0, miRNAs annotation from miRBase-v22.1, classic analysis with P-value threshold 0.05 and FDR correction)

Rank	Term name	Target genes (Term genes)	P-value	FDR
1	Salmonella infection	55 (277)	4,61866E-08	1,35605E-05
2	Regulation of actin cytoskeleton	47 (224)	7,95338E-08	1,35605E-05
3	Cell cycle	32 (129)	1,90504E-07	2,16539E-05
4	Hippo signaling pathway	37 (164)	2,92858E-07	2,49661E-05
5	AMPK signaling pathway	30 (130)	2,35738E-06	0,000133978
6	Proteoglycans in cancer	43 (220)	2,14044E-06	0,000133978
7	ErbB signaling pathway	22 (86)	9,08219E-06	0,000334678
8	Adherens junction	21 (79)	7,56732E-06	0,000334678
9	HIF-1 signaling pathway	26 (112)	9,81461E-06	0,000334678
10	Central carbon metabolism in cancer	20 (74)	9,47091E-06	0,000334678
…
49	PI3K-Akt signaling pathway	52 (372)	0,002003942	0,014067807
…
69	mTOR signaling pathway	27 (177)	0,007431441	0,036726398

Obviously, differentiation of Treg lymphocytes also relies on proper miRNA signaling. Among those, whose expression pattern generally distinguishes Treg cells from other CD4 + T lymphocytes [314], the most attention, taking into account the current considerations, should be drawn to upregulated miR-155 and miR-146a. Accordingly, the overexpression of pro-inflammatory miR-155 in Treg cells could be quite surprising. However, early in cellular development, FoxP3 transcriptional activity triggers miR-155 expression in order to ensure sufficient IL-2 responsiveness and tolerogenic phenotype of proliferating Treg cells by diminishing SOCS1 activity, whilst miR-155-depleted Tregs are capable of preventing colitis in mice. In contrast, the regulatory function of Treg cells seems to strongly depend on miR-146a [298], which is reduced in psoriatic CD4 + T lymphocytes. Instead, augmented miR-146a activity facilitates Treg cell maturation and functioning by affecting STAT5b [315], and, importantly, protects them from acquiring the Th1-like phenotype along with limiting the STAT1-driven effector activity of Th1 lymphocytes [296, 298, 316]. Besides, among other miRNAs, miR-146a is enriched, whereas miR-155 is absent in Treg cell-derived EVs [250], which appear to potentiate immune regulatory activity in an endocrine manner. On the other hand, there is a significant gap in the current knowledge about the miRNA signature of Tr1 lymphocytes with one study implying that miR-92a-3p and miR-125a may inhibit their differentiation [317].

From another perspective, the epigenetic regulation of cellular activity at the molecular level has outstanding therapeutic potential in autoimmune diseases [318]. In this context, non-coding RNAs, miRNAs especially, emerge as the most promising tools for such therapeutic approaches. As reviewed elsewhere [319–321], topical application of miRNAs may be the most tolerated route of their therapeutic administration by patients, and their encapsulation in EVs directed towards the desired acceptor cells may become the most effective and safest formulation. Along these lines, topical application of miR-126-5p and miR-320-3p in modified EVs has been demonstrated to alleviate psoriatic inflammation in mice by diminishing IL-17 signaling circuit [322]. Similarly, therapeutic administration of miR-340 has been found to block IL-17 production by Th17 lymphocytes in mouse model of psoriasis [323].

In general, targeting the cytokine axis is currently the leading strategy in the biological treatment of autoimmune diseases, and miRNAs are emerging as promising candidates for the sustained epigenetic regulation of cytokine production. However, as elegantly summarized by Salvi et al. [324], the effects of miRNAs on cytokine synthesis pathways are multifaceted. Specifically, an individual miRNA may block cytokine synthesis by binding to its corresponding mRNA or reducing the activity of transcription factors, whereas in other circumstances even the same miRNA may increase cytokine release by stimulating TLR7/8 receptors or degrading repressor mRNA. This is a perfect example to highlight that the use of miRNAs in biological therapy requires tailored strategies to deliver them directly to the desired cells in a specific differentiation/activation state.

Accordingly, another suggested formulation for miRNA-based therapeutics is to deliver them as spherical nucleic acids, i.e. miRNAs coated on a dense core to ensure their bioavailable spherical conformation [325]. However, the most commonly considered means is the use of naturally generated EVs or synthetic liposomes. Interestingly, the use of lipid vesicles to facilitate miRNA penetration through the skin has recently been proposed [326], which is currently attracting interest in the topical treatment of psoriasis, as mentioned above. Conversely, some studies also suggest the possibility of dissolving therapeutic miRNAs in reagents originally intended for transfection before intradermal injection, which supposedly may increase the chance of effective incorporation of miRNAs by acceptor cells. This method was used when administering miR-146a to mice, which alleviated the symptoms of psoriasis [300]. Similar effects were attributed to framework nucleic acids, precisely designed three-dimensional RNA nanostructures with high biocompatibility, bioavailability, and tissue permeability, which have been used to topically deliver miR-125b to mice with IMQ-induced psoriatic inflammation [294]. On the other hand, intradermal administration of miRNAs enclosed in naturally generated EVs also offers an interesting therapeutic opportunity with high biocompatibility and bioavailability.

Accordingly, our previous studies have shown that the immune regulatory function of CD8 + T suppressor (Ts) lymphocytes in mouse delayed-type hypersensitivity results from the release of miRNA-enriched EVs [327–329]. These EVs are then coated with antigen-specific antibody light chains to specifically target antigen-presenting cells thereby modulating CD4 + T cell activation [330–334]. Of note, among the EV-enriched miRNAs, miR-150 was proven to mediate the inhibitory function of Ts cells in vivo and after therapeutic delivery of Ts cell EVs via different routes, including intradermal administration [327, 329, 331, 333, 335]. This miRNA has already been shown to impair CD4 + T cell maturation by targeting AKT3/BIM signaling cascade [336]. Moreover, we demonstrated that miR-150 restrains self-reactive CD4 + T lymphocytes in a mouse model of delayed-type hypersensitivity induced by syngeneic red blood cells [337].

Noteworthy, prior studies, including those involving psoriatic patients, have uncovered that miR-150 is enriched in Treg cell-derived EVs [250], and in this form is transferred to DCs, which in turn increase its transcription and develop an anti-inflammatory phenotype [272]. Strikingly, we observed the same effect in antigen-presenting macrophages pretreated with miR-150-carrying EVs from Ts cells [333]. It can therefore be speculated that miR-150 is released in EVs by regulatory/suppressor T lymphocytes in order to induce tolerogenic phenotype in antigen-presenting cells to prevent the activation of self-reactive T lymphocytes at the immunological synapse (Fig. 3b) [334]. This speculation is of great interest to our ongoing research focused on the possibility of inhibiting the CD4 + T lymphocytes involved in the pathogenesis of IMQ-induced psoriatic inflammation by Ts cell-derived EVs. Nevertheless, because the pathomechanisms underlying autoimmune effector responses are much more complex than the modeled delayed-type hypersensitivity reaction, we hypothesized that other miRNAs present in Ts cell EVs may also be involved in regulating autoreactive T lymphocytes in order to enhance miR-150 action.

To initially validate this hypothesis and search for possible molecular targets in silico, we performed the contextual functional analysis [238] of the ten miRNAs most enriched in Ts cell EVs (please refer to supplementary Table E1 in [327]). The analysis revealed five of them that cooperatively modulate the vast majority of modulated KEGG pathways. Those include mmu-let-7b-5p, mmu-miR-150-5p, mmu-miR-33-5p, mmu-miR-484, and mmu-miR-486a-5p (Table 2). Of particular note is the fact that all five miRNAs affect the KEGG pathways “Th17 cell differentiation” and “Th1 and Th2 cell differentiation” as well as “FOXO signaling pathway” discussed above. Among the target molecules involved in these pathways, IRF4 seems to be the most interesting, being influenced by let-7b, miR-150 and miR-33. As discussed above, this transcription factor is essential for Th17 lymphocyte differentiation [338] due to its ability to bind to the E1A binding protein p300 (EP300) promoter, which triggers RORγt transcription and increases IL-17 A secretion [206]. Besides, IRF4 augments miR-155 production by Th17 cells [236] and supports Th9 cell development [279]. Importantly, therapeutic blockade of IRF4 significantly ameliorates IMQ-induced psoriasis inflammation in mice [206]. These activities makes transcription factor IRF4 a promising candidate for epigenetic modulation by miRNAs, including Ts cell-derived miR-150, let-7b and miR-33. It can be assumed that such treatment of CD4 + T cells will prevent Th17 cell differentiation and reverse the harmful effects of miR-155 in psoriatic inflammation.

