Caulerpin Mitigates Helicobacter pylori-Induced Inflammation via Formyl Peptide Receptors

The identification of novel strategies to control Helicobacter pylori (Hp)-associated chronic inflammation is, at present, a considerable challenge. Here, we attempt to combat this issue by modulating the innate immune response, targeting formyl peptide receptors (FPRs), G-protein coupled receptors that play key roles in both the regulation and the resolution of the innate inflammatory response. Specifically, we investigated, in vitro, whether Caulerpin—a bis-indole alkaloid isolated from algae of the genus Caulerpa—could act as a molecular antagonist scaffold of FPRs. We showed that Caulerpin significantly reduces the immune response against Hp culture filtrate, by reverting the FPR2-related signaling cascade and thus counteracting the inflammatory reaction triggered by Hp peptide Hp(2–20). Our study suggests Caulerpin to be a promising therapeutic or adjuvant agent for the attenuation of inflammation triggered by Hp infection, as well as its related adverse clinical outcomes.


Introduction
Helicobacter pylori (H. pylori) is a Gram-negative bacterium colonizing the gastric mucosa of over 50% of the humans worldwide [1]. Despite the fact that the infection is often asymptomatic, H. pylori represents one of the primary causes of gastric cancer, contributing to 75% of all gastric cancer cases [2,3]. While remaining a local pathogen, H. pylori may exert systemic effects and contribute to the occurrence of clinical extra-gastric manifestations. H. pylori infection, in fact, has been reported to increase the risk of iron deficiency anemia, as well as neurological, cardiovascular, dermatological and metabolic disorders [4][5][6][7]. The clinical outcomes of H. