**2. Immune cells in the development of psoriasis**

As already stated, Ps is an immune-mediated inflammatory skin disorder based on the pathological crosstalk between KCs and immune cells sustained by a complex array of pro-inflammatory cytokines/chemokines. Disturbances in both innate and adaptive immune responses, as well as hyper-proliferative KCs, have important roles in triggering the early psoriatic events and in sustaining the chronic inflammation that follows. Immune cells are highly involved in both phases of Ps pathogenesis: innate immune cells (PDCs, MDCs, NK cells, Mɸ, neutrophils) play a major role in the initiation of the psoriatic events, while adaptive immune cells (Th-cell subsets: Th17, Th22, Th1) are the main actors in maintaining the inflammatory status of the disease.

#### **2.1 Tissue resident immune cells**

Dendritic cells, professional antigen-presenting cells (APCs), are crucial in the early stages of the disease due to their ability to recognize the antimicrobial peptides (AMP) (LL-37, β-defensins, S100 proteins) released by damaged KCs in response to

various triggering factors [19]. LL-37, recognized as an autoantigen in Ps, binds to self-DNA and RNA from other damaged cells and stimulates toll-like receptor (TLR) 9, respectively TLR8 in PDCs. Activated PDCs will secrete IFN-α thus promoting MDCs activation. These activated cells will migrate into draining lymph nodes and by secreting high amounts of IL-12, IL-23 and TNF-α will further guide the differentiation of naïve T-cells in Th1, Th17, and Th22 populations [15]. These effector subsets will produce TNF-α, IFN-α, IL-17, IL-22 thus promoting KCs proliferation. Activated KCs will perpetuate the inflammatory cycle by generating AMP (LL-37, β-defensins, S100 proteins) and by secreting cytokines (TNF-α, IL-6, IL-1β) and chemokines (CXCL1, CXCL2, CXCL3, CXCL5, CXCL8, CXCL9, CXCL10, CXCL20) [14]. LL-37 secreted by KCs will be recognized by LL-37-activated PDCs which will perpetuate the pathogenic mechanism. IL-17, CXCL1, CXCL2, and CXCL8 released by Th17 and KCs mediate the recruitment of neutrophils which will migrate to the psoriatic lesions, release large amounts of reactive oxygen species (ROS), granules and form neutrophil extracellular traps (NETs) [20].

MDCs can be also PDCs-independent activated, by CCL20 released by KCs in response to the skin microbiome, drugs, and injury [21]. Once activated, MDCs will initiate the inflammatory state in Ps. Two subpopulations of dermal MDCs have been described in psoriatic lesions: CD11c<sup>+</sup> BDCA-1+ cells, which are also found in normal skin, and CD11c+ BDCA-1− cells, characterized by the ability to produce inflammatory cytokines. CD11c+ BDCA-1+ cells are comparable in number to those found in normal skin, while CD11c+ BDCA-1− cells, named "inflammatory MDCs", are increased 30-fold in psoriatic lesions. Both subpopulations of MDCs induce T cell proliferation and cause differentiation into Th1 cells. In addition, inflammatory MDCs are responsible for the IL-23-induced Th17 stimulation [22].

Neutrophils, important representatives of innate immune cells, are considered to be regulators between innate and adaptive immune systems. Neutrophils are attracted to the psoriatic plaque by chemokines (CXCL1, CXCL2) and cytokines (IL-8) released by activated KCs, forming Munro microabscesses, an important histopathological feature of Ps [23]. Respiratory burst, degranulation, and NETs formation, the main offensive function of neutrophils, are correlated with Ps' development and progression.

During activation, neutrophils mobilize and release granular components thus inducing their own migration and activating their antimicrobial activities. NE (neutrophil elastase), MPO (myeloperoxidase), and LL-37 (recognized as an autoantigen in Ps) were reported to be highly involved in Ps. NE and cathepsin G activate IL-36 leading to an exacerbated tissue inflammation [24]. In Ps patients' circulatory neutrophils have increased MPO and NOX2 (reduced nicotinamide adenine dinucleotide phosphate-NADPH oxidase) activities and enhanced respiratory burst, releasing more ROS compared to healthy individuals [25]. Overproduction of ROS induces proliferation and differentiation of Th1, Th17, and Th22 cells, and inhibits regulatory T cells (Tregs). Inflammatory cytokines secreted by these T cell subsets along with VEGF, stimulate KCs proliferation and angiogenesis [26, 27]. In psoriatic plaque and pustules, NETs are overexpressed and IL-17 releasing cells will be activated. Accordingly, the synthesis of inflammatory mediators will be stimulated, leading to auto-amplification of neutrophil's overall activities [20]. Inhibition of neutrophils degranulation and suppression of respiratory burst can become new therapeutical targets for alleviating Ps symptoms.

Macrophages, cells with phagocytic and antigen-presenting properties, are important innate immune sentinels. Mɸ is abundant in psoriatic lesions and are involved in

the inflammatory process due to their ability to produce cytokines and inflammatory mediators. The most representative monokines are IL-23, responsible for the differentiation of Th17 [28], and TNF-α responsible for Th22 differentiation [29].

NK cells, recognized for their ability to kill virally infected cells and cancer cells, appear to be involved in the pathogenesis of Ps, but their role still remains controversial. Thus, Ottaviani et al. identified a subpopulation of NK cells with the CD56brightCD16− CD158+ phenotype able to produce large amounts of IFN-γ, and these cells are recruited by KC activated through chemokine secretion (CXCL10, CCL5) [30]. Thus recruited, NK cells can contribute to the inflammatory environment of the skin. On the other hand, Dunphy et al. noted the presence of NK cells in skin lesions of Ps patients, but these cells were characterized by poor degranulation potency and reduced secretion of proinflammatory cytokines (IFN-γ and TNF-α) [31].

NKT cells are a distinct cellular subset that exhibits both T-cell receptor (TCR) and NK cells lineage markers and can produce large amounts of cytokines in response to various stimuli, such as lipids and cytokines. NKT cells have been identified in psoriatic lesions and were reported a decreased following treatment [32]. Bonish et al. have shown that NKT cells (CD161<sup>+</sup> ) can produce large amounts of IFN-γ that induce CD1d overexpression on KCs in psoriatic lesions. These KCs subsequently activate NKT cells to produce IFN-γ. Bonish et al. also suggested that the interactions between NKT and KCs could provide a relevant mechanism in the pathogenesis of Ps [33]. Several NKT cells can secrete IL-17 and IL-22 after IL-1β and IL-23 stimulation. These cells, named NKT17, express high levels of RORγT, and were found in the lung, liver, marginal lymph nodes, and skin [34], but their role in the skin is not yet well defined.

Other innate immune cells involved in Ps pathogenesis are innate lymphoid cells (ILCs) and γδT cells, which are activated in Ps and may represent important sources of Il-22 and IL-17.

ILCs are members of lymphoid lineage and are involved in the early response to infections in the skin, lung, and gastrointestinal tract. In the absence of an antigenspecific receptor, they are activated through signals from cytokine and NK receptors and secrete high levels of cytokines. Within the three types of known ILCs, ILC3 type seems to be involved in Ps due to its ability to produce IL-17 and IL-22. Depending on the presence or absence of natural cytotoxicity receptor (NCR), ILC3 were classified into NCR+ILC3 (increased in psoriatic lesions), NCR− ILC3 (commonly in normal skin), and lymphoid tissue inducer cells (LTi). When they are activated, NCR<sup>+</sup> ILC3 secrete IL-22, NCR-ILC3 produce IL-17, IL-22 and IFN-γ, LTi-lymphotoxin, IL-17, and IL-22 [35]. Vilanova et al. reported an increased amount of IL-22 and IL-17 producing ILC3 in psoriatic skin, correlated with disease severity [36]. Another study showed that NCR<sup>+</sup> ILC3 isolated from psoriatic skin, stimulated by IL-2, IL-23, and IL-1β, produce large amounts of IL-22, but not IL-17 [37], thus suggesting that ILC3 contribute to the development of Ps via IL-22.

γδT cells are involved in the pathology of allergic and autoimmune diseases in mice and humans and contribute to the development of Ps by IL-17 production. Unlike αβT cells, γδT cells express TCR that consists of γ and δ chains. They interact with both innate and adaptive immune cells, and with non-immune tissue cells, and can promote tissue repair and wound healing [38]. γδT cells are able to recruit inflammatory myeloid cells and modulate classical T cells functions by increasing Th17 inflammatory responses and/or by reducing the activity of Treg cells [39]. In addition to IL-17, they also secrete IFN-γ, IL-22, and GM-CSF. Cai et al. reported that both human and murine dermal innate γδT cells are an important source of IL-17 in the skin upon IL-23 stimulation, and may represent a novel target for the Ps' treatment [40].