Table 2.

The most important KEGG pathways collectively modulated by mmu-let-7b-5p, mmu-miR-150-5p, mmu-miR-33-5p, mmu-miR-484, and mmu-miR-486a-5p, that emerged during analysis of the top 10 miRNAs enriched in Ts cell-derived EVs using DIANA-miRPath v4.0 (direct target prediction based on TarBase v8.0, miRNAs annotation from miRBase-v22.1, classic analysis with P-value threshold 0.05 and FDR correction)

Rank	Term name	Target genes (Term genes)	P-value	FDR
1	MicroRNAs in cancer	65 (164)	1,0746E-14	3,6214E-12
2	Proteoglycans in cancer	65 (207)	1,6896E-09	2,8469E-07
3	FoxO signaling pathway	48 (136)	3,5207E-09	2,9662E-07
4	Autophagy - animal	50 (144)	3,1471E-09	2,9662E-07
5	AGE-RAGE signaling pathway in diabetic complications	37 (103)	1,3019E-07	8,7749E-06
6	MAPK signaling pathway	79 (302)	2,739E-07	1,5384E-05
7	Adherens junction	28 (71)	4,8752E-07	2,347E-05
8	EGFR tyrosine kinase inhibitor resistance	30 (82)	1,2764E-06	5,3768E-05
9	Human T-cell leukemia virus 1 infection	65 (244)	1,6046E-06	5,8514E-05
10	Lysine degradation	25 (63)	1,7363E-06	5,8514E-05
11	HIF-1 signaling pathway	37 (116)	3,657E-06	0,00011204
12	AMPK signaling pathway	40 (130)	4,0879E-06	0,0001148
13	Th17 cell differentiation	34 (105)	6,2094E-06	0,00016097
14	Protein processing in endoplasmic reticulum	48 (172)	9,6436E-06	0,00023213
15	Ubiquitin mediated proteolysis	42 (145)	1,2791E-05	0,00028736
…
18	PI3K-Akt signaling pathway	84 (364)	2,57573E-05	0,000482233
…
22	mTOR signaling pathway	44 (163)	5,4223E-05	0,000830597
…
39	Th1 and Th2 cell differentiation	26 (89)	0,000477843	0,004129055

In addition, Ts cell EV-enriched miRNAs can ameliorate psoriatic inflammation by modulating “MAPK signaling pathway” (Table 2), in which JNK2 kinase is involved in Th1 lymphocyte maturation and p38 MAPK is essential for optimal IL-17 production by self-reactive Th17 cells [339, 340]. Furthermore, all five miRNAs modulate AMPK, HIF-1α and PI3K/Akt/mTOR pathways (Table 2), which are involved in Th1 and Th17 cell differentiation, as discussed above. Thus, it seems likely that miRNAs from Ts cell EVs compensate for reduced expression of miR-99a-5p, miR-125b-5p, miR-138-5p, and miR-146a-5p in psoriatic CD4 + T cells (Fig. 4). It is worth noting herein that miR-150 is known to cooperate with miR-99a to activate Treg cell differentiation and repress Th17 cell development by affecting mTOR and HIF-1α signaling cascade [341]. However, some conflicting data suggest that miR-150 depletion impairs spontaneous proliferation of colitogenic Th17 lymphocytes thereby ameliorating intestinal inflammation [342]. These potential contradictions clearly indicate that induced expression of miR-99a enables miR-150 to exert its anti-inflammatory effects, confirming again the complexity and interdependence of miRNA-triggered regulatory mechanisms. Moreover, this seems to support our assumption that miR-150 alone is not able to fully inhibit effector function of autoreactive CD4 + T cells.

Fig. 4.

Molecular pathways modulated by miRNAs differently expressed in psoriatic CD4 + T lymphocytes. Characteristic overexpression of miR-21, miR-148a and miR-155 disrupts FOXO signaling, while repression of miR-99a, miR-125b, miR-138 and miR-146a promotes activation of AMPK, PI3K/Akt/mTOR and HIF-1α pathways, all resulting in CD4 + T cell differentiation towards Th1 and Th17 phenotypes. Conversely, miRNAs enriched in suppressor T cell-derived extracellular vesicles (EVs), including miR-150, let-7b, miR-33, miR-484 and miR-486a, collectively affect Th1 and Th17 differentiation pathways along with AMPK, PI3K/Akt/mTOR and HIF-1α signaling cascades to prevent psoriasis-related autoimmune responses. Created with BioRender.com under a license for the Faculty of Medicine, Jagiellonian University Medical College

Along these lines, apart from lineage-specific RORγt transcriptional activity, IL-23-triggered Th17 cell differentiation involves the activation of STAT3, which enables cytokine production, as well as the Rho-associated protein kinase (ROCK), which promotes cell migration [343]. It is worth noting that, according to TargetScan database, RORγt (RORC) and STAT3 mRNAs are directly targeted by let-7b, while ROCK mRNA is affected by miR-150. In addition, miR-150 has already been shown to target STAT1 [344], which may prevent IFNγ-driven functional maturation of Th1 lymphocytes. Another known target of miR-150 is NOTCH1 [345], which has been shown to influence the Th17/Treg balance in patients with psoriasis [346]. Additionally, as mentioned, together with miR-99a, miR-150 represses mTOR activity, which in turn drives Treg cell differentiation and prevents polarization towards Th17 phenotype [341]. Finally, miR-150 deficiency has been correlated with decreased expression of TGFβRII by intraepithelial lymphocytes [347], while this signaling pathway has been found to determine IL-10 production by non-pathogenic Th17 cells [348]. Therefore, miR-150 should be considered as a potential candidate for anti-inflammatory regulation of Th17 cell activity.

On the other hand, let-7b has been found to promote anti-inflammatory macrophage polarization [349], which may have a beneficial effect on psoriatic inflammation [1]. Especially that Ts cell EV-transmitted miR-150 has been demonstrated to target antigen-presenting macrophages, which inhibited CD4 + T cell activation [331–333]. Besides, since mTOR-driven signaling cascades shape macrophage functioning in autoimmune responses [350], their control by miRNAs, including miR-150, appears as another potential epigenetic mechanism comprehensively regulating inflammation in psoriasis [351]. Aside from that, miR-33 also seems to induce an anti-inflammatory phenotype of macrophages [352], which together provides new evidence that macrophages play a critical intermediary role in Th1 and Th17 cell suppression. At the same time, the immunomodulatory functions of miR-484 and miR-486a are still insufficiently investigated, but preliminary data indicate their involvement in macrophage antimicrobial defense [353, 354]. Nevertheless, miR-486 has been suggested to target SITR1 in macrophages [353], which may also link this miRNA with complex helper T cell differentiation pathways and FOXO signaling.

Taken together, these data strongly suggest that miRNAs transmitted by Ts cell-derived EVs, miR-150, let-7b and miR-33 especially, are collectively capable of inhibiting Th1 and Th17 cell differentiation, thereby efficiently ensuring immune tolerance by modulating multiple signaling pathways. However, these findings require experimental confirmation.

miRNA Networks Regulating CD4 + T Cell Fate in Psoriasis – Biological Insights

Based on the above considerations, three main groups of miRNAs regulating CD4 + T cell activity in psoriasis can be distinguished: (i) pathogenicity-promoting miRNAs, which enhance the inflammatory response of CD4 + T cells, such as miR-155, miR-148a and miR-210; (ii) immune tolerance-related miRNAs, involved in maintaining Treg function and preventing pathogenic effector T cell responses, including miR-146a, miR-150, miR-99a and miR-125b; as well as (iii) “context-dependent” miRNAs, whose activity varies depending on the cellular microenvironment and disease stage, like miR-21, miR-20b, miR-142-3p, miR-31 and also miR-146a. Interestingly, many of these miRNAs are now considered promising biomarkers for diagnostic and prognostic monitoring of the disease and therapeutic responses, as reviewed elsewhere [355]. Moreover, depending on the cellular source and disease state, these miRNAs may be considered as casual regulators, and/or biomarkers of inflammation. For instance, while miR-146a is a leading epigenetic regulator of immune homeostasis, miR-155 can serve as a marker of NF-κB pathway activation and inflammation [356, 357]. Thus, due to the extremely complex and heterogeneous course of psoriasis, determining the individual miRNA profile and its interpretation in the context of current knowledge will thus be crucial for its effective management and prediction of possible treatment resistance or disease relapse.

