The hybrid peptide YTP was designed by combining the peptides YW12D and TP5. The complete amino acid sequences of the hybrid peptide YTP and its parental peptide are listed in Table 1 . The hybrid peptide YTP and its parental peptides YW12D and TP5 were synthesized (95% purity) by KangLong Biochem Ltd. (Jiangsu, China) and their molecular weights were determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The peptides were stored at -80°C until analysis.

CD spectroscopy was used to analyze the secondary structure of the hybrid peptide YTP. The peptide was dissolved in sterile water and 25 mM sodium dodecyl sulfate (SDS) at a concentration of 0.1 mg/mL. CD measurements were performed over an ultraviolet (UV) spectral range of 190–250 nm at 25°C using a Jasco J-810 spectropolarimeter.

Murine macrophage cells (RAW264.7) were purchased from the Shanghai Cell Bank, the Institute of Cell Biology, China Academy of Sciences and cultured in Dulbecco’s modified Eagle’s medium (DMEM; HyClone, UT, USA). The DMEM medium was supplemented with 10% (v/v) fetal bovine serum (Bioscience) and 1% (v/v) penicillin/streptomycin (HyClone) and maintained at 37°C in a humidified atmosphere (5% CO₂, 95% air).

RAW264.7 viability was measured using a Cell Counting Kit-8 (CCK-8) Assay Kit (Dojinbo, Japan). Cells were seeded in 96-well plates at a density of 2 × 10⁴ cells/well and treated with or without YW12D, TP5 and YTP peptides (10–80 μg/mL). After incubation for 24 h, the cells were then incubated in CCK-8 reagent for 4 h at 37°C. The absorbance of each well was measured at 450 nm using a 96-well microplate reader. Cell viability was evaluated as follows:

RAW264.7 cells were stimulated with or without 10 μg/mL peptides for 24 h at 37°C. Levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in the cell culture supernatant were qualified using ELISA kits (eBioscience, San Diego, USA) according to the manufacturer’s instructions.

Rat plasma was collected by centrifuging whole blood from healthy adult rats. YW12D, TP5, and YTP (10 µg/mL) were incubated for different time at 37°C in rat plasma. The samples were collected into prechilled tubes containing 1 mL of acidic acetone (hydrochloric acid/acetone/H₂O, 1:40:5) and centrifuged at 2, 000 × g for 20 min at 4°C. The obtained precipitates were dried in vacuum. Then, the dried precipitates were dissolved in 0.5 mL of 1 M acetic acid. The peptide analysis was conducted using HPLC. The half-lives of the peptides were calculated by a logarithm-linear regression analysis of the peptide concentrations.

Sixty Balb/c female mice (6-8 weeks of age) were purchased from Charles River (Beijing, China) and maintained in cages under specific-pathogen-free (SPF) conditions. Throughout the experimental period, feed and fresh water were provided ad libitum to all mice. All procedures and experiments were performed in accordance with guidelines provided by the Institutional Animal Care and Use Committee of China Agricultural University.

All animals were randomly divided into the following 5 groups (12 mice in each group): control group; CTX group, treated with CTX (80 mg/kg mouse weight); CTX+YW12D group, mice pretreated with CTX followed by 10 mg/kg YW12D treatment; CTX+TP5 group, mice pretreated with CTX followed by 10 mg/kg TP5 treatment; and the CTX+ YTP group, mice pretreated with CTX, followed by 10 mg/kg YTP treatment. CTX was intraperitoneally (i.p.) injected into the CTX, CTX+YW12D, CTX+TP5, and CTX+YTP groups once daily for 3 days. From days 4 to 10, 10 mg/kg mouse weight peptides were i.p. injected into mice (CTX+YW12D, CTX+TP5, and CTX+ YTP) each day. An equal volume of sterile saline (HyClone) was administered i.p. to the control group each day. From day 4 to 10, the CTX group was administered an equal volume of sterile saline as a control. Twenty-four hours after the last dose, the mice were sacrificed, and their blood and tissues were collected. The mouse body weights were recorded at the beginning and end of the experiment. Spleen and thymus indices were calculated as follows:

Spleens were collected, ground, and passed through 40-μm-mesh cell strainers to harvest the single cell suspension. The cells were labeled with peridinin-chlorophyll-protein complex (PerCP)-conjugated anti-mouse CD3⁺, allophycocyanin (APC)-conjugated anti-mouse CD4⁺, and fluorescein isothiocyanate (FITC)-conjugated anti-mouse CD8⁺ (BD Pharmingen, America) for 30 min at 4°C. The T lymphocyte subpopulations were determined by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Mouse peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density gradient centrifugation. Cells were suspended in PBS containing 5% FBS and then incubated with 10% (v/v) normal goat serum for 15 min at 4°C. The cells were labeled with FITC-conjugated antibodies specific for major histocompatibility complex class-II (MHC-II; BD Pharmingen, America). The surface expression of MHC-II was determined by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA).

Mouse whole blood was centrifuged at 1,000 × g for 20 min and serum was collected. Levels of TNF-α, IL-6, IL-1β, IgG, IgA, and IgM in the serum were measured by ELISA (Solarbio, Beijing, China).

The three-dimensional (3D) structure of the hybrid peptide YTP was built using I-TASSER (https://zhanglab.ccmb.med.umich.edu/I-TASSER/). The constructed 3D structure mode of YTP was next subjected to molecular docking and visualized by PYMOL software. The crystallographic structure of TLR2 was obtained from the Protein Data Bank (PDB ID: 1FYW). For protein-protein docking, Zdock 3.0.2 was employed to acquire the initial complex structure of TLR2-YTP. Altogether, 3600 decoy structures were obtained from the Zdock binding prediction, from which the best decoy structure with the lowest energy was chosen for the following flexible docking analysis. One thousand decoy structures were acquired by flexpepdock (http://flexpepdock.furmanlab.cs.huji.ac.il/), among which the plausible TLR2-YTP docking structure with the lowest binding energy score was selected for analysis.

GROMACS 2020.6 software was used to perform MD simulations of the YTP-TLR2 complex with the AMBER99SB-ILDN force field (31). The complex was placed centrally in a dodecaherdron box with a size of 1.2 nm and dissolved in water (32). Na⁺/Cl^- ions were added to keep the system in a neutral environment (32). Energy minimization was performed using the steepest descent algorithm and sustained until the maximum force < 1000 kJ/mol/nm with a step size of 0.01 (33). NVT and NPT ensembles were simulated using the leap-frog algorithm for 1 ns, and the temperature and pressure were set to 310 K and 1 bar, respectively (34). At the end a 200 ns MD simulation was performed. After the stimulation, the trajectory file was analyzed using GROMACS, and the root mean square deviation (RMSD) and radius of gyration (Rg) of the system were obtained.

RAW264.7 cells were treated with PBS or an anti-mouse mAbTLR2 complex (MTS510 Ab) (eBioscience, San Diego, CA, USA), followed by treatment with or without 10 μg/mL YTP peptide and further incubated at 37°C for 24 h. TNF-α, IL-6, and IL-1β levels in the culture supernatant were detected by ELISA.

Surface plasmon response (SPR) experiments were conducted using a ProteOn XPR36 instrument (Bio-Rad, Hercules, CA, USA) with a ProteOn GLH sensor chip (Bio-Rad) at 25°C. The running buffer (PBS with 1% Tween 20) was continuously passed into the reaction chamber at 30 μL/min. The SPR sensing chip with recombinant TLR2 (R&D Systems) was immobilized by amino coupling to capture YTP. The binding affinity of YTP to TLR2 was examined using peptide concentrations of 0, 1.25, 2.5, 5, and 10 mM. The running buffer was injected into the empty channels as a reference. Sensor chip regeneration and desorption were achieved by injecting 10 mM Gly-HCl buffer (pH 2.5) before the next round of analyses. The experimental data were processed using ProteOn manager software (version 2.0). The binding curves were processed for the start injection alignment and baseline. The reference-subtracted sensorgram was then fitted to the curves labeling a homogeneous 1:1 model. Protein surface data were grouped to fit the association (Ka) and dissociation (Kd) rate constants. The symmetry dissociation constant (KD) for the peptide-TLR4/MD2 interaction was evaluated by using the following equation:

Whole protein in the serum was collected using a whole-protein extraction kit (KeyGEN Biotech, Nanjing, China). The concentration of protein was determined with a BCA protein assay kit (KeyGEN Biotech, Nanjing, China). Proteins were fractionated by 10% polyacrylamide gels and transferred to polyvinylidene difluoride membranes. After blocking with 5% nonfat dried milk for 2 h at room temperature, membranes were immunoblotted with primary antibodies against phospho-IKKα/β, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB; p65), phospho-NF-κB (p-NF-κB; p-p65), inhibitor of κB (IκB)-α, phospho-IκB-α (p-IκB-α), and β-actin-specific monoclonal antibodies (Santa Cruz Biotechnology, CA, USA; 1:1000) overnight at 4°C. Afterwards, membranes were incubated with HRP-conjugated secondary antibody (Santa Cruz Biotechnology; 1:1000) for 1 h at room temperature. The density of the specific proteins was quantified using the ChemiDoc MP Imaging System (Bio-Rad, Hercules, CA, USA).

Statistical analysis was performed using GraphPad Prism v7.0. Statistical comparisons were performed using Student’s t test. All data are expressed as the means ± SEM from at least 3 independent experiments. Significance was claimed with P values ≤ 0.05. NS: P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, ****: P ≤ 0.0001.

The sequences of the hybrid peptide YTP and its parental peptides YW12D and TP5 are shown in Table 1 . The 3D structure of YTP was generated through I-TASSER and analyzed by PYMOL software ( Figure 1A ). In addition, structure determination by CD spectroscopy showed that YTP possesses an α-helical structure in 50% (v/v) TFE and a random coil structure in water ( Figure 1B ), which is consistent with the structure obtained through I-TASSER.

The cytotoxic activity of YTP against RAW264.7 macrophages was appraised by CCK-8 assay ( Figure 2 ). The cytotoxicity of YTP was significantly lower than that of its parental peptides, YW12D and TP5, at all concentrations tested. In addition, YTP exhibited no significant cytotoxicity even at a high dose (80 µg/mL).

The plasma concentrations of YW12D, TP5, and YTP peptides over time are shown in Figure 3 . TP5 showed a very short half-life, and its concentrations decreased nearly 95% after incubation in plasma for 5 min. The half-life of YTP in plasma was slightly more than 2 h, which was significantly longer than that of YW12D (P ≤ 0.01) and TP5 (P ≤ 0.0001) ( Figure 3 ).

As shown in Figure 4 , the body weight of immunosuppressed mice in the CTX group were significantly reduced compared with those in the control group. In contrast to the CTX-induced group, mice in the CTX + YTP groups rapidly recovered their bodyweight. In addition, mouse body weight in the CTX + YTP group was significantly increased compared with that in the CTX + YW12D group ( Figure 4A ). As anticipated, the thymus ( Figure 4B ) and spleen ( Figure 4C ) indices were significantly decreased in the CTX-treated group. However, the administration of peptides remarkably improved the spleen and thymus indices, and the immune organ indices in the YTP-treated group were significantly increased compared with those in YW12D-treated and TP5-treated groups.

To investigate the effect of YTP on serum DC maturation, the expression levels of DC phenotype factor (MHC-II) were determined by flow cytometry ( Figure 5 ). The expression of MHC-II decreased significantly in the CTX group ( Figure 5A-b ) compared with that of the control group ( Figures 5A-a , B ). Treatment with YTP ( Figure 5A-e ) significantly increased the expression of MHC-II ( Figure 5B ). In addition, the expression of MHC-II in the YTP-treated group ( Figure 5A-e ) was significantly increased compared with that in the YW12D-treated group ( Figure 5A-c ) and similar to that in the TP5-treated group ( Figures 5A-d , B ).

To characterize the immunomodulatory activities of YTP, the counts of CD4⁺ and CD8⁺ T lymphocytes in spleen were determined by flow cytometry. Compared with the control group ( Figure 6A-a ), CTX ( Figure 6A-b ) remarkably reduced the proportions of CD4⁺ and CD8⁺ T lymphocytes ( Figure 6B ), and treatment with peptides ( Figure 6A-e ) reversed this effect. In addition, the proportions of CD4⁺ and CD8⁺ T lymphocytes in the YTP-treated group ( Figure 6A-e ) was significantly higher than that in the YW12D-treated group ( Figure 6A-c ) and similar to that in the TP5-treated group ( Figures 6A-d , B ).

To investigate the immunomodulatory activity of YTP in CTX-treated mice, serum IL-1β, IL-6, and TNF-α levels were evaluated by ELISA ( Figure 7 ). As shown in Figure 7A , the IL-1β, IL-6, and TNF-α levels in the CTX + TP5 and CTX +YTP groups were significantly increased compared with the CTX-induced group. In addition, mice in the CTX +YTP group exhibited markedly increased IL-1β, IL-6, and TNF-α expression compared with the CTX + YW12D and CTX + TP5 groups ( Figure 7A ).

Furthermore, CTX treatment resulted in significant reductions in IgA, IgG and IgM levels ( Figure 7B ). The administration of YTP caused a marked increase in all these immunoglobulin levels in the serum of mice. Notably, treatment with YTP resulted in higher IgA and IgM levels than in the CTX + YW12D and CTX + TP5 groups ( Figure 7B ).

To investigate the immunomodulatory mechanism of YTP, YTP binding to TLR2 was examined. RAW264.7 cells were incubated with PBS or TLR2 mAb (C9A12) for 1 h followed by treatment with or without 10 μg/mL YTP for 24 h. Then, IL-1β, IL-6, and TNF-α levels in the cell culture supernatant were quantified by ELISA ( Figure 8A ). YTP caused a significant increase in the production of IL-1β, IL-6, and TNF-α. Interestingly, pretreatment with TLR2 mAb significantly inhibited the TNF-α, IL-6, and IL-1β production induced by YTP, indicating that the YTP-TLR2 interaction is required for YTP immunomodulatory signaling activation.

Furthermore, to analyze the interaction of YTP and TLR2, an SPR assay was performed to evaluate the binding kinetics of ligand-receptor interactions in detail ( Figure 8B ). Five different concentrations of YTP (0, 1.25, 2.5, 5, and 10 μM) were passed over immobilized TLR2. As indicated in Figure 7B , YTP binding to the chip-bound protein exhibited a dose-dependent increase. The calculated Ka and Kd values for YTP binding to TLR2 were 4.31 × 10⁶ s^–1 and 1.62 × 10^-2 M^–1s^–1, respectively, and the KD value was 3.76 × 10^-3 μM.

To better understand how YTP interacts with the TLR2 receptor, we used molecular docking and molecular dynamics simulations to analyze the binding pattern of the two molecules ( Figures 8C–E ). The complex trajectory analysis showed a stable RMSD value at 6 Å after approximately 100 ns ( Figure 8D ). The Rg value also stabilized at around 1.62 nm after 100 ns ( Figure 8E ). Key parameters of the interaction between YTP and TLR2 were analyzed, including electrostatic interactions (e.g., attractive charge), hydrogen bond, and hydrophobic interactions (e.g., Pi-Pi T-shaped, alkyl and Pi- alkyl) ( Figure 8C ; Supplementary Table 1 ). Fifteen pairs of interactions were formed between YTP and TLR2, in which the main amino acids involved in the interactions on YTP were Arg13 and Tyr17. Arg13 of YTP formed hydrogen bonds with G738 of TLR2 and formed both hydrogen bonds and an attractive charge with Asp651 of TLR2. As for Tyr17 of YTP, it formed a hydrogen bond and a hydrophobic bond with Leu737 of TLR2, two Pi-Pi T-shaped interactions with Trp764, and an attractive charge and two hydrogen bonds with Arg771, respectively. Both Arg13 and Tyr17 of YTP are derived from TP5, suggesting that the major contributor to the interaction between YTP and TLR2 is the parental peptide TP5. However, a few amino acids in YTP derived from YW12D (e.g., Lys9) also interacted with TLR2, which may have contributed to the slightly higher immunoreactivity of YTP than TP5 ( Figure 8C , Supplementary Table 1 ).

Next, to investigate the mechanism underlying the immunomodulatory activity of YTP, we studied the downstream signaling pathway of TLR2 ( Figure 9 ). IkK, IкB-α and P65 phosphorylation were reduced significantly after induction by CTX ( Figure 9 ). However, levels of p-IkK, p-IкB-α and p-P65 were significantly elevated in mice treated with YTP after CTX induction ( Figure 9 ). These data indicated that YTP exerts its immunomodulatory activity by activating the TLR2-NF-кB signaling pathway.