In vitro Impact of Yeast Expressed Hybrid Peptide CATH-2TP5 as a Prophylactic Measure Toward Sepsis and Inflammation

CATH-2TP5 is a linear cationic hybrid peptide, consequent from naturally occurring antimicrobial peptide (AMPs) Cathelicidin-2 (CATH-2) and Immunomodulatory peptide Thymopentin (TP5) having dynamic and potent anti-inflammatory activities without hemolytic effect. The biocompatible mechanism of CATH-2TP5 is favored to explore new methodologies in the direction of biomedical applications. In this retrospectively study, an antiendotoxin and anti-inflammatory hybrid peptide CATH-2TP5 was emulated into pPICZα-A and successfully expressed in Pichia pastoris (P. pastoris). The recombinant CATH-2TP5 was purified through the Ni-affinity column and reversed-phase HPLC. The purified CATH-2TP5 peptide exhibited robust anti-endotoxin activity and significantly (p < 0.05) neutralized the effect of lipopolysaccharide (LPS). Furthermore, the down-regulated effect of CATH-2TP was more pronounced (p < 0.05) on LPS-induced cytotoxic effects, nitric oxide secretion and pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) in murine RAW264.7 macrophages. As associated to control and parental peptide the number of apoptotic cells was also contracted with the treatment of CATH-2TP5. Thus, we concluded that CATH-2TP5 peptide may be used in various biomedical applications as a therapeutic drug.


INTRODUCTION
Antimicrobial peptides (AMPs) act as an imperative component of the innate immune response in vertebrates and invertebrates against microbial infections (Golec, 2007;Mardirossian et al., 2016). As a major aspect of their defense system, mostly animals produce a variety of peptides, which have both immunomodulatory, and antimicrobial properties (Nikaido, 2003;Mansour et al., 2014). AMPs peptides capable of being applied to treat various microbes as they act on the bacterial membrane through several processes including pore-formating, barrel-stave, and carpet model which were completely different from traditional drugs (Wu et al., 2014). Antibiotic resistance is a major global issue from both an economic and social perspective (Laxminarayan et al., 2013).
Despite the accessibility of drugs and antibiotics, microbial infections are the quite main cause of death due to the incapability of the particular therapeutics to kill microbes and bind their pathogenic effect like LPS (Lu et al., 2010;Hwang et al., 2013). Gram-negative bacteria contain LPS in the outer leaflet of membrane that usually plays a key role in the pathogenesis of respiratory diseases, chronic illness and septic shock (Golec, 2007;Pompilio et al., 2012;Mardirossian et al., 2016). Gram-negative bacteria release LPS which is recognized by toll-like receptor 4 (TLR4) on macrophages ultimately triggering the activation of cytokines that ultimately leads to inflammation and sepsis (Nagaoka et al., 2001). AMPs have been determined as a potential source in applied medicine as antibacterial, immunomodulatory and anti-inflammatory agents. Accordingly, there is an instantaneous and urgent need for the development of new peptides that have both antibacterial and LPS neutralizing activities.
Cathelicidin-2 (CATH-2) is a highly effective cationic peptide that is under investigation for its potential therapeutic use against bacterial and fungal infections. CATH-2 inhibits LPSinduced inflammatory cytokines production such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β) and induces expression of immune cells (Solmajer, 1990;Bals et al., 1999;Pompilio et al., 2011;Hancock et al., 2016). Systemic immune activation by LPS can be lethal, so inhibition of inflammatory reactions during sepsis is essential to protect the host from over-secretion. The other peptide Thymopentin (TP5) includes the amino acids (Arg-Lys-Asp-Val-Tyr) and represents residues 32-36 of the nuclear protein thymopoietin (Goldstein et al., 1979). TP5 shows likewise organic action as thymopoietin in-charge of phenotypic separation of T-cells and the regulation of the immune system (Singh et al., 1998). It has been perceived as an immune modulator for the treatment of essential immune deficiency diseases such as AIDS (Sundal and Bertelletti, 1994), rheumatoid arthritis (Shah et al., 2017), and immune system infections.
The application of naturally occurring AMPs displays higher nephrotoxicity, promoting hemolysis, toxicity, and neurotoxicity (Cereghino and Cregg, 2000). Subsequently, it is challenged to develop synthetic peptide based on naturally occurring AMPs to successfully treat infectious diseases without its harmful effects. However, hybridizing different peptides have taken rising attention to the development of new agents having antibacterial, anti-inflammatory properties with less cytotoxicity (Solmajer, 1990;Pompilio et al., 2011). Furthermore, a costeffective and active production-based method is required for commercialization of hybrid peptides whereas, due to the high expense of peptide synthesis the heterologous expression of the recombinant peptide is the best choice. Methylotrophic yeast expression system has been an excellent host for the large-scale production of the hybrid peptides (Sreekrishna et al., 1997). The advantages to this system include the stable integration into the expression plasmid at a specific site, extra-cellular secretion of the recombinant protein into the medium, and a simple purification procedure of secreted proteins (Sreekrishna et al., 1997). Therefore, several anti-microbial peptides have been produced on a large scale and have been successfully expressed in P. pastoris (Li et al., 2005;Jin et al., 2006b).
Moreover, our previous laboratory designed and expressed hybrid peptide LL-37Tα1 in P. pastoris depicted its immunomodulatory, anti-inflammatory, and antiendotoxin activities (Ahmad et al., 2019). In the current study, we sought to assess that the combination of CATH-2 (16 amino acid) and Thymopentin (5 amino acid) may have improved endotoxin binding, anti-inflammatory activity along with tiniest cytotoxic effects. Consequently, we synthesized and expressed the hybrid peptide CATH-2TP5 in methylotrophic yeast and explored its biological activities.

Cloning and Expression of the Recombinant Plasmid
The amino acid sequence of the hybrid CATH-2TP5 peptide was optimized through the JAVA codon adaptation tool (JCAT) 1 . The full-length gene of 96 bp was synthesized by Sangon Biotech and expressed in vector pPICZαA at the restriction sites of Kpnl and XbaI with 6 × Histidine tag (His-tag). The transformation of expressed plasmid was carried out in E. coli strain DH5α. The positive transformant colonies were selected on low-salt LB plates and confirmed by PCR using specific primers as P00-1 F: 5 -CCAGATGGGGTAGATTCTT-3 P00-2 R 5 -GTAACGTCCTTTCTCTTTG-3 and then sequenced (Tsingke Biotech). The reaction mixture was incubated at 95 • C for 5 min, followed by denaturation for 30 s at 95 • C, annealing at 52 • C for 30 s, extension at 72 • C for 50 s and a final extension at 72 • C for 10 min. The efficacious recombinant strains were then cultured overnight and the plasmid was attained. The SacI linearized pPICZαA plasmids were transmuted into P. pastoris strain (X-33) by electroporation method as specified by the Pichia expression guide. The positive cells were further selected on YPD agar plates containing antibiotics (Zeocin 100 µg/mL). The addition of the aim genes into the P. pastoris genome was confirmed by PCR using both (sense and antisense) specific primers. For negative control, a vacant expression vector pPICZαA was prepared. The confident colonies of P. pastoris were cultured in 5 mL tubes of BMGY medium at 28 • C, 200 rpm for 12 h until the OD 600 reached to 4-6. After centrifugation (at 3,000 × g) for 5 min and the collected cells were resuspended in 50 mL of BMMY containing 1.34% YNB and biotin (4.0 µg/mL) in a 250 mL shake flask and incubated at 28 • C. Pure methanol was consequently added to the culture medium at the final concentration of 1% every 24 h to maintain induction (Ausubel et al., 1991;Ahmad et al., 2019). All induction experiments were performed in triplicate.

Purification and Mass Spectrometry Analysis of the Recombinant CATH-2TP5
The expressed recombinant CATH-2TP5 peptide was purified after the removal of cell debris from culture supernatant via centrifugation at 12,000 rpm for 20 min at 4 • C. The supernatant (containing CATH-2TP5 peptide with 6 × His-tag) was sieved and laden to 1 ml His Trap Chelating Ni-affinity column (Bio-Beads TM, Sweden). The column was equilibrated with 1 × phosphate buffer (PB) and 10 mM imidazole. The adsorbed hybrid recombinant peptide was extracted by using various concentrations of a linear gradient of imidazole (50-500 mM). The eluted samples were sized-separated by Tricine-SDS-PAGE. The Bio-Rad dye agent with bovine serum albumin (BSA) was used as a standard for the evaluation of protein concentration (Ausubel et al., 1991). After early purification, the peptide was additional purified by using reverse-phase high-performance liquid chromatography (RP-HPLC) on a Kromasil C18 column (4.6 × 250 mm, 5 µm) with a linear gradient of acetonitrile (0-100% for 30 min) containing 0.1% Trifluoroacetic acid (TFA) at a flow rate of 1.0 ml/min. The elution points were detected at 220 nm and the active fraction was verified by using quantitative Chromogenic limulus amebocyte lysate (LAL) (Ahmad et al., 2019). The RP-HPLC purified CATH-2TP5 was diluted with Milli-Q water and filtered through 0.22 µm and further subjected to electrospray ionization mass spectrometry ESI-MS/MS.

Lipopolysaccharide (LPS) Neutralization Assay
The endotoxin neutralization of parental (CATH-2) and hybrid (CATH-2TP5) peptide were assessed using a Chromogenic limulus amebocyte lysate (LAL) according to manual instruction. Different concentrations of parental and hybrid peptides (15 to 40 µg/mL) were treated with a constant concentration of LPS (1 EU/mL) at 37 • C for 15 min in the wells of a pyrogenic sterile microliter plate. Furthermore, 50 µL of the aliquots were then supplemented to equal volumes of LAL reagent and incubated for 6 min at 37 • C. A reaction was suspended with the addition of HCl solution and analyzed on OD 545 nm (Ahmad et al., 2019).

Hemolytic Activity Assay
Mouse red blood cells isolated from heparinized blood as described previously (Ahmad et al., 2019) were used to analyze the hemolytic activity of parental and hybrid peptides. A 4 ml mice RBCs were centrifuged at 1,500 rpm for 10 min. The cells were washed three times with diluted 10% hematocrit and incubated with various concentrations (15 to 40 µL) of hybrid CATH-2TP5 peptide for 1 h at 37 • C. The sample was further centrifuged at 3,500 rpm for 5 min and absorbance of the supernatant was analyzed on 414 nm.

LPS-Induced Cytotoxicity Detection by Lactate Dehydrogenase Activity (LDH) Assay
Lactate dehydrogenase activity assay was used to assess the cytotoxic effect of hybrid peptide on murine RAW264.7 macrophages infected with or without LPS (1 µg/mL) incubated at 24 h as described previously (Anton et al., 2012;Ghonime et al., 2014). Additionally, LPS-induced cells (1 × 10 5 cells/mL) were exposed to parental and hybrid peptide (15 to 40 µg/mL) to plaid the comparative effect. The supernatants were collected and the cytotoxic level was measured according to the kit instructions (Dojingdo Laboratories, Kumamoto, Japan).

Quantification of Nitric Oxide (NO) and
Pro-inflammatory Cytokines Production LPS-Induced Mouse RAW264.7 Macrophages Mouse RAW264.7 cells (5 × 10 5 /well) were incubated with (1 µg/mL) LPS in the presence and absence of parental and hybrid (15 to 40 µg/mL) peptide in DEME medium in a humidified chamber with 5% CO 2 atmosphere. The culture supernatant was assorted with Griess reagent and incubated for 10 min at room temperature. The secretion of NO was measured on a microplate reader at 540 nm (Green et al., 1982).
The effect of parental and hybrid peptide on the expression of TNF-α, IL-6, and IL-1β in LPS-stimulated mouse RAW 264.7 macrophages was quantified by enzyme-linked immunosorbent assay (ELISA; Cloud-clone corp, Houston, United States) as described previously (Ahmad et al., 2019).

Determination of Apoptosis via Flow Cytometry
Lactate dehydrogenase activity-induced apoptosis in RAW264.7 macrophages was interpreted by annexin V-FITC (V conjugated to green-fluorescent FITC dye) and PI (Propidium iodide) staining procedure. The mouse RAW264.7 macrophages (1 × 10 5 cells/mL) were cultured as described above. The cultivated cells were infected with LPS (10 µg/mL) in the presence and absence of parental and hybrid peptides (40 µg/mL) for 4, 12, and 24 h. After treatment, the cells were washed thrice with icecold PBS (washing buffer) and stained with FITC annexin V/PI apoptosis detection kit (Becton Dickinson, Franklin Lakes, NJ, United States) as described previously (Shahid et al., 2017;Ahmad et al., 2019). Apoptosis rate was expressed using a FACSA by flow Cytometry (Becton Dickinson, United States).

Statistical Analysis
The three independent experiment data were expressed as mean ± standard deviation (SD). Statistical data were estimated by the one-way analysis of variance (ANOVA) according to the least significant difference (LSD) test for multiple comparisons. A post hoc test using SPSS 19.0 (SPSS, Inc., Chicago, IL, United States). For all analysis, p < 0.05 and p < 0.01 were considered significant.

Optimization and Expression of the Recombinant CATH-2TP5
Yeast expression vector pPICZαA-CATH-2TP5 that contains a 96 bp DNA fragment encoding a recombinant C-terminal 6 × His-tagged that facilitate purification and analysis of expressed proteins. The restriction enzyme KpnI and XbaI were attached at its 5 and 3 ends and fragment was cloned in the frame of α-factor signal, downstream promoter alcohol oxidase (AOXI) of the expression vector pPICZαA and the result in recombinant pPICZαA-CATH-2TP5 after double digested with a restriction enzyme (Supplementary Figure S1). The adjustment of the inserted peptide sequence was confirmed by PCR. The recombinant plasmid pPICZαA-CATH-2TP5 was linearized with SacI and altered into the competent cells of P. pastoris (X-33) by electroporation. The zeocin (100 µg/mL) resistant P. pastoris transformants were obtained and further subjected to PCR and sequencing for the confirmation of positive transformants. Our result exhibited that pPICZαA-CATH-2TP5 was successfully assimilated into host cells. Moreover, the expression conditions were optimized according to the method described above. The optimized factors included initial pH value (pH 5-6), final methanol concentration (0.25-2.0%) and incubation time (24-120 h). The optimal conditions for recombinant peptide expression were pH 6, and incubation for 120 h with 1% methanol. The expression level of recombinant CATH-2TP5 peptide was 119, 110, and 123 µg/ml, respectively (Figures 1A-C).
The culture supernatant was separated on Tricine-SDS-PAGE and silver staining. The expression of the recombinant CATH-2TP5 in P. pastoris indicated the apparent molecular weights of 2.6 kDa (Figure 2A).

Purification and Mass Spectrometry Analysis of Recombinant CATH-2TP5
After centrifugation of the cultured media, supernatant was purified by Ni-NTA affinity chromatography column. The pure CATH-2TP5 peptide was eluted with 500 mM imidazole. As shown in Figure 2B, SDS-PAGE of the recombinant purified peptide appeared as a single band with molecular weight of 2.6 kDa. The peptide was further applied to RP-HPLC and resultant product was highly purified (yielded 4 mg) which presented in Figure 2C. Furthermore, the purified recombinant CATH-2TP5 was diluted in milli-Q water, filtered and analyzed by ESI-MS/MS. Mass spectrometry of the purified CATH-2TP5 expressed a single non-dispersed signal ( Figure 2D)

CATH-2TP5 Neutralizes Lipopolysaccharide (LPS)
LAL test was used to asses the aptitude of parental and hybrid peptide to neutralize the LPS. The CATH-2TP5 peptide has a net charge of + 9 under the physiological conditions and it can neutralize and bind with LPS. Our results exhibited that CATH-2 (35 and 40 µg/ml) was able to neutralize LPS (62.321% ± 2.321, 75.331% ± 2.456, respectively) and CATH-2TP5 peptide (70.656% ± 4.382, 80.1% ± 4.395, respectively) in a dose-dependent manner. However, as compared to parental peptide, neutralization of LPS by hybrid peptide was more pronounced (p < 0.05) ( Figure 3A).

CATH-2TP5 Ameliorates the Cytotoxic Effects of LPS in Mouse RAW264.7 Macrophages
Lactate dehydrogenase activity release into cell medium was used as an indicator of cell death with the NAD + reduction. The cells along with only LPS infection showed higher level of LDH at 24 h (3.06 ± 0.067) as associated with co-infection of LPS plus parental and hybrid peptide (15 to 40 µg/mL), and control group. This describes that LPS significantly (p < 0.05) damaged the mouse RAW264.7 macrophages. However, the LPS-induced cytotoxicity significantly (p < 0.01) decreased 2.3 ± 0.077 to 2.03 ± 0.067, respectively with a concentration of LPS (35 µg/mL), while the cytotoxic effect of LPS at a concentration of 40 µg/mL lowered from 2.13 ± 0.048 to 1.81 ± 0.032, respectively when cells were treated with CATH-2 and CATH-2TP5 peptides. Furthermore, CATH-2TP5 decreased the LDH level as compared with CATH-2. These results elaborated that CATH-2TP5 peptide neutralizes the LPS and significantly lowered the cytotoxic effects of LPS in a dose dependent manner ( Figure 3B).
The hybrid CATH-2TP5 peptide exhibited pronounced anti-endotoxin activity. The hemolytic activity of the parental and recombinant peptide was detected by lysing mouse red blood cells ( Figure 3C). Comparatively, with control cells 0% (p < 0.001) hemolysis was observed in treated cells and hybrid peptide triggered less hemolysis than CATH-2. Conversely, Our result exhibited that recombinant FIGURE 1 | Effects of pH value, methanol concentration, and inductive time and inductive on the peptide expression. (A) Recombinant peptide expression was conducted at different pH values (pH 5-6.5), induced by 1% final methanol concentration at 28 • C for 120 h. (B) Recombinant peptide expression was conducted at pH 6, inducted by various methanol concentration (0.5-2.5%) at 28 • C for 120 h. (C) Recombinant peptide expression was conducted at pH 6.0, inducted by 1% methanol concentration at 28 • C for 24-120 h. Data are presented as mean ± SD, n = 3. While **p < 0.01, ***p < 0.001 vs. Controls. hybrid CATH-2TP5 peptide does not have cytotoxic and hemolytic activities.

CATH-2TP5 Down-Regulates LPS-Induced Inflammatory Response of RAW264.7 Macrophages
The ability of CATH-2TP5 peptide to neutralize LPS provoked us whether CATH-2TP5 could conquest the inflammatory response elicited by LPS. To further investigate the query, we measured the effect of recombinant CATH-2TP5 on LPS-stimulated secretion of NO, and proinflammatory cytokines TNFα, IL-6, and IL-1β from mouse RAW 264.7 macrophages. As shown by the results in Figure 4A, LPS significantly increased the NO secretion compared with control cells (61 µM vs. 8 µM, respectively), but the response was most efficiently inhibited (34 and 21 µM) after FIGURE 3 | Endotoxin neutralization, cytotoxicity, and hemolytic activity test of parental and recombinant CATH-2TP5 peptide. (A) LPS neutralization by CATH-2 and CATH-2TP5 indomitable by using an endotoxin quantitation kit. Mean values accessible; n = 3 ± SD ( # p < 0.05 and ## p < 0.01 revealed contrast of CATH-2 vs. CATH-2TP5). (B) CATH-2TP5 diminished cytotoxicity in the supernatant of endotoxin-infected murine RAW264.7 macrophages. Data signified as mean ± standard deviation (SDs) of independent experiments. **p < 0.01 vs. LPS. While, # p < 0.05 and ## p < 0.01 specifies significant variance related with parental CATH-2 peptide. (C) Hemolytic activities of CATH-2TP5 in contradiction of mouse RBCs. The data resemble to the mean values of three independent experiments and are expressed as a % age of hemolysis ± standard deviation (**p < 0.01, ***p < 0.001 vs. Triton X-100). Whereas, # p < 0.05 and ## p < 0.01 indicates significant difference compared with parental CATH-2 peptide.

CATH-2TP5 Reduces LPS-Induced Apoptosis in Murine RAW264.7 Macrophages
To explore the role of CATH-2 and CATH-2TP5 peptide in LPS-stimulated apoptosis, mouse RAW264.7 macrophages were cultivated. After 95% of confluency, macrophages were treated with only LPS and LPS plus CATH-5TP5 peptide for 4, 12, and 24 h. the macrophages were stained with annexin V-FITC and PI as per instructions of the manual. The number of apoptotic cells was significantly increased in LPS treated group in both early (annexin V-positive cells) and late-stage (annexin V-and PI-double positive cells) at 4, 12, and 24 h post-infection ( Figure 5A). The percentage of apoptotic cells was increased in a time dependent manner 8.99% ± 3%, 39.1% ± 11% and 47.3% ± 13% upon LPS infection after 4, 12, and 24 h, respectively. However, the addition of CATH-2TP5 along with LPS infection significantly reduced (p < 0.01) the number of apoptotic cells as compared to LPS infection alone ( Figure 5B). Our results provide evidence that CATH-2TP5 peptide could neutralize the LPS and ultimately reduces the LPS-induced apoptosis in RAW 264.7 macrophages.

DISCUSSION
Hybridization of the natural peptides isolated from different sources is a common technique to construct novel peptides against the pathogenic microbes exhibiting antimicrobial resistance (Bulet et al., 2004;Jenner et al., 2019). AMPs have a positive net charge that allows them to interconnect with a bacterial membrane which has a negative charge, help to bind with endotoxin (Nagaoka et al., 2001). LPS, also known as endotoxin which is released during lysis of Garm-negative bacteria and plays a key role in pathogenesis of endotoxin shock (Nagaoka et al., 2001). In the previous study, the experts have struggled to amend the amino acid sequence of the parental peptides to enhance the expression and activities against infections. Moreover, the well-preserved amino acid sequence of the peptides has an inordinate impact on immunity and anti-inflammatory activities and also the proper replacement or addition in the peptide sequence does not affect its function but also improve the response of hybrid peptides (Liu et al., 2018). However, the development of the novel peptides through hybridizing of various parental peptides is an operative method to enhance the high immunomodulatory and anti-inflammatory activities along with LPS neutralizing ability with least adverse effects (Jang et al., 2006;Lee et al., 2016). Therefore, more attention has been focused on the expression of hybrid peptides with enhanced therapeutic efficacy as it could be beneficial in the treatment of several infectious diseases. The intention of hybrid peptides is based on the amino acid sequence of the parental AMPs such as cecropin, cathelicidin, magainin II, LL37, and melittin (Lee et al., 2002;Jin et al., 2006a;Wu et al., 2014).
There are some methods for the manufacture of hybrid peptides: (i) extraction from natural resources, (ii) chemical synthesis, and (iii) expression in yeast or bacteria (Van Harten et al., 2018). Due to the high expense, the yeast expression system delivers an opportunity for the production of hybrid peptides on a commercial scale. According to the best of our knowledge, we for the first time have optimized the expression conditions like temperature, PH, and concentration of methanol induction in culturing medium of the novel hybrid peptide (CATH-2TP5) in p. pastoris. We expressed anti-inflammatory hybrid peptide with the optimum condition. The concentration of recombinant CATH-2TP5 peptide was a maximum of 50 mg/L in the culture medium. Our SDS-PAGE results showed that the size of the recombinant peptide is 2.6 kDa. The expression yield is higher than earlier described such as LL-37Tα1 (Ahmad et al., 2019), ceropinAD (Parachin et al., 2012), and CA-MA (Jin et al., 2006a). In the current study, we also reported the benefits of expression in P. pastoris as compared to bacterial expression system concerning cost-effectiveness and heterologous expression of novel active CATH-2TP5 for commercial usage. There are many advantages of a eukaryotic expression over bacterial expression systems such as protein glycosylation, post-translational modifications, folding and processing which is probably absent in the E.coli system (Gupta and Shukla, 2017). Comparatively, the bacterial expression system is difficult for the recovery of the small peptides to be recovered so this system could not be used directly to express the peptide with the toxicity to bacteria (Parachin et al., 2012). For pure hybrid peptide, we performed the purification method as described before with slight modification (Jin et al., 2009). For the two steps purification, we used Ni-NTA affinity chromatography column (Zhang et al., 2015) and HPLC, and we attained 4 mg quite pure recombinant CATH-2TP5 peptide from 200 ml culture medium.
In the current study, purified CATH-2TP5 peptide was exposed to endotoxin binding, cytotoxicity, and hemolytic activity reflected as imperative advantages of recombinant peptide to be used as capable drugs. LPS comprises three parts; lipid A, O-antigen and polysaccharide core. Lipid A is unambiguously a physiologically active component of endotoxin and principally involved in stimulating the inflammatory response (Iwanaga et al., 1992). Lipid A is also accountable FIGURE 5 | Apoptosis of LPS-induced murine RAW264.7 macrophage at 4, 12, and 24 h with FITC-conjugated annexin-V (Green) and PI (Red). (A) The marked RAW264.7 macrophage was observed and analyzed by flow cytometry, the control signifies normal cells, the middle panel LPS discloses demonstrative RAW264.7 macrophage treated with LPS only, and the lower panel reveals RAW264.7 macrophages treated with LPS plus parental and hybrid peptides. (B) Observe the effects of LPS with CATH-2TP5 in RAW264.7 macrophages apoptosis. and **p < 0.01, ***p < 0.001 indicate the significance and highly significance difference vs. LPS. While # p < 0.05 indicated significance divulges between parental and hybrid peptide.
for the instigation of LAL reagent (Gutsmann et al., 2010a). In the present study, LAL depicted that CATH-2TP5 peptide binds to lipid A part of LPS and neutralizes the biological effects of endotoxin. However, based on previous reports, positive charge and hydrophobicity are essential for the peptide to have LPS neutralizing and anti-inflammatory characteristics (Sun and Shang, 2015;Zhang et al., 2018). In the current study, Our hybrid peptide comprises an N-terminal region along with polar active part of CATH-2 (16 amino acid) and a C-terminal TP5 having a net charge of +9 with reducing hydrophobicity as reported in the previous study (Gutsmann et al., 2010b;Kaconis et al., 2011;Ahmad et al., 2019). It sustained our assumption that the amalgamation of active parts of dissimilar parental peptides conferred strong electrostatic interactions between AMPs and LPS. AMPs have revealed cytotoxic and hemolytic effects on mammalian cells (Zhang et al., 1999;Kaconis et al., 2011;Lee et al., 2016). In the current study, our hybrid peptide showed less cytotoxicity and hemolytic activity as compared with parent peptide due to reduced hydrophobicity which in line with the earlier study (Ahmad et al., 2019).
In the present study, we also detected LPS-induced production of NO, TNF-α, IL-6, and IL-1β in mouse RAW264.7 macrophages. LPS activates macrophages to release NO and potent inflammatory mediators which involved in several acute and chronic diseases containing hemorrhagic shock, endotoxin shock and atherosclerosis (Bertolini et al., 2001;Lind, 2003). Approximately, antibiotics are recognized to kill the bacteria but at the same time they also stimulate the release of LPS and may aggravate the endotoxic shock (Nagaoka et al., 2001) owing unable to neutralize the LPS. So, a drug that subdues the creation of NO, inflammatory cytokines and could neutralize the LPS may be a sensible additional therapy for endotoxin shock. Consequently, preventing these inflammatory cytokines may be potentially effective for averting inflammatory diseases. As compared with previously reported recombinant peptides lunasin-4  and SPHF1 (Ahn et al., 2012), we originate that CATH-2TP5 more proficiently inhibits the production of proinflammatory cytokines. These features predict that CATH-2TP5 peptide is an attractive drug candidate for the treatment of inflammation and endotoxin shock produced by Gram-negative bacterial infection.
Our findings implicate that CATH-2TP5 treatment effectively reduces the endotoxins and proinflammatory mediator, thus it might have potency for inhibiting inflammatory diseases. Furthermore, our recombinant hybrid peptide depicted high immunomodulatory and anti-inflammatory activities, and also neutralized the bioactivity of endotoxin without cytotoxic and hemolytic effects as modeled in Figure 6. These data are in agreement with the previous study reporting high antiinflammatory and LPS neutralizing activity of CATH-2 without hemolytic activity (van Dijk et al., 2016;Banaschewski et al., 2017). Moreover, endotoxin up-regulates adhesion molecules (Lush et al., 2000) and induces apoptosis (Bannerman and Goldblum, 2003). In the present study, we used PI and annexin-V FITC staining which expounded that LPS significantly induces apoptosis in murine RAW264.7 macrophages. Under some circumstances, macrophages are responsible for severe systemic inflammatory response triggered by LPS which has deleterious effects and leads to septic shock (Bone, 1991;Guo et al., 2019). We identified that CATH-2TP5 neutralizes LPS and reduces both early and late apoptosis. Generally, these investigations designate that CATH-2TP5 is a propitious peptide that could be a potential candidate for application in the medical industry as therapeutic condidate.

CONCLUSION
For the first time, we report a successful expression method for the hybrid CATH-2TP5 peptide in P. pastoris with expression plasmid PpICZαA. CATH-2TP5 potently neutralized LPS with no cytotoxic and hemolytic activity. Additionally, CATH-2TP5 novel peptide exhibited immunomodulatory, and antiinflammatory activity by preventing cytokines release including NO, TNF-α, IL-6, and IL1β, and also decreased the number of apoptotic cells in LPS-infected RAW264.7 macrophages. These results deliver a potential strategy for recombinant production of bioactive CATH-2TP5 in the industry and might help prevent inflammatory diseases.

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.