Moreover, as discussed in this article, miRNAs are currently considered a promising tool in precision therapy of psoriasis [234], although their implementation in clinical practice is still far from routine due to many limitations and challenges. However, the use of miRNA mimics and/or inhibitors for epigenetic control of disease-involved immune cells, CD4 + T cells especially, may significantly complement or even replace currently used biological therapies due to the greater possibility of individual adjustment of both the composition of mimics and inhibitors and their targeting to selected cells. The latter could be achieved by delivering miRNAs and/or their inhibitors with the use of nanoparticle- or EV-based platforms [233], administered via the most effective routes and targeted to the selected cell, for instance using specific antibodies [319]. Overall, miRNA-based therapeutic approaches appear to have significantly fewer side effects compared to currently available treatments.

In this regard, next-generation miRNA-based precision therapies offer specific advantages over currently available biologics, such as anti-IL-23, anti-TNF, and anti-IL-17 monoclonal antibodies. Accordingly, it can be assumed that combination miRNA therapies selectively delivered to specific immune cells will act by modulating entire multi-gene inflammatory pathways rather than neutralizing a single cytokine, potentially lowering the risk of generalized immunosuppression and allowing for more comprehensive intracellular tuning of pathogenic immune responses with simultaneous restoration of immune tolerance. In other words, therapeutic miRNA modulation is likely to selectively reprogram the activity of pathogenic CD4 + T cells without impairing immune surveillance after employing a cell-specific miRNA delivery systems to overcome current translational barriers. Furthermore, before miRNAs are introduced into routine therapy, changes in their profile can also be used to predict a patient’s response to biologics and thus support the selection of a specific drug that best suits a given individual [358, 359].

However, therapeutic regulation of miRNA post-transcriptional activity also poses significant challenges. These include, among others, the full understanding of miRNA sorting and packaging into EVs by living cells [360, 361], which is being attempted to be replicated in vitro for therapeutic purposes [362], as well as reciprocal interactions with other non-coding RNAs, such as lncRNAs and circRNAs that strongly influence miRNA action, as recently reviewed in detail elsewhere [363, 364]. Therefore, the biological outcomes of therapeutic miRNA activity are still a subject of uncertainty, and thus, despite their enormous potential, such therapeutics have yet to be implemented in routine clinical practice. However, the ever-increasing knowledge in this field should enable the refinement of miRNA-based therapies for the benefit of humanity in the foreseeable future owing to the fast development of modern research technologies.

Furthermore, it should be remembered that the direct translation of individual experimental results into complex mechanisms operating in a living organism, which are subject to continuous adaptation, is burdened by significant discrepancies. Therefore, the overall impact of one miRNA observed biologically results from the dominance of one effect over the others and depends on many contexts.

Finally, existing discrepancies between the postulated roles of individual miRNAs may also result from differences in experimental procedures and conditions, including heterogeneity between patient groups, different animal models, variable cytokine stimulation conditions, and cellular heterogeneity. These aspects should also be considered when holistically interpreting the data.

Therefore, the introduction of personalized therapies containing miRNA mimics and/or inhibitors requires continuous research to ultimately standardize and validate our understanding of miRNA biology in psoriasis and overcome many technological limitations, which is still associated with enormous financial outlays.

Conclusions and Future Persepctives

To conclude, our comprehensive summary of the latest knowledge regarding molecular dysregulation and miRNA profile of CD4 + T lymphocytes in plaque psoriasis highlighted IRF4 as the most relevant target for future precision therapies. Whereas, contextual functional analysis identified miR-150 as the most promising candidate for epigenetic regulation of Th1 and Th17 lymphocytes involved in the pathogenesis of psoriasis.

