Neuroblast Differentiation-Associated Protein Derived Polypeptides: AHNAK(5758-5775) Induced In ammatory Reaction in Skin By Mast Cells Activation

Delu Che Xi'an Jiaotong University Second A liated Hospital Xiangjin Song Xi'an Jiaotong University Second A liated Hospital Lei Zhang Xi'an Jiaotong University Xueshan Du Xi'an Jiaotong University Second A liated Hospital Yi Zheng Xi'an Jiaotong University Tao Jia Xi'an Jiaotong University Second A liated Hospital Tong Zhou Xi'an Jiaotong University Second A liated Hospital Songmei Geng (  gsm312@yahoo.com ) Xi'an Jiaotong University Second A liated Hospital https://orcid.org/0000-0001-8731-6267


Introduction
Psoriasis vulgaris (PV) is a chronic and systemic in ammatory skin disorder. Clinically manifests as welldemarcated erythematous plaques covered with silver thick scales, it affects 2-3% of the world's population nowadays. The onset of PV is antimicrobial peptides (AMP) combine with self-nucleic acids and then activate plasmacytoid DC (pDC), who act as antigen presenting cells and secrete cytokines to promote the differentiation of Th17 and Th22 cells. Most of the current studies focus on the IL-23/Th17 axis and suggest that adaptive immune represented by T cells plays an important role. However, accumulating evidences show that innate immune have an unexpected critical status in PV pathogenesis.
Due to the complex in ammatory molecular and cellular network in psoriasis, we cannot separate the innate and adaptive immunity completely when discussing the pathogenesis. Mast cells (MCs) have been reported to play an irreplaceable role in chronic in ammatory diseases. Studies discovered an increased in ltration of activated MCs and neutrophils in psoriatic lesions, and MCs have been activated in the early stage of the lesions [1][2][3]. MCs are the main source of IL-22 in psoriasis [3]. Neutrophil in ltration is one of the downstream reactions of MCs activation, secretion of enzymes, cytokines and chemokines like CXCL1/CXCL2, expression of cell-surface molecules by MCs could promote the aggregation of neutrophils [1].
Psoriasis requires the presence of autoantigens to initiate in ammatory responses as an autoimmune skin disease. Endogenous substances such as LL37 and ADAMTSL5 were successively discovered as triggers of psoriasis [4]. Other works demonstrated that peptides such as leptin, catestatin, and some isolated human albumin fragments could activate MCs and then lead to downstream in ammation [5][6][7].
AHNAK mainly express in skin and other organs. Basically, AHNAK is involved in the differentiation of neurons and participates in the formation of cytoskeletal structure muscular regeneration, and calcium homeostasis process [9]. In skin, keratinocytes and broblasts in the basal layer are main producers. Present researches mainly focused on its role in tumor invasion, tumor metastasis and tumor inhibition effect [10,11]. AHNAK-derived peptides are effective regulators of the cardiac L-type Ca 2+ channel [12].
AHNAK is highly expressed in MCs and participates in TLR4-mediated MCs activation signaling pathway as well [13]. However, there is no research of AHNAK or AHNAK-derived peptide in psoriasis or other in ammatory diseases, more related content is waiting for our exploration.
In this study, we found an increase of AHNAK-derived peptides. Interestingly, it could activate mast cells via ST2 and participate in neutrophil in ltration. We hypothesized that this might be part of the PV pathogenesis 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 1).

Samples of Psoriasis Vulgaris (PV) Patients and Polypeptide Omics Analysis
A single-center and case-control study was adopted. 3 psoriasis vulgaris without no other diseases samples were from Department of Dermatology, Second A liated Hospital of Xi'an Jiaotong University. 3 control samples were from Department of orthopedic, Second A liated Hospital of Xi'an Jiaotong University, diagnosed as 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 nish by MH BioTech Co.Ltd (Shanghai, China).

Animals
Adult male C57BL/6 mice ageing 8 weeks were purchased from the Experimental Animal Center of Xi'an Jiaotong University. 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 sacri ced through CO 2 inhalation. Ears samples were xed in 4% paraformaldehyde and embedded in para n. Tissue sections were stained with hematoxylin and eosin (H&E). Avidin-FITC was used for marking mast cells and anti-Ly6G antibody was used for marking neutrophil.
Cytokines Analysis in Mice Skin 10 µl AHNAK(5758-5775) (20 µM) prepared in saline were injected subcutaneously into mice ears for 3 days. 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. ELISA Kits were purchased from Sino Biological Inc (Beijing, China).

Mouse Peritoneal Mast Cell (MPMC) Puri cation and Media Analysis
Adult male C57BL/6 mice were sacri ced 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 puri cation 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 by ELISA.

Preparation of Neutrophils From Human Blood and Activation Assay
Blood samples were collected from different authors of this paper. Peripheral neutrophils were separated using a magnetic-activated cell-sorting method. 1 × 10 6 neutrophils were incubated in a 96-well plate. The culture medium was removed, AHNAK(5758-5775) prepared by serum-free RPMI 1640 culture medium (10, 20, 40 µM) was added at the indicated concentrations for 6 h for cytokines assay. LPS (200 ng/mL) was set as a positive control and only serum-free RPMI 1640 culture medium was set as a negative control. The cytokine release analysis by ELISA.
For neutrophils chemotaxis analysis, neutrophils were diluted with serum-free RPMI 1640 medium to a concentration of 1 × 10 6 cells/ml. 500 µl of serum-free 1640 medium with AHNAK(5758-5775) (10, 20, 40 µM) and LPS were added into the lower chamber, and 200 µl neutrophils were added into a 3 µm aperture size transwell chamber for 2 h. The cells on the membrane were counted under a microscope, and cells in the lower chamber were counted using a Cellometer Mini (Nexcelom, San Mateo, CA, USA).
Mast Cells Medium Release Assay 1 × 10 6 LAD2 cells were incubated in a 96-well plate overnight. 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 for 1 h for tryptase, histamine, and β-hexosaminidase assay at 37°C with 5% CO 2 , while 8 h for cytokines assay. 10 µg/ml C 48/80 were set as positive control.

Small interfering (si)RNA Transfection of LAD2 Cells
Speci c knockdown was achieved using small interfering (si)RNAs targeting ST2, and non-targeting siRNAs as negative control (NC). The siRNA sequences were as follows: forward, 5'-GGCAUCACAAAUAGCCAAATT-3' and reverse, 5'-UUUGGCUAUUUGUGAUGCCTT-3' for ST2; and forward, 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 eld 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 le, 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
Data were expressed as mean ± S.E.M. and analyzed one-tail paired Student's t-test. An independent samples analysis of variance was used to determine statistical signi cance in comparisons of the data using the SPSS software. For the paired comparison samples, the treated groups were compared with the negative control group. Differences were considered signi cant at * p < 0.05, ** p < 0.01, and *** p < 0.005. For the multiple doses samples, the treated groups compared respectively with the negative control group were calibrated by Bonferroni's test.

Results
AHNAK(5758-5775) Signi cantly Increased in Psoriasis Patients and Induced Skin In ammatory Reaction AHNAK(5758-5775) is derived from neuroblast differentiation-associated protein, which contains amino acids at positions 5758-5775. After removing the proteins, The results of polypeptide omics by LC-MS showed that AHNAK(5758-5775) signi cantly increased in psoriasis patients than the control samples ( Figure 1A). AHNAK(5758-5775) caused in ammatory cells in ltration in the lesions ( Figure 1B). It was further found by labeling neutrophils that AHNAK(5758-5775) could recruit neutrophils ( Figure 1C). The results of cytokines analysis showed that AHNAK(5758-5775) induced the rise of TNF-α and CXCL2, while showed little in uence on IL-23 or IL-17 ( Figure 1D). The activation effect of AHNAK(5758-5775) on human neutrophils was further studied in vitro. The results showed that AHNAK(5758-5775) had little activation effect on human neutrophils, and did not induce TNF-α, IL-8, IL-1β or IL-6 release and neutrophils chemotaxis (Figure 2).
Further studies on the effect of AHNAK(5758-5775) on the activation of human mast cells found that AHNAK(5758-5775) activated LAD2 cells and induced release of IL-8, TNF-α and MCP-1, while showed little effect on tryptase, histamine, β-hexosamine, IL-6 or IL-1β release (Figure 4).Mast cells-neutrophils co-cultured was used to analyze chemotaxis neutrophils mediated by AHNAK(5758-5775). The results showed that AHNAK(5758-5775) induced co-cultured neutrophils release TNF-α, IL-8, IL-1β and IL-6, and promoted neutrophil migration ( Figure 5). AHNAK(5758-5775) Might Activate Mast Cells via ST2 ST2 can mediated MCs activation but without degranulation reaction. AHNAK(5758-5775) could bind to the pocket of ST2.Ten hydrogen bonds were formed with the residues LYS22, GLN23, ARG35, GLN39, TYR119, THR121, THR135, and ARG198 in the pocket. It was also found that 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 6A). Root Mean Square Deviation (RMSD) was used to indicate the degree of molecular structure change and to measure the stability of the complex system. RMSD uctuation of AHNAK(5758-5775)-ST2 systems is less than 0.6 nm. Root Mean Square Fluctuation (RMSF) showed the uctuation and structural exibility of amino acid residues of receptor protein. The RMSF value of the amino acid residues binding AHNAK(5758-5775) in ST2 was less than 1.5 nm, re ecting that AHNAK(5758-5775) binding can make the structure of the binding region more stable to a certain extent. Radius of gyration (Rg) measures for compactness of protein. Rg values of 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 there is conformation transition in the simulation process. The molecular dynamics simulation results indicated that the complexes remained stable with favorable conformations throughout 100 ns. Above all, it suggested that AHNAK(5758-5775) were stable at the binding site of receptors during the interactions ( Figure 6B).
Knocking down the expression of ST2 by siRNA showed that the release of IL-8, TNF-α and MCP-1 by NC-LAD2 cells was signi cantly higher than that of Knockdown-LAD2 cells ( Figure 7A). Moreover, activated co-culture NC-LAD2 cells caused more TNF-α and IL-1β release and neutrophil migration than Knockdown-LAD2 cells ( Figure 7B).

Discussion
In addition to T cell-mediated adaptive immune abnormalities, psoriasis also depends on the role of innate immune cells. Neutrophil aggregation in the stratum corneum of psoriasis lesion is called the Munro's microabscesses, which is one of the cardinal histopathological sign of PV. Neutrophils are the most abundant cells of the innate immunity system, new sights think neutrophil extracellular traps (NETs) may be the early source of self-DNA, RNA and LL37 that activate dendritic cells, and then produce in ammatory mediators such as IFN-γ [14] [15]. Though it is not generally believed that neutrophils are speci c in psoriasis vulgaris, some studies detected that they were recruited early before the appearance of skin lesions [16].
MCs are considered of linkers of innate and adaptive immunity, they could release cytokines, enzymes, chemokines and histamines when activated. Expressing cell surface molecules to interact with other cells such as endothelial cells, neutrophils, keratinocytes and T-cell subsets to participate in in ammation. The number of MCs, especially those that produce IL-8, TNF-αand IFN-γ, was found increased in psoriasis lesions [17]. Generally, it is believed that IL-22 mainly originated from TH17, TH22, and TC22 cells in psoriasis, but researchers proposed that MCs are major producers of IL-22 and can also secrete IL-17 in 2015 [3]. They can also release mast cell extracellular traps (MCETs) in psoriasis, playing a role similar to that of NETs [18,19]. Apparently, increasing effort has made to identify MCs' function as proin ammatory cells in psoriasis.
Suppression of tumorigenicity 2(ST2), is a member of the interleukin-1 receptor-like protein-1 (IL1RL1) family. It plays roles in both tumors and in ammatory diseases. As an immune response molecular, it exists mainly in: T-lymphocytic cell line (LyT), macrophages, primary mast cells, dendritic cells [20]. IL-33 is an important ligand of ST2, it is de ned as the IL-33/ST2 pathway and participates in many autoimmune diseases, and the current studies on ST2 and psoriasis are mainly limited to this pathway. Notably, elevated serum IL-33 levels in PV patients are effective activators of MCs, and then the releasement of IL-1, IL-6, IL-13, TNF-α, CCL2, and CCL3 could cause neutrophil in ltration. In the phorbol ester-induced psoriasis model, the ST2 -/mouse model showed less in ammation than the wild type [21][22][23]. These results suggest that MCs activation via ST2 is of great signi cance in PV.
We detected the psoriatic lesions and found that the AHNAK-derived peptides signi cantly increased in the lesions compared with the normal skin. It has low tissue speci city and can be produced by keratinocytes in the skin [24]. As we discovered, neutrophil in ltration could be nd in the mice ears after injection the peptide solution. However, our experiment proves this effect is not direct, and there may be other involved procedures. The immuno uorescence con rms that mast cells indeed activated in mice skin lesions. The activation of mast cells induced by antimicrobial peptides, complements, neuropeptides, cytokines and chemokines belongs to IgE-independent pathway [25]. AHNAK(5758-5775) could activate mast cells through an IgE-independent way. Co-cultured of MCs and neutrophils showed several in ammatory mediators and chemokines released by mast cells could lead to the recruitment of neutrophils. The known ST2 is the target receptor of the peptide on mast cells. In spite that few studies mentioned or con rmed whether mast cells are directly involved in neutrophil in ltration in psoriasis, it is not di cult to speculate the molecular network and interactions between them.
In summary, we propose a novel peptide derived from neuroblast differentiation-associated protein (AHNAK) from the psoriatic lesions, it may act as an autoantigen in the rst step of psoriatic in ammatory activation. AHNAK(5758-5775) activate MCs via ST2 receptor and participate in neutrophil in ltration. The discovery of those kinds of endogenous peptides will make better understanding of the pathogenesis of psoriasis and focus attentions on the role of innate immune cells in psoriasis.   AHNAK(5758-5775) induced mast cells release cytokines. A: AHNAK(5758-5775) showed little effect on tryptase, histamine, β-hexosamine, IL-6 or IL-1β release. B: AHNAK(5758-5775) activated LAD2 cells and induced IL-8, TNF-α and MCP-1 release. (data are expressed as mean ± S.E.M. and were calibrated by Bonferroni's test. Differences were considered signi cant at * p < 0.0125, ** p < 0.0025, and *** p < 0.00125.)
(data are expressed as mean ± S.E.M. and and were calibrated by Bonferroni's test. Differences were considered signi cant at * p < 0.0125, and ** p < 0.0025).

Supplementary Files
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