**3. Immune molecules as biomarkers in psoriasis**

Ps is a polygenic skin disease with immunological etiology. The immune system interacts with KC and a complex network of cells is generated, where dendritic cells, T-lymphocytes, neutrophils, and mast cells communicate through immune-related molecules inducing the complex pathology of Ps. The main immune molecules involved in the development of this disease are IL-23, IL-17, IL-12, IL-22, IL-23, IL-6, IL-10, IFN, TNF, TGF-β1 [91]. All these molecules have cellular sources mainly Th17, Th22, and Tregs cells [92]. The presence of these molecules induces an inflammatory process that sustains the proliferation of epidermal cells, neo-angiogenesis, and infiltration of DCs in the skin. Recently, new players were identified in the Ps development, such as the skin microbiome [18] and the skin's serotonergic system [93]. For example, it was recently reported that the microbiome variations are associated with the level of inflammatory cytokines receptors, especially with the IL-2 receptor [94, 95].

A recent study has shown that Ps deregulated genes were found. These genes are involving cytokine-cytokine receptor interaction, cell cycle and cell adhesion molecules and these candidate genes regulating important immune pathways can be new therapeutic targets in Ps [96]. There are more than 50 genetic susceptible biomarkers associated with the risk of Ps. The strongest association is with the presence of the HLA-C\*06 gene and HLA-B27 [97]. The other genetic Ps risk, single nucleotide polymorphisms (SNPs) are near the genes encoding molecules functioning in the adaptive and innate immunity [98].

### **3.1 Tissue immune molecules as biomarkers in psoriasis**

The first cellular line of skin's defense is KCs that upon injury secrete an array of alarmins. These are molecules that induce a rapid innate immune response against danger signals. Any deregulation in the physiology of KCs can lead to chronic inflammation, hyper-proliferation, and eventually a psoriatic lesion. Keratins (KRTs), major structural intermediate filament proteins of KCs were shown to be involved in Ps. Hence, up-regulation of KRT6/16/17 induces hyper-proliferation and innate immune activation followed by autoimmune T cells activation [99]. Transcriptomic studies have shown that KCs from Ps skin harvested from patient's skin have a high expression of mRNA encoding for SERPINB [100]. Moreover, SERPINB is regulated by TEA domain family member 4 (TEAD4) and affects the secretion of chemokines in Ps, hindering the normal cross-talk between KCs and T cells [101]. Other important cells within the skin are dermal fibroblasts that are in close contact with immune cells. A recent proteomic study has shown that Ps fibroblasts have upregulated proinflammatory factors and downregulated other factors that are involved in transcription/ translation processes, glycolysis/ adenosine triphosphate synthesis. All these deregulations contribute to the promotion of epidermal cell hyper-proliferation [102]. Another major cell involved in Ps is the Langerhans cell (LCs), the only DCs residing

in the epidermis that is physically linked to KCs through E-cadherin. A recent study has shown that in Ps E-cadherin does not regulate LCs maturation, migration, and function [103].

There are a series of surface molecules appending to immune cells that are highly involved in Ps' appearance and maintenance of immune tolerance. Therefore, neuropilin-1 (NRP1), PD-1, and HLA-G are the main tissue players in Ps. These molecules were found significantly lower in Ps compared to normal skin and were similar in Ps variants like PsA and Ps vulgaris patients [104]. Another tissue immune marker relevant for Ps is the TLR superfamily. When TLRs become aberrantly activated, T cellmediated autoimmune activation will take place, leading to several diseases including Ps [105]. In Ps' skin samples low levels of thrombospondin-1 (TSP-1) and CD47 were found inversely correlated with disease severity. The TSP-1/CD47 signaling pathway impacts Th17 and Tregs differentiation, favoring disease initiation [106]. Recently in an IMQ-induced psoriatic animal model, it was shown that IL-17A, IL-23, TNF-α, and STRA6 levels were found significantly increased in tissue along with their circulatory counterparts. RBP4 and STRA6 were found upregulated and involved in the experimental Ps [107]. The inflammatory Th17 response in Ps was reported to be modulated by IL-33 produced by the inflamed skin tissue, adding to the chronic status of Ps [108]. CDC6 is an essential regulator of the complex (pre-RC) assembly on chromatin. CDC6 expression was found upregulated in Ps lesions and probably the main route is induced by IL-22/STAT3 signaling, a key signaling pathway in Ps [109]. In a genetic mouse model, it was demonstrated that IL-23 produced by KCs induces chronic skin inflammation displaying an IL-17 pattern. In KCs from Ps' lesions, a decrease in H3K9 demethylation is correlated with IL-23 increased expression [110]. In a mouse experimental model harboring mutations in the gene encoding for CARD14 (KCs signaling molecule) it was shown that the animals spontaneously develop Ps, their skin has increased expression of anti-microbial peptides, chemokines, and cytokines, therefore Ps' pathogenesis in this model being driven by the IL-23/IL-17 axis. A diagram of the main immune molecules involved in Ps' pathogenesis is depicted in **Figure 3** [111].

In the IL-23/IL-17 axis, T lymphocytes, namely the Th17 subpopulation are the main cells that secrete IL-17A, IL-17F, and IL-22 [112], but also mast cells and neutrophils were shown to produce IL-17 in Ps' lesions [113].

#### **3.2 Circulatory immune markers**

Cytokines/chemokines that appear in the psoriatic lesion are essential for KCs activation. In immune homeostasis, if genetic and/or non-genetic factors interfere in the KC-T cell cross-talk the relation between resident T cells and KCs will be altered and a chronic inflammatory response will sustain the Ps lesions [114, 115]. All these alterations in terms of immune and non-immune molecules will be mirrored by the circulatory pattern of cytokines, chemokines, and various other molecules that can become biomarkers in Ps. A proteomic approach of Ps patients' plasma has shown that several proteins are decreased while others are increased. Hence apolipoprotein M, and proteins involved in vitamin D metabolism were found to decrease, while proteins involved in signaling molecule secretion were found to increase favoring cellular proliferation [116].

The complement system is essential in host defense against various pathogens, but in the last years, it was shown that due to its regulation properties of inflammation is involved in Ps development. As autoantibodies can activate the complement system, any deficiency of this system induces an impaired immune complex clearance and would sustain Ps lesions [117].

#### **Figure 3.**

*IL-23/Th17 axis in Ps. DC are activated by various cells (KCs, M*ɸ*, PDC, NKT cells) and stimuli. When activated DC will secrete IL-23 activating Th17 lymphocytes that will activate KCs through IL-17, IL-22, IL-21. Activated KCs will express specific Ps' genes (DEFB4 gene which encoded human β-defensin 2) and secrete an array of molecules (S100 protein, lipocalin (LCN), CCL20, CXCL1-3, 5, 6, 8, IL-1β) that will induce chronic inflammation of the skin. Created with BioRender.com.*

As already stated, the main deregulated axis in Ps is IL-22/IL-23/IL-17 axis. Memory Tregs are the main source of IL-17 in Ps [118]. IL-17A and F, when highly abundant, strongly induce S100-alarmins expression during KCs maturation [119]. IL-17A and IL-22 were found elevated in the serum of Ps patients, in accordance with increased IL-17 mRNA levels in the skin [120, 121]. IL-17A would further stimulate KCs to secrete chemokine CCL20, IL-8, and AMP, promoting inflammation [122]. Additionally, in Ps IL-17A synergizes with TNF and IL-22 and this cytokine cocktail upregulates IL-36 contributing to the inflammatory status [123]. IL-17F is found elevated in the serum of Ps [124] and is promoted by IL-23 [125], inducing further IL-6 and IL-8 secretion by KCs [126].

CD100-plexins were found elevated in Ps serum and were reported to increase the production of chemokines (CXCL1, CCL20) and cytokines (IL-1β, IL-18) when KCs activate NLRP3 inflammasome [127].

A recent study has shown that surviving, a protein belonging to the apoptosis inhibitor family was found to significantly increase in Ps patients suggesting its role in the pathogenesis of Ps [128].