However, before miRNA-based therapy can be implemented into routine clinical practice, many challenges and limitations must be overcome. The most influencing obstacles include delivery efficiency and in vivo stability, tissue distribution and cell specificity, off-target effects and eventual immunogenicity. All of them raise general safety concerns in terms of context-dependent multi-target pleiotropy, possible tissue accumulation, and dosage restrictions to induce the expected effect while avoiding saturation of the cellular machinery. These limitations are being solved in various ways. Along these lines, systemic administration of miRNAs exposes them to ribonuclease digestion and rapid renal excretion, which significantly shortens their plasma half-life. Therefore, various chemical modifications, recently described in detail by Shi and colleagues [365], and protective delivery platforms, ranging from vectors to lipid-based nanoparticles and cell-derived EVs [366], have been proposed to date in order not only to increase the bioavailability of miRNAs but also to ensure dosing control and selective targeting [367]. In this regard, our observations so far indicate that EVs transporting miRNAs can be delivered specifically to selected cells by coating them with specific antibodies and/or their light chains [319]. It should be noted that such selective delivery would also limit the overall immunogenicity and tissue toxicity of miRNAs, while chemical modifications may reduce their TLR-stimulating activity as well as off-target effects resulting from miRNA binding to the 5’ non-coding region or coding sequence of the mRNA to significantly increase the overall safety of this therapeutic approach [368]. However, all these aspects still require intensive investigation and solutions that may be available in the near future thanks to the rapid development of high-resolution and precision technologies, which, however, are currently extremely expensive.

The above considerations clearly indicate that activation of molecular pathways involved in the pathogenesis of psoriasis exerts different biological effects depending on the cell type. Therefore, potential therapeutic regulation of these pathways requires precise and selective targeting of biologic drugs to specific cell populations. However, directing these drugs to CD4 + T cells is not sufficient, because although it inhibits the effector functions of helper T lymphocytes, it also blocks the expected activity of Treg cells. Therefore, when considering potential other targets for the delivery of such drugs, we propose the IL-23 receptor. As comprehensively reviewed by Mezghiche and coauthors [369], it is expressed by conventional and unconventional T cells, NK cells, macrophages and dendritic cells, as well as by B lymphocytes. In all these populations, its ligation enhances effector functions, which can be considered pathogenic in psoriasis. In contrast, IL-23 receptor signaling destabilizes the suppressive activity of Treg cells [370]. Thus, the use of an IL-23 receptor antagonist in order to selectively target IL-23R+ cells with a biologic should elicit beneficial therapeutic effects while significantly reducing systemic adverse reactions. However, the therapeutic delivered in this way should be selected precisely, because the effects of miRNA on myeloid and lymphoid cells can be opposite. Although with respect to the miR-150-IRF4 regulatory axis suggested in our considerations, macrophages exposed to IL-23 do not appear to overexpress IRF4 [371], miR-150 should therefore induce tolerogenic effects in both myeloid and lymphoid populations.

Nevertheless, several important questions remain without clear answers. Firstly, are we able to unequivocally determine the miRNA profiles that characterize the cells involved in the pathogenesis of psoriasis at a given stage of the disease? Will this knowledge be capable of translating into clinical applications for monitoring and predicting disease progression and treatment? Finally, do miRNA-based therapeutics have a chance of widespread clinical use? Positive answers depend on how precisely we will be able to understand the signaling pathways and functions of individual miRNAs in cells involved in the pathogenesis of psoriasis, including CD4 + T lymphocytes, through coordinated basic, preclinical, and clinical research. This requires time and financial resources, the involvement of scientists from around the world and various disciplines, including medicine, immunology and bioinformatics, cooperation with the technology industry and increasing patient awareness. Therefore, the more we know, the more likely it is that our knowledge will be implemented in clinical practice.

Altogether, since CD4 + T lymphocytes play such a crucial role in the pathogenesis of plaque psoriasis, and the incredible development of research techniques allows for increasingly precise identification of the molecular mechanisms underlying their pathogenic function, future research should focus on establishing the therapeutic efficacy of targeted therapies, starting from basic research involving laboratory animals and ending with full-scale clinical trials.

Authors’ Contributions

Conceptualization, Y.D. and K.N.; formal analysis, Y.D. and K.N.; investigation, Y.D. and K.N.; writing—original draft, Y.D. and K.N.; writing—review and editing, K.N.; visualization, Y.D. and K.N.; supervision, K.N.; project administration, K.N.; funding acquisition, K.N. All authors have read and agreed to the published version of the manuscript.

Funding

Our preliminary studies on the role of miR-150 in Th1 and Th17 cell suppression are supported by a subsidy from the Polish Ministry of Science and Higher Education for the Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland under grant number N41/DBS/001623 for K.N. Open access funding was provided by Springer Open Publishing Program under a Polish National License.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Competing Interests

The authors declare no competing interests.