Peptides Regulating Proliferative Activity and Inflammatory Pathways in the Monocyte/Macrophage THP-1 Cell Line

Innate immunity represents a finely controlled network of physiological processes that synchronize immunocompetent cells to control and patrol the integrity of organs when exposed to injuries that may alter their functions [ 1 , 2 , 3 , 4 ]. Leukocytes are a qualified group of immune cells that are mostly involved in recognizing foreign organisms and in developing proper phagocytic activities [ 5 , 6 , 7 ]. In particular, monocytes are circulating leukocytes that move to sites of infection attracted by cytokines and chemokines, a specialized set of molecules that cooperate to induce terminal differentiation of monocytes into macrophages with intense phagocytic activity [ 8 , 9 , 10 , 11 ]. These large phagocytes are found in all tissues [ 12 ], where they patrol for potential pathogens through amoeboid movement. Besides phagocytosis, they play a critical role in nonspecific defense (innate immunity) and help in activating adaptive immunity functions by recruiting other immune cells, such as lymphocytes, by presenting antigens to T cells. In humans, dysfunctional macrophages cause severe diseases, such as chronic granulomatous disease, which result in frequent infections [ 13 ]. In addition to increasing the inflammation and stimulation of the immune system, macrophages play an important anti-inflammatory role, and can decrease immune reactions through cytokines release. Macrophages which promote inflammation are called M1 macrophages, while those that reduce inflammation and encourage tissue repair are called M2 macrophages [ 14 ]. This difference is reflected in their metabolism: M1 macrophages have the unique ability to metabolize arginine to the “killer” molecule nitric oxide, whereas M2 macrophages have the unique ability to metabolize arginine to the “repair” molecule ornithine [ 15 , 16 ]. Monocyte-derived cell lines with varying degrees of differentiation are often used to describe macrophage function, since they show clear advantages for ease of acquisition over primary macrophage in vitro cultures. In particular, the monocytic leukemic THP-1 cell line represents a suitable model for studying Khavinson Peptides ® effects on cellular proliferation and inflammatory activity [ 17 ]. These peptides propose a new challenge for understanding how natural and partially digested proteins can generate signaling molecules participating in the regulation of cell physiology. In 1983, V. Morozov and V. Khavinson demonstrated that cells produce low molecular weight peptide compounds, and that these peptides can mediate information transfer by regulating proliferation, differentiation and intercellular interactions [ 18 , 19 ]. These substances were discovered and identified in various tissues and called short peptides [ 20 , 21 ]. The isolated substances were all short-chain peptides with low molecular weight of up to 10,000 Da. During the last forty years, over twenty complexes of physiologically active peptides were extracted and characterized. Peptides were found to participate in gene-expression regulation and protein synthesis, according to the peptide cascade model, modulating multiple physiological functions of the organism [ 22 , 23 , 24 ]. These processes are aimed at preventing DNA damage, or its eventual suppression, and stimulating its repair, with the aim of restoring cellular homeostasis. Their main function is a normalizing effect on specific organs, replacing or supporting the activity of substances secreted by these morphological structures. They also increase the lifespan of mesenchymal stem cell (MCS) and induce molecular mechanisms that contrast senescence [ 25 ]. They modulate neuronal activity and hinder cognitive degeneration, probably by improving the integration of cellular functions, and thus counteracting the accumulation of free radicals. Furthermore, they implement the action of telomerase by promoting anti-aging effects that characterize many cell types [ 26 ]. Most of these peptides perform their function specifically on the organs from which they have been isolated. Several studies report that thymus involution is responsible for promoting harm in the elderly due to the reduction of immune reactions and the development of immune dysfunctions [ 27 , 28 ]. According to the reports of V. Khavinson, this observation set the stage for the use of therapeutic compounds with natural or synthetic thymic factors as immune modulators. Those natural thymic factors (NTFs) have been isolated from calf thymus by mild acid extraction (V. Morozov and V. Khavinson, 1991) and pharmaceutical products containing NTF (Thymalin) are used in clinical practice for the prevention and treatment of immunodeficiency [ 29 , 30 ]. Pharmaceuticals containing one of the immunomodulatory molecules (L-Glu-L-Trp) was derived from Thymalin by reversed-phase high performance liquid chromatography (RP-HPLC), termed Thymogen [ 31 ]. A large-scale study was conducted to isolate biologically active peptides from different h

Since peptides have been described as inducers of anti-senescence mechanisms in stem cells [ 45 ]. The possibility that they can influence the proliferation of THP-1 cells in culture was investigated. In particular, the hypothesis that they can induce phosphorylation of cytoplasmic kinase sets, which are activated at various levels during mitogenic stimuli, was tested. As shown in Figure 2 , cell extracts from THP-1 cells, treated with standard concentrations of peptides, show an increased phosphorylation of ERK1/2, a MAP kinase activated during the mitogenic process. All the peptides, except for Vilon (P2), can increase the phosphorylated level of ERK1/2, suggesting that they could influence the transcriptional or translational regulation of important cellular signaling molecules. The dipeptide Vilon (P2), on the other hand, shows a small modulatory capacity. When THP-1 cells were incubated with the bacterial-derived lipopolysaccharide (LPS), which is an inducer of pro-inflammatory pathways, either alone or in the presence of the peptides, an addictive effect on tyrosine phosphorylation of ERK½ was only observed on extracts co-treated with Epitalon (P1) or Vilon (P2) ( Figure 2 A). No significant coordinated action between LPS and peptides influenced the activation of other kinases as the mTOR dependent p70 S6 ( Figure 2 C). Interestingly, a similar pattern of increased tyrosine phosphorylation was observed when specific anti-phospho antibodies raised against the stress related c-Jun N-terminal kinase (JNK) were used in a Western Blot assay of extracts derived from the peptide-treated THP-1 monocytes ( Figure 2 B). Surprisingly the co-incubation downregulated the activity of JNK with a reproducible exception of Thymalin (P4). This would suggest that when peptides are used as co-stimulators with pro-inflammatory inducers, a complex network of synchronized signaling events take place inside the cells.

In order to better investigate the molecular mechanisms that could influence the anti-inflammatory abilities of bio-regulators, Western Blot analyses were conducted to evaluate the activation status of the signal transducers and activators of transcription, namely STATs. STATs are a series of cytoplasmic signaling molecules mainly involved in cytokine activation, proliferative induction signals and inflammatory response modulation. As shown in Figure 3 B, the treatment of macrophages with the pro-inflammatory inducer LPS activates STAT1 phosphorylation on a time course manner with maximum induction between four and eight hours after treatment. Co-incubation with standard amounts of peptides determines an increase in the phosphorylation process with the same temporal pattern, suggesting that the peptides may cooperate within the same transduction axis, or through an independent mechanism. This experiment confirms that at least Epitalon (P1), Vilon (P2) and the bronchial peptide Chonluten (P5), activate STAT1 phosphorylation, functioning possibly via a receptor-independent mechanism, not involving IFN-α/IFN-R interactions, as documented by no effect on IFN-α production ( Figure 3 A). As a matter of fact, several evidences suggest that the chemical structure of such peptides allows direct penetration into the cellular compartment regulating gene expression [ 22 ]. The possibility that peptides could regulate other STAT molecules involved in cytokine-mediated signaling, specifically those acting in the acute phase of inflammation, was also investigated. We tested several transducers of the STAT family, in particular the activator of transcription STAT3. STAT3 is mostly involved in IFN-α and IFN-γ mediated signaling, but it is also a major player in the IL-6 production and IL-6 mediated activity. Surprisingly, co-incubation of LPS and peptides do not determine any additive or synergistic effect on STAT3 phosphorylation. In particular the level of phosphorylated STAT3 slightly decrease and attenuates the activation mediated by the pro-inflammatory LPS ( Figure 4 ). Moreover, the incubation of the THP-1 macrophages with the P1 peptide does not activate the phosphorylation status of the latent cytoplasmic transducers, since the anti-phospho-STAT3 antibodies did not show any significant increase in activated molecules. In addition, the P1 time–course treatment seems to further downregulate the phosphorylation level of phospho-STAT3 as compared to the control ( Figure S4 of Supplementary Data ).

A clear shifting of the latent cytoplasmic STAT1 molecules into the nuclei of peptide-treated macrophages was observed through confocal microscopy. As evident in Figure 6 , the control differentiated THP-1 cells did not show any significant transfer of the STAT1 into the nuclei. Whereas, upon peptide incubation, most of the peptides were able to activate cytoplasmic transducers passage into the nuclei as marked in Figure 6 . The phosphorylation of the STAT1 molecules allows the unmasking of the nuclear localization site (NLS), determining the translocation into the cell nucleus. The conventional DAPI staining, that specifically binds strongly to adenine–thymine-rich regions in DNA, was used. Subtraction of the background staining was from incubation of fluorescence secondary specific antibody.

In this study, we have investigated the proliferative and anti-inflammatory activity of Khavinson Peptides ® on the THP-1 human monocytic cell line, whose monocytes can switch phenotype differentiating into macrophages upon incubation with PMA. Monocytes are blood-circulating leukocytes acting as a primary line of defense in various areas of the body. Using THP-1 cells as an immune system cellular model, both mechanisms of replicative induction and of modulation of the inflammatory response were evaluated in relation to peptide influence on these functions at molecular level [ 47 , 48 ]. Since it has been shown these nano peptides may act as anti-senescence factors able to prolong the life span of mesenchymal stem cells in culture [ 33 ], monocytes were treated with a standard dose of peptides to investigate some cellular functions. Initially, treating THP-1 monocytic cultures with specific peptides, we reported an increase in the growth and cell proliferation with a balanced distribution along the various cell cycle phases [ 49 ], and only the bronchogenic peptide Chonluten (P5) appears to increase the treated cells apoptosis level, doubling the value in relation to other peptides ( Supplementary Data ). Underlying this observation may be a unique behavior of Chonluten (P5) in the specific regulation of monocytic function through increased proliferation and, at the same time, modulation of apoptotic activity. [ 50 ]. As a matter of fact, since monocyte recruitment is quite active on bronchial and alveolar compartments during the extension of inflammation, the peptide itself could massively modulate the release activity of extracellular vesicles as a sign of cell viability associated with the proliferation index. These observations suggest that, under cell duplication conditions, peptides influence the duplication rate without causing dysfunction in the cell cycle phases. To better understand the degree of monocytes proliferative induction, the level of activation of the MAP kinase complex depending on the peptide treatment was studied. As widely demonstrated, MAP kinases are key elements in the phosphorylation and dephosphorylation processes, acting in a critical interplay in macrophage biology [ 51 ]. In particular, the ERK1/2, which represents the goal of the mitogenic signaling inside the cell, showed various degrees of phosphorylation, possibly modulating the transcription of target genes. Indeed, as largely evidenced in recent years, extracellular signal-regulated kinases (ERKs) or classic MAP kinases are expressed protein kinases, which are involved in signal transduction mechanisms including the regulation of meiosis, mitosis and post-mitotic phases in differentiated cells [ 52 ]. Many different stimuli can activate the ERK pathway, including growth factors, cytokines, virus infection, ligands for heterotrimeric G protein-coupled receptors, transforming agents, and carcinogens. Therefore, it is reported here that both ERK1/2 phosphorylation on peptide-treated cells and specific activation of downstream signaling lead to the promotion of protein synthesis, as documented by the specific induction of the mTOR-dependent p70S6 kinase. It is important to underline how the in vitro peptide treatment of proliferating cells affects the phosphorylation level of kinases, such as ERK1/2, involved in the mitogenesis process. Sure enough, this signal axis is important in mediating the proliferative effect through the increase in glucose-dependent metabolism, as it is widely demonstrated in mTOR-dependent signaling. The activation of the c-Jun N-terminal JNK kinase by peptide regulators also seems to play an important role [ 53 ]. JNK represents a class of cytoplasmic kinases involved in signal transduction pathways activated during stress and increased exposure to oxidant species. Since several reports have stated that most peptides synchronously regulate and sustain antioxidant activity, it is quite interesting to observe that the same molecules promote JNK activation, suggesting a possible mechanism related to detoxification programs which are active on monocytes during intense cell proliferation. It is of interest to highlight ( Figure 2 ) a significant activation of the p70-S6 kinase, whose coordinated signaling is downstream with respect to the activated molecules induced by mitogenic factors. The p70-S6 kinase is activated by mTOR-dependent transduction mechanisms to satisfy the greater glucose-dependent energy demand seen during cell proliferation. JNK activation also reflects this mechanism, with relevance to stress induced activity, such as the ability to counteract the negative effects of increased oxidation and free radical induced cell damage already seen in other cell types. Whereas it would be very important to analytically investigate molecular mechanisms of gene activation to better understand the action of peptides in vivo, it is here reported how most of the investigated Khavinson Peptides are a

Human leukemia monocytic cell line (THP-1) was purchased from ATCC. Cells were grown in RPMI 1640 (Sigma–Aldrich; Merck Millipore, Darmstadt, Germany) supplemented with 10% FBS, 2 mM L-glutamine and 1% Pen–Strep (Thermo Fisher Scientific, Inc., Waltham, MA, USA), and were maintained at 37 °C in a humidified atmosphere of 5% CO 2 in air. Two series of experiments were performed when the cells reached 80% confluence. THP-1 monocytic cells (~2 × 10 5 cells/mL) were plated as a control group w / o treatment or treated overnight with 100 ng/mL per peptide (P1–P5) alone or supplemented by 100 ng/mL lipopolysaccharide (LPS). LPS derived from E. coli was purchased from Sigma–Aldrich, Saint Louis, MO, USA. Subsequently monocytes were collected by centrifuging for 8 min at 1000 rpm and cell extracts were made according to standard procedures Cell extracts and differentiation of THP-1 cells into macrophages were achieved using the methods described by Daigneault et al. [ 56 ]. THP-1 cells (~2 × 10 5 /mL) were incubated with 100ng/mL PMA (Sigma–Aldrich, Saint Louis, MO, USA) for 3 days at 37 °C, in order to induce monocyte–macrophage differentiation. The PMA-containing media was removed, and cells were incubated for a further four days in conventional medium, before treating THP-1-derived macrophages on a time–course experiment (2, 4, 8 and 12 h) with 100 ng/mL of every peptide alone or supplemented with 100 ng/mL LPS, according to a specific time–course. THP-1-derived macrophages were detached incubating into Trypsin-EDTA 1X (Euro Clone) for 10 min at 37 °C. Monocytes and macrophages were counted using 0.4% of Trypan–blue solution (Sigma–Aldrich, Saint Louis, MO, USA) in a Burker chamber and tested for viability. For Amino Acid treatment, a mixture of L-isoleucin, L-leucin, L-alanine and L-valine was used where indicated (Freliver Sport, Dompé Co., L’Aquila, Italy). HUVECs were provided as a donation by the research group of Professor Mario Romano of the University of Chieti. These cells were isolated by collagenase-1 treatment on umbilical veins collected from women undergoing cesarean section in the department of Obstetrics and Gynecology, obtained from randomly selected healthy mothers delivering at the Chieti University Hospital (Chieti, Abruzzo, Italy), using 0.1% collagenase at 37 °C (with ethical clearance and written informed consent) [ 57 ]. Separated cells were grown in appropriate medium (DEM low glucose and supplemented with 10% FCS and 1% Pen–Strep).

Cell cycle investigations were performed according to published protocols [ 58 , 59 ]. Cell samples from the T25 flasks were collected, washed with PBS (phosphate buffered saline) and centrifuged (400× g , 10 min at room temperature, RT). Cell pellets (5 × 10 5 cells/sample) were fixed by adding 500 µL of 70% cold ethanol and then stored at 4 °C. After at least one week, cell pellets were washed, re-suspended and stained by adding 500 µL of a solution containing 50 µg/mL of propidium iodide (PI, Sigma–Aldrich, Milan, Italy) and 200 µg/mL of RNase A (Sigma–Aldrich). Samples were incubated overnight at 4 °C in the dark and then acquired by flow cytometry (FACS Canto II, BD Biosciences, San Jose, CA, USA). PI fluorescence data were collected using linear amplification. Debris was excluded from the analysis by gating on morphological parameters. A minimum of 10 4 events were recorded for each sample. Each sample was prepared and analyzed in triplicate. Data were analyzed using FlowJo X v 10.0.7 (TreeStar, now Becton, Dickinson and Company, Ashland, OR, USA). Aggregates were excluded [ 58 , 59 ], and the percentages of cells in G0/G1, S, and G2/M phases of the cell cycle, as well as apoptotic and necrotic populations, were calculated. In order to identify the activated compartment, THP-1 cells (500 × 10 3 /sample), after centrifugation at 400× g for 10 min, were washed in PBS and stained using the PE-conjugated anti-CD25 (BD Biosciences, San Jose, CA, USA). After a 30-min incubation at 4 °C with the antibody, the cells were washed, re-suspended in 0.5 mL PBS, and 100 × 10 3 events/sample were acquired using a flow cytometer (FACS Canto II; BD Biosciences, San Jose, CA, USA). The gates of CD25+ cells were placed based on the relative unstained sample. A forward scatter area (FSC-A) vs. side scatter area (SSC-A) dot plot was used to identify and gate THP-1 cells. The cytometer setup and tracking module (CS&T) (BD Biosciences, San Jose, CA, USA) was used to test instrument performances and data reproducibility; these were further validated by the acquisition of rainbow beads (BD Biosciences, San Jose, CA, USA) and compensation was calculated using comp beads (BD Biosciences, Franklin Lakes, NJ, USA). Before each analysis, the flow cytometer was cleaned according to the manufacturer’s instructions, and carryover between samples was prevented using the FACSCanto II SIT Flush device. The FACSDiva v6.1.3 or FACSuite v 1.0.6.5230 (BD Biosciences, San Jose, CA, USA) were used to analyze the data after the acquisitions.

Total proteins were extracted from monocytes and macrophages in 50 mM Tris-HCl pH 7.8, 1% Triton X100, 0.1% SDS, 250 mM NaCl, 5 mM EDTA lysis buffer in the presence of the mini protease inhibitor cocktail at 150 µL/mL (Roche Diagnostics, Mannheim, Germany) as described [ 65 ]. Cell lysates were separated on 12% or 15% SDS-PAGE depending on molecular weight of target proteins and transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). Membranes were incubated overnight at 4 °C with specific primary Abs (diluted 1/100, Santa Cruz Biotechnology, Dallas, TX, USA). Antibodies directed against their protein target were purchased from Cell Signaling Technologies (Danvers, MA, USA). Membranes were incubated overnight at 4 °C on a shaker with specific primary antibodies to phospho-SAPK/JNK, phospho-p70 S6 kinase, phospho-p44/42 MAPK (ERK1/2), IFN alpha, and β-actin as a control. Specific secondary goat anti-rabbit IgG-HRP conjugated antibodies (1:2000, Santa Cruz) (dilution according to Cell Signaling Technology specific protocol) were used for detections in all experiments, incubating membranes for one hour at RT with gentle shaking. Immunocomplexes were visualized using the ECL detection system (Amersham Pharmacia Biotech, Little Chalfont, UK). Western Blot densitometry band quantification was by ImageJ software (Rasband, 1997–2014).

Concentration of several cytokines, released during biopeptide treatment, was determined using flow cytometry (Becton Dickinson, NJ, USA) [ 65 , 66 ]. The release of cytokines (IL-2, IL-4, IL-6, IL-10, TNF, INF-γ, IL17-A) in supernatants was measured by flow cytometry using the commercial Cytometric Bead Array (CBA) Human Th1/Th2/Th17 Cytokine Kit (BD Bioscience catalogue 560484), following the manufacturer’s instructions. Samples acquisition was performed on FACSVerse (BD Biosciences) with FACSuite v 1.0.6.5230 (BD Biosciences) and data analysis by FCAP Array Software v3.0 (BD Bioscience) as suggested.

After removing the unbound monocytes with PBS (3×), the HUVEC and bound monocytes were fixed in paraformaldehyde (2%) for 30′ and then wash in PBS (3×) After nuclear counterstain using DAPI cells were visualized using a ZEISS LSM 800 confocal laser scanning microscope [ 67 ].