Neuroblast Differentiation-Associated Protein Derived Polypeptides: AHNAK(5758-5775) Induces Inflammation by Activating Mast Cells via ST2

ABSTRACT Psoriasis is a chronic inflammatory skin disease. Mast cells are significantly increased and activated in psoriatic lesions and are involved in psoriatic inflammation. Some endogenous substances can interact with the surface receptors of mast cells and initiate the release of downstream cytokines that participate in inflammatory reactions. Neuroblast differentiation-associated protein (AHNAK) is mainly expressed in the skin, esophagus, kidney, and other organs and participates in various biological processes in the human body. AHNAK and its derived peptides have been reported to be involved in the activation of mast cells and other immune processes. This study aimed to investigate whether AHNAK (5758–5775), a neuroblast differentiation-associated protein-derived polypeptide, could be considered a new endogenous substance in psoriasis patients, which activates mast cells and induces the skin inflammatory response contributing to psoriasis. Wild-type mice were treated with AHNAK(5758–5775) to observe the infiltration of inflammatory cells in the skin and cytokine release in vivo. The release of inflammatory mediators by mouse primary mast cells and the laboratory of allergic disease 2 (LAD2) human mast cells was measured in vitro. Molecular docking analysis, molecular dynamics simulation, and siRNA transfection were used to identify the receptor of AHNAK(5758–5775). AHNAK(5758–5775) could cause skin inflammation and cytokine release in wild-type mice and activated mast cells in vitro. Moreover, suppression of tumorigenicity 2 (ST2) might be a key receptor mediating AHNAK(5758–5775)’s effect on mast cells and cytokine release. We propose a novel polypeptide, AHNAK(5758–5775), which induces an inflammatory reaction and participates in the occurrence and development of psoriasis by activating mast cells.


Introduction
Psoriasis vulgaris (PV) is a chronic, systemic, and immune-mediated inflammatory skin disorder that manifests clinically as well-demarcated erythematous plaques covered with thick silvery scales. The prevalence of PV is approximately 0.1-2.0%, varying among different regions and countries (Parisi et al. 2020). The most prominent feature of psoriasis is epidermal hyperproliferation accompanied by mixed infiltration of inflammatory cells in the epidermis and dermis, including T cells, neutrophils, dendritic cells, macrophages, and mast cells. Psoriasis has a complex network of inflammatory factors, and both endogenous factor-induced innate immune hyperactivation and T cell-driven adaptive immune disorders play important roles in pathogenesis. It is generally accepted that adaptive immune disorders predominate in psoriasis, with the IL-23/Th17 inflammatory pathway being the core axis (Griffiths et al. 2021).
Accumulating evidence has shown that innate immune cells have an unexpected critical role in PV pathogenesis. Neutrophils and dendritic cells are reported to upregulate oxidative stress by secreting inflammatory and oxidative mediators, which is concomitant with psoriatic inflammation in mice (Al-Harbi et al. 2020). In addition, mast cells are activated in the early stages of psoriatic lesions, and increased infiltration of activated mast cells and neutrophils has also been discovered (Gong and Wang 2021;Harvima et al. 2008;Mashiko et al. 2015). IL-17A was previously thought to be mainly derived from Th17 cells, but recent studies have shown that mast cells are an important source of IL-17A (Lin et al. 2011).
Several studies indicate that they may act as pro-inflammatory cells that participate in the local inflammation of psoriasis.
It should be noted that mast cells can be activated by some endogenous or exogenous peptides or proteins, including complement products C3a and C5a, neuropeptides such as substance P (SP), vasoactive intestinal peptide (VIP), as well as β -melanocyte hormone (Harvima et al. 2008). Other studies have demonstrated that peptides such as leptin, catestatin, and isolated human albumin fragments can activate mast cells, leading to downstream inflammation (Aung et al. 2011;Karhu et al. 2017;Zelechowska et al. 2018). Mast cells express several peptide-mediated activated receptors, including MrgprX2, ETA receptor, NK1 R, NTR, and FPRL1 (Lu et al. 2017). Suppression of tumorigenicity 2(ST2), a member of the interleukin-1 receptor-like protein-1 (IL1RL1) family, also occurs on the surface of mast cells. As an immune response molecule, it is also expressed in the T-lymphocytic cell line (LyT), macrophages, dendritic cells, and activated polymorphonuclear leukocytes (PMNL) (Homsak and Gruson 2020). To date, there are few reports on endogenous small molecular ligands of mast cells or their receptors in psoriasis, and their role in the inflammatory axis of psoriasis remains to be explored.
The neuroblast differentiation-associated protein AHNAK is considered the largest protein in the human body and is expressed in the skin (mainly in keratinocytes), central nervous system, esophagus, and other organs. It has diverse functions, including cell signaling and cell contacts, regulation of calcium channels, membrane repair, tumor metastasis, and the formation of cytoskeletal structure muscular regeneration, which allows it to participate in various biological processes in the human body (Choi et al. 2019;Davis et al. 2014). Furthermore, AHNAK is highly expressed in mast cells and participates in the toll-like receptors (TLR)4-mediated mast cell activation signaling pathway (Martin-Avila et al. 2016). Skoldberg et al. discovered that AHNAK could function as an autoantigen in lupus erythematosus (Skoldberg et al. 2002). In addition to macromolecules, small molecular peptides derived from AHNAK have also been considered as regulators of cardiac L-type Ca 2+ channels in adult mammals (Skoldberg et al. 2002).
The inflammatory waterfall in psoriasis can be caused by both endogenous and exogenous factors. Short-chain fatty acids from gut are reported to activate GPR43 on immune cells and induce dual oxidase 2/reactive oxygen species and IL-6 signaling in psoriasis-like inflammation . Other endogenous substances, such as polypeptides and cathelicidin, can also trigger and enhance psoriatic inflammation. Therefore, it remains to be determined whether AHNAK or AHNAK-derived peptides act as ligands that activate innate immune cells and participate in the psoriatic inflammatory response.
In this study, we discuss the role of AHNAK-derived polypeptides AHNAK(5758-5775) in psoriatic inflammation and its downstream immune mechanism. We revealed a significant increase in AHNAK(5758-5775) in psoriatic lesions. Moreover, we assessed in vivo and in vitro and found that the polypeptides could interact with mast cells, which contribute to downstream neutrophil recruitment.