However, recent studies have shown that γδT cells account for up to 1% of T cells in Ps, the majority of IL-17-producing T cells being αβT cells and not γδT emphasizing that human Ps is mainly driven by αβT cells [41]. However, more studies are needed to accurately determine the involvement of γδT cells in Ps.

T lymphocytes, cellular exponents of the adaptive immune system, are the key players in the phase of maintaining the inflammatory status in Ps. The pathogenic interactions between T-cells subsets (T-CD8+ , autoreactive T cells Th1, Th17, Th22,), DCs, and KCs, result in a self-maintaining inflammatory status with TNFα/IL-23/IL17 axis as a key point [42]. Although the involvement of T cells in Ps has been extensively studied, the gradual scaling of T-cell mediated events is not yet fully elucidated. Casciano et al. pointed out the four T-dependent stages consisting of a skin T cell activation phase, the onset of chronic inflammation, the maintenance of the inflammatory status, and the migration of specific subsets of T cells outside the skin possibly involved in Ps extra-cutaneous manifestations [43]. Various T cell sub-populations have been recognized as involved in Ps pathogenesis.

T-CD8 cells have a crucial role in Ps plaque formation and strongly sustain the autoimmune nature of the disease. DCs recognize and present epidermal autoantigens (LL-37, ADAMTS-like-protein 5, keratin 17) to IL-17-producing T-CD8 cells, in an MHC-restricted manner. Human leukocyte antigen (HLA)-C\*06:02, a specific MHC allele is involved in the autoimmune T-cell response in Ps [44]. Recent studies have shown that T-CD8 cells accumulate in active and resolved psoriatic lesions as tissue-resident memory T cells (TRM) with αβTCR [41] and have an IFN-γ/IL-17/ IL-22 cytokine profile (thus maintaining the inflammatory status) [45]. Autoantigens recognition by Tc1/Tc17 cells induces the secretion of cytokines that will cause the onset of KCs hyper-proliferation. Activated KCs will contribute to the propagation and maintenance of inflammation through the secretion of antimicrobial peptides, cytokines, and chemokines [46].

Activated DCs also secrete IL-12 and IL-23, inducing the differentiation in Th1 and Th17 subsets of naive T lymphocytes. In response, these effector subsets will produce TNF-α, IFN-α, IL-17, IL-22 thus promoting KCs proliferation.

Initially, Ps was considered a predominantly Th1-mediated disease, being reported as an imbalance between Th1 and Th2 cells. This imbalance was due to the increasing of IFN-γ expressions as compared to low levels obtained for IL-4, IL-5, or IL-10, which are specific Th2 cytokines [47]. Th1 cells, a subset of T-CD4+ cells, are defined by activation of transcription factors STAT4 and T-bet and the secretion of proinflammatory cytokines (IFN-γ, IL-2, and TNF). Th1 cells and IFN-γ levels were found elevated in psoriatic lesions [48]. IFN-γ, along with TNF-α, IFN-α, IFN-β, IL-1β, and IL-6 activate IL-12 and IL-23 – producing MDCs in the initial phase of the disease, and under the influence of Th1 cells, KCs secrete pro-inflammatory mediators (IL-1β and IL-18) that are further involved in the differentiation of Th1 and Th17 cells [49]. Although the involvement of IFN-γ-producing Th1 cells has been extensively studied, the results of clinical trials based on anti-IFN-γ antibodies did not have the expected results [50].

Extensive literature in recent years has proven the clinical efficacy of IL-17 inhibitors, highlighting the important role of IL-17-secreting lymphocytes—Th17 and Tc17 cells—in Ps. Th17 cells are a T-CD4+ subset defined by mainly IL-17A, IL-17F, IL-22, IL-21, and IL-26 secretion, and express retinoic acid receptor-related orphan receptor-γt (ROR-γt) and IL-23 receptor (R) [51]. Th17-derived proinflammatory cytokines have a critical role in the pathogenesis of many autoimmune and inflammatory diseases, including Ps. Th17 cells activated by IL-1β and IL-23, trigger inflammation and autoimmunity, while activation through IL-6 and transforming growth factor (TGF)-β is involved in tissue defense and homeostasis [52]. Although NK and γδT cells also synthesize IL-17, Th17 remains the main source of IL-17.