Materials and methods
Reagents AHNAK(5758-5775) was purchased from Nanjing Peptide Biotech Ltd. (Nanjing, China) and was detected by HIGH performance liquid chromatography and mass spectrometry (supplementary Figure S1). IL-33 and lipopolysaccharide were purchased from MCE (Shanghai, China). Tyrode´s solution buffer was prepared fresh on the day of use (6.954 g/l NaCl, 0.353 g/l KCl, 0.282 g/l CaCl 2 , 0.143 g/l MgSO 4 , 0.162 g/l KH 2 PO 4 , 2.383 g/l HEPES, 0.991 g/l glucose, and 1 g/l BSA, pH = 7). And all the reagents are prepared before use.

Samples of psoriasis vulgaris (PV) patients and polypeptide omics analysis
A single-center and case-control study was adopted. 3 case skin samples were from Department of Dermatology, Second Affiliated Hospital of Xi'an Jiaotong University, who was diagnosed psoriasis vulgaris without no other diseases. Samples were obtained during a skin biopsy on the patient's limbs or trunk, and the patient was not undergoing any medical treatment at the time. 3 skin control samples were from Department of orthopedic, Second Affiliated Hospital of Xi'an Jiaotong University, who were diagnosed fracture patients with no infection and immune diseases. The samples were frozen with liquid nitrogen to extract peptides and the protein was removed. Polypeptide segments were detected by liquid chromatography-mass spectrometry. And the polypeptide omics analysis was finish by MH BioTech Co., Ltd (Shanghai, China).

Ethical approval and informed consent
This study was registered at Chinese Clinical Trail Registry, and the registration number is ChiCTR2100052365. The ethical approval (2021-1517) was approved by Ethics Committee at Xi'an Jiaotong University and conformed to the ethical standard. All the specimen in this study had signed informed consent.

Animals
The 8 weeks male C57BL/6 mice used in this study, were purchased from the Experimental Animal Center of the Xi'an Jiaotong University (Xi'an, China). Animals were housed in individual cages in a large colony room, with free access to water, and fed a standard dry chow twice a day. The breeding environment was 20-25°C, with a relative humidity of 40% on a 12 h light/dark cycle. Nine mice were used per group.

Histology
10 μl AHNAK(5758-5775) (20 μM) prepared in saline were injected subcutaneously into mice ears for 3 days. And saline was set as negative control. Mice were sacrificed through CO 2 inhalation. Ears samples were fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin (H&E). After dried at 37 ºC for 30 min and pre-incubated in blocking solution (10% normal goat serum (v/v), 0.2% Triton X-100 (v/v) in PBS, pH 7.4) for 2 h at 25 ºC, avidin-FITC was used for marking mast cells.

Cytokines analysis in mice skin
10 μl AHNAK(5758-5775) (20 μM) prepared in saline were injected subcutaneously into mice ears for 3 days. And saline was set as negative control. Mice were sacrificed through CO 2 inhalation. The samples were frozen with liquid nitrogen and added 0.5 ml saline. Then the ears were cut into pieces. The supernatant was obtained by centrifugation after 30 min ultrasound treated. Mouse TNF-α, IL-17, IL-23, CXCL2 and IL-33 ELISA Kit were purchased from Sino Biological Inc (Beijing, China). All steps were performed strictly according to the manufacturers' instructions.

Mouse peritoneal mast cell (MPMC) purification and media analysis
Adult male C57BL/6 mice were sacrificed through CO 2 inhalation. A total of 12 ml mast cell dissociation medium (MCDM) (15 ml of MCDM: 1.5 ml of 10×HBSS, 450 μl of fetal bovine serum, 150 μl of 1 M HEPES, 12.9 ml of sterile water, pH = 7.2) was used for two to three sequential peritoneal lavages, then centrifuged at 200 g for 10 min at 4°C. Anti-mouse CD117 and Anti-R-Phycoerythrin (PE) Magnetic (BD Biosciences, New York, USA) was used for isolation and purification mouse peritoneal mast cell (MPMC). MPMC in the pellet were retrieved, and purity was >95% as assayed by morphology. Then MPMC resuspended in DMEM with 100 ng/ml recombinant mouse stem cell factor (SCF), and used in 4 h.
1 × 10 5 MPMC per well, were incubated in a 96-well plate. The culture medium was removed, AHNAK(5758-5775) prepared by Tyrode solution (10, 20, 40 μM) was added at the indicated concentrations, and the cells were incubated for 1 h for tryptase, histamine, and β-hexosaminidase assay at 37°C with 5% CO 2 , while 8 h for cytokines assay. Mouse TNF-α, MCP-1 CXCL2 and IL-33 ELISA Kit were purchased from Sino Biological Inc (Beijing, China). Mouse tryptase, histamine and β-hexosaminidase ELISA Kit were purchased from Shanghai MLBIO Biotechnology Co. Ltd. (Shanghai, China). 300 pg/ml IL-33 were set as positive control. All steps were performed strictly according to the manufacturers' instructions.

Ethics statement
Experimental protocols involving mice were approved by Animal Ethics Committee at Xi'an Jiaotong University, Xi'an, China (Permit Number: XJTU 2021-1025). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health.

Mast cells medium release assay
The laboratory of allergic disease 2 (LAD2) human mast cells were kindly provided by A. Kirshenbaum and D. Metcalfe (NIH, USA). LAD2 cells are one of the first reported human mast cell lines established from a mast cell sarcoma/leukemia patient, which closely resemble primary cultures of CD34+-derived human mast cells and have the ultrastructural features. 1 × 10 6 LAD2 cells were incubated in a 96-well plate overnight at 37°C with 5% CO2. The culture medium was removed, AHNAK(5758-5775) prepared by Tyrode solution (10, 20, 40 μM) was added at the indicated concentrations, and the cells were incubated for 1 h for tryptase, histamine, and β-hexosaminidase assay at 37°C with 5% CO 2 , while 8 h for cytokines assay. Human TNF-α, MCP-1 and IL-8 ELISA Kit were purchased from Sino Biological Inc (Beijing, China). Human tryptase, histamine and β-hexosaminidase ELISA Kit were purchased from Shanghai MLBIO Biotechnology Co. Ltd. (Shanghai, China). 10 μg/ml C 48/80 were set as positive control. 300 pg/ml IL-33 were set as positive control. All steps were performed strictly according to the manufacturers' instructions.

Small interfering (si)RNA transfection of LAD2 cells
Specific knockdown was achieved using small interfering (si)RNAs targeting ST2, and nontargeting siRNAs as negative control (NC). The siRNA sequences were as follows: forward, 5'-GGCAUCACAAAUAGCCAAATT-3' and reverse, 5'-UUUGGCUAUUU GUGAUGCCTT-3' for ST2; and forward, 5'-UUCUCCGAACGUGUCACGUTT-3' and reverse, 5'-ACGUGACACGUUCGGAGAATT-3' for the control. For transfection, siRNA was delivered at a final concentration of 80 nM using the Lipofectamine® 3000 reagent according to the manufacturer's instructions. Cells were incubated for 48 h to allow knockdown of ST2. ST2 knockout efficiency was detected by RT-qPCR, and the knockdown-LAD2 cells were used in mast cells medium release assay.

Molecular docking analysis and molecular dynamics simulation
Autodock Vina v. 1.1.2 (Scripps Research Institute, San Diego, CA, USA) was used to perform a molecular docking analysis. The Research Collaboratory for Structural Bioinformatics (RCSB) protein data bank was used to obtain target protein crystal structures. Discovery Studio v. 2016 was used to optimize the crystal structures by removing redundant ligands, adding charge, adding hydrogens, and removing water before running docking experiments. Discovery Studio v. 2016 were employed to visualize the docking model.
The GROMACS 2019 software was used for molecular dynamics simulations. The docking complex was used as the initial conformation for all-atomic molecular dynamics simulation. Amber 99sB-ILDN field parameters were used for both protein and EB molecules with the aid of ACPYPE Server (https://www.bio2byte.be/acpype/) Server generates UNK molecular topology file, select dodecahedron solvation box, set the system boundary and compounds in recent distance of 1.0 nm, use TIP3P water model and based on method of VERLET cut random Na+ and Cl-added to the complex system to counteract the charge of the protein. Then, the system energy was minimized, the temperature was controlled by NVT and the pressure was controlled by NPT. The system temperature was 300K and the pressure was constant at 101.325kPa. Based on the above equilibrium, the free dynamics of the system was simulated for 100ns.

Statistical analysis
Experiments were repeated 3 times. Data are expressed as mean ± S.E.M. and analyzed onetail paired Student's t-test. An independent samples analysis of variance was used to determine statistical significance in comparisons of the data using the SPSS 13.0 software. For the paired comparison samples, the treated groups were compared with the negative control group. Differences were considered significant at * p < .05, ** p < .01, and *** p < .005. For the multiple doses samples, the treated groups compared respectively with the negative control group were calibrated by Bonferroni's test.

AHNAK(5758-5775) significantly increased in psoriasis patients and induced skin inflammatory reaction
AHNAK(5758-5775) is derived from a neuroblast differentiation-associated protein that contains amino acids at positions 5758-5775. After removing the proteins, the results of polypeptide omics by LC-MS showed that AHNAK(5758-5775) was significantly increased in psoriasis patients compared to the control samples (Figure 1a,b). Several studies have confirmed that polypeptide compounds participate in the inflammatory response in psoriasis. AHNAK(5758-5775) was injected into the skin of mice to verify its role in skin inflammation. After hematoxylin and eosin (H&E) staining, mice treated with AHNAK (5758-5775) showed significant inflammatory cell infiltration and vasodilation in the ear dermis compared to the control group (Figure 1c). Activation of the TNF-α/IL-23/IL-17 inflammatory axis plays an important role in the inflammatory response in psoriasis, and that of CXCL2 in chemotactic neutrophils in psoriasis lesions. Cytokine analysis showed that AHNAK(5758-5775) induced an increase in TNF-α and CXCL2 but had little influence on IL-23, IL-17, or IL-33 (Figure 1d).

AHNAK(5758-5775) activated mast cells in vivo and in vitro
Mast cells were significantly increased in mouse lesions ( Figure 2a); therefore, we studied the effect of AHNAK(5758-5775) on mast cells. The results showed that AHNAK(5758-5775) did not induce mast cell degranulation. MPMC did not release tryptase, histamine, or significantly increased in psoriasis patients than the control samples analysis by LC-MS. c: AHNAK(5758-5775) caused inflammatory cells to infiltrate the lesions after hypodermic injection in mice. d: AHNAK (5758-5775) induced up-regulated the level of TNF-α and CXCL2 in mice skin, while showed little influence on IL-23, IL-7 or IL-33 release. (n = 9, data are expressed as mean ± S.E.M. and analyzed onetail paired Student's t-test. Differences were considered significant at * p < .05, and *** p < .001).
GLN23, ARG35, GLN39, TYR119, THR121, THR135, and ARG198 in the pocket. In addition, the negative part of peptide AHNAK(5758-5775) binds to the positively charged part of the binding pocket, and the electrostatic force formed can further increase the stability of the complex (Figure 4a). Docking complexes were selected for molecular dynamics simulation experiments. The root mean square deviation (RMSD) was used to indicate the degree of molecular structure change and measure the stability of the complex system. AHNAK(5758-5775) was stable during the surgery. The RMSD fluctuation of the AHNAK(5758-5775)-ST2 system was less than 0.6 nm. Root mean square fluctuation (RMSF) showed the fluctuation and structural flexibility of the amino acid residues of receptor proteins. RMSF analysis showed that the RMSF value of the amino acid residues binding AHNAK(5758-5775) in ST2 was less than 1.5 nm, indicating that AHNAK(5758-5775) binding can stabilize the structure of the binding region to a certain extent. The radius of gyration (Rg) measures the compactness of the protein. The Rg values of the AHNAK (5758-5775)-ST2 systems were less than 3.5 nm, which may be related to the fact that ST2 contains more loop structures, or that there is a conformational transition in the simulation process. The molecular dynamics simulation results indicated that the complexes remained stable with favorable conformations throughout 100 ns. Therefore, this suggests that AHNAK(5758-5775) was stable at the binding site of the receptors during the interactions (Figure 4b).

Discussion
Psoriasis is an autoimmune disease with complex pathogenesis. Exploring and solving doubts regarding the early triggering factors, crucial effector cells, and immune network interactions in psoriasis could contribute to breakthroughs in its accurate therapeutic strategies. This study aimed to explore the role and possible mechanisms of AHNAKderived polypeptides AHNAK(5758-5775) in the pathogenesis of psoriasis. We found that AHNAK(5758-5775) could activate mast cells via ST2 through the MAPK and NF-kB signaling pathways, as well as release inflammatory mediators participating in psoriatic inflammation and immune disorders.
We detected psoriatic lesions and found that AHNAK-derived peptides were significantly increased in the lesions compared to the normal skin. AHNAK has a molecular weight of 700kD and is described as a nucleoprotein in neuroblastoma cell lines. Related to the differentiation of neurons, AHNAK is thought to be involved in myelination in the peripheral nervous system and the formation of the blood-brain barrier in the central nervous system. It has been well established that AHNAK is involved in physiological processes such as cardiac L-type calcium channel function, nerve cell differentiation, and T-cell calcium signal transduction (Chen et al. 2017). It has low tissue specificity and is produced by keratinocytes in the skin. However, there are relatively few studies on its function in the skin, and current research mainly focuses on its role in tumors (Lee et al. 2014;Sheppard et al. 2016;Sudo et al. 2014).
We have tried to elucidate the relationship between AHNAK, immunity, and inflammation. Previous studies have confirmed that AHNAK is involved in immunoregulation. It plays a role in calcium signal transduction in CD8+ and CD4+ T cells, and AHNAKknockout mice have shown impaired Th1 and Th2 immune functions (Choi et al. 2019;Li et al. 2022;Zardab et al. 2022). AHNAK was identified as a novel autoantigen in systemic lupus erythematosus, suggesting that AHNAK is a novel target of autoreactive B cells in autoimmune disease (Skoldberg et al. 2002). We also noticed a previous study suggesting that AHNAK's large size and highly repetitive structure in the central domain are conducive to promoting the formation of immune complexes (Skoldberg et al. 2002). Psoriasis requires the presence of autoantigens to initiate an inflammatory cascade as it is an autoimmune skin disease. Endogenous substances, such as cathelicidin LL-37, melanocytic ADAMTSL5, lipid antigen PLA2G4D, and keratin 17 have been identified as triggers of psoriasis (Chiricozzi et al. 2018;Ten Bergen et al. 2020). Thus, we speculate that AHNAK- Figure 5. The activation effect of AHNAK(5758-5775) on mast cell reduced after knockdown the expression of ST2. a: The efficiency of knockdown ST2 analysis by  induced the release of IL-8, TNF-α and MCP-1 by NC-LAD2 cells was significantly higher than that of knockdown-LAD2 cells. (Data are expressed as mean ± S.E.M. and and were calibrated by Bonferroni's test. Differences were considered significant at * p < .0125, and ** p < .0025).
derived peptides may act as endogenous peptides and play a role in psoriasis. This is also the first study to investigate AHNAK alongside psoriasis.
The onset of PV occurs when antimicrobial peptides (AMP) released by keratinocytes form complexes with self-nucleic acids and then activate TLRs on plasmacytoid DC (pDC), which act as antigen-presenting cells and secrete cytokines to promote the differentiation of Th17, Th1, and Th22 cells (Takahashi and Yamasaki 2020). The subsequent production of cytokines, including IL-17, IL-22, TNF, and IFN-γ, activates keratinocytes to amplify and maintain inflammation (Parisi et al. 2020;Rendon and Schakel 2019). According to the classical theory, T cell subsets are the main culprits of psoriasis. Inflammatory factors such as IL-17 and IL-22 secreted by them act on keratinocytes and promote their recruitment of inflammatory cells, resulting in chronic inflammation in psoriasis (Lanna et al. 2019;Takahashi and Yamasaki 2020). In addition to Th1, Th2, and Th17 cell-derived cytokines, innate immune cells such as dendritic cells, neutrophils, mast cells, plasma cells, and macrophages respond rapidly to internal or external stimuli and secrete inflammatory mediators. This interaction leads to the amplification and maintenance of inflammation in psoriasis patients.
Neuroimmune mechanisms are exist in psoriasis. Receptors on immune cells are the material basis, and dysfunctions of receptors could alter immune cells response and cause dysregulation. Enhanced psoriasis-like inflammation was observed in BTBR T+ tf/J (BTBR) autistic mice due to the enhanced TLR7/IL-23/IL-6 and Th17-related signaling responses in dendritic cells, and resveratrol was found to improve neuroimmune dysregulation through M. and were calculated by Bonferroni's test. Differences were considered significant at * p < .0125, *** p < .00125, **** p < .0001).