IL-12 and IL-23 secreted by activated MDCs cause differentiation into Th1, Th17, and Th22 effector cells. IL-23 secreted by MDCs induces the differentiation of Th17 cells, and by activation of IL-23R expressed on Th17 cell, maintain the local inflammation [53]. IL-17A released by Th17 cells will promote the KCs activation, which will maintain the chronic inflammatory status through the secretion of antimicrobial peptides, cytokines, and chemokines. Activated Th17 cells are present in psoriatic lesions [54], and elevated levels of IL-23, a Th17-driving cytokine, were reported in lesional skin [55]. These data sustain the importance of IL-23/T17 axis in the development of Ps.

Although it is recognized that Th17 cells play a critical role in the Ps pathogenesis, and the secreted cytokines are strongly involved not only in the onset and development of the disease but can cause complications of other associated diseases, more research is needed regarding the pathogenicity of Th17 cells in Ps.

Another subset of T-CD4<sup>+</sup> lymphocytes involved in Ps pathogenesis is represented by Th22 cells. Th22 differentiate from naive T cells in the presence of IL-23, IL-6, and TNF-α, and upon activation, these cells will produce IL-22, TNF-α, IL-13, and IL-26, but not IFN-γ or IL-17. IL-22 can induce specific chemokines that will increase specific effector responses mediated by the IL-23 / Th17 axis [56], the network leading to epidermal hyperplasia and hypergranulosis. In a recent study, Cheuk et al. demonstrated that epidermal Th22 and Tc17 cells are retained in healed psoriatic skin and can induce the recurrence of the disease in previously affected areas through the secretion of specific cytokines [57].

Another significant role in Ps' pathogenesis is played by Tregs lymphocytes, cells that suppress the autoimmune responses and other aberrant or excessive immune responses against non-self-antigens [58]. Tregs play an important role in maintaining homeostasis and can cause local suppression of the activity of other immune cells, including Th1 and Th17 cells. In Ps, the suppressive function of Tregs is altered, resulting in a Th17/Tregs imbalance and an upregulation of pro-inflammatory cytokines [59]. The decreased suppression function of Tregs can be a result of elevated levels of IL-6 produced by DCs, Th17, and endothelial cells, which inhibit per se Tregs' activity. Furthermore, Tregs cells can differentiate into a Th17 phenotype in Ps. IL-17A+ Foxp3+ CD4+ cells were found in psoriatic lesions from patients with severe Ps, thus highlighting Tregs' potential to differentiate into an IL-17A-producing phenotype [60]. Thus, in Ps Tregs behave in a Th17 cells manner and hence are unable to exert their suppressor functions. Nussbaum et al. suggested several ways of restoring Th17/ Tregs balance: induction of Tregs (using anti-TNF-α, folic acid analogs, phototherapy, vitamin D, retinoids, anti-IL-17, and anti-IL-23), downregulation of Th17 cells (using phototherapy, folic acid analogs, retinoids, and anti-IL-17) and inhibition of Tregs plasticity (using pan-PKCs inhibitor and anti-IL-23) [59].Although the involvement of B lymphocytes in Ps pathogenesis has been not as much studied compared to T lymphocytes, recent years' research has highlighted the possible role of IL-10 secreting regulatory B cells (Bregs) in disease's attenuation. Bregs are a subset with regulatory properties exerted mainly by the production of cytokines like IL-10, TGFβ. These subpopulations are associated with the regulation and control of excessive inflammatory responses. Recently was underlined the heterogeneity of Bregs cells, suggests that this population may develop from any subset of B cells in the context of adequate stimulation [61]. IL-10-secreting B cells seem to have an inhibitory effect in Ps development. Thus, Yanaba et al. have shown that Bregs cells suppress Ps-like skin

inflammation induced by imiquimod (IMQ ), a potent agonist for TLR7 and − 8 in a murine experimental model [62]. Although recombinant IL-10 was one of the first biologic agents used in Ps, the clinical trials have not been completed to be introduced in the clinical management of Ps patients, thus more studies are needed to determine Bregs involvement in the course of Ps.