the inhibition of proinflammatory mediators and TLRs/NF-κB transcription factor signaling Nadeem et al. 2018). Elevated levels of adenosine was detected in psoriasis lesions, and the activation of the Adenosine A2A receptor(A2AR) signaling was reported to attenuate disease progression or correct neuroimmune dysfunction in psoriasis and autism Welihinda et al. 2022). Psoriasis is also related to the increased activity of tyrosine kinases, inhibition of its activity could selectively suppress signal transduction pathways in a range of immune cells such as mast cell. In rheumatoid arthritis, another T cell mediated autoimmune disease, inhibition of protein tyrosine kinase activity was found to reduces responses of inflammatory cells and decreases the disease progression (Ahmad et al. 2016). Taken together, focusing on the proinflammatory and anti-inflammatory effects of these small molecules and associated receptors could help open up the possibility of uncovering the inflammatory bypasses and provide inspiration for treatment in immune diseases.
Mast cells are considered linkers of innate and adaptive immunity. They are a source of many proinflammatory factors. They release cytokines, enzymes, chemokines, and histamines when activated; express cell surface molecules to interact with other cells such as endothelial cells, neutrophils, keratinocytes, and T-cell subsets to participate in inflammation, and play an important role in the neuroimmune regulation of psoriasis. Current research has shown that the number of mast cells that produce IL-8, TNF-α, and IFNγincreases in psoriasis lesions (Conti et al. 2021). Generally, it is believed that IL-22 mainly originates from Th17, Th22, and Th22 cells in psoriasis; however, researchers have proposed that mast cells are major producers of IL-22 and can also secrete IL-17 in 2015. Moreover, IL-1, IL-33, IL-36, and vascular endothelial growth factors (VEGFs) could also participate in inflammation (Conti et al. 2021;Mashiko et al. 2015).
Mast cells can be activated by IL-33 to amplify inflammation (Conti et al. 2021). They release mast cell extracellular traps (MCETs) in psoriasis and play a role similar to that of NETs (Lin et al. 2011;Takahashi and Yamasaki 2020). Notably, elevated serum IL-33 levels in PV patients are effective activators of mast cells, and the release of IL-1, IL-6, IL-13, TNFα, CCL2, and CCL3 can cause neutrophil infiltration. ST2 plays a role in both tumors and inflammatory diseases. IL-33 is an important ligand of ST2, which is defined as the IL-33/ ST2 pathway and participates in many autoimmune diseases. Current studies on ST2 and psoriasis are mainly limited to this pathway. In the phorbol ester-induced psoriasis model, the ST2-/-mouse model showed less inflammation than the wild-type (Hueber et al. 2011;Mitsui et al. 2016;Saluja et al. 2015). These results suggest that mast cell activation via ST2 is of great significance in PV.
Immunofluorescence confirmed that mast cells were activated in mouse skin lesions. Mast cell activation can be divided into two pathways: an IgE-dependent pathway and an IgE-independent pathway. In the former, mast cells immediately degranulate and release presynthesized mediators, such as tryptase, chymase, and TNF-α. The activation of mast cells induced by antimicrobial peptides, complements, neuropeptides, cytokines, and chemokines belongs to the IgE-independent pathway (Yu et al. 2016). Our research revealed that after adding AHNAK(5758-5775) to mast cells in vitro, the release of MCP-1, TNF-α, CXCL2, and IL-8 in the supernatant increased significantly. This indicates that the AHNAK-related peptide could activate mast cells in an IgE-independent manner. Similarly, several inflammatory mediators and chemokines released by mast cells can induce inflammatory responses in the skin. ST2 is the target receptor of this peptide in mast cells.
It has been discovered that neutrophil infiltration is one of the downstream reactions of mast cell activation. Secretion of enzymes, cytokines, and chemokines, such as CXCL1/ CXCL2, as well as the expression of cell-surface molecules by mast cells, could promote the aggregation of neutrophils (Harvima et al. 2008). In psoriatic lesions, IL-1 produced by mast cells can increase the number of infiltrating neutrophils (Mashiko et al. 2015). In our experiment, we observed local inflammatory cell infiltration after subcutaneous injection of AHNAK (5758-5775). According to previous studies, we speculated that activated mast cells secrete inflammatory factors that recruit neutrophils and other cells. Mast cells participate in the inflammatory response, however the specific mechanism of mast cell aggregation in psoriatic lesions is unclear.
However, many aspects of our study could still be developed further. For example, it is worth exploring the cell types activated by mast cell activation induced by AHNAK (5758-5775) downstream. The mouse model has various components of infiltrating cells; however, according to previous studies, neutrophils should be emphasized. In vivo and in vitro tests can be performed to obtain further relevant information. In addition, as AHNAK(5758-5775) showed a weaker activating effect on mast cells in psoriasis than IL-33, and it is rarely mentioned in inflammatory skin diseases, more mechanisms are still waiting to be explored.

Conclusion
In conclusion, we propose novel peptides derived from neuroblast differentiationassociated protein (AHNAK), which are increased in psoriatic lesions and activate mast cells via ST2. AHNAK(5758-5775) participates in immune cell activation and downstream inflammation in psoriasis. Overall, the discovery of these endogenous peptides will improve our understanding of the pathogenesis of psoriasis and help to elucidate the role of mast cells in psoriasis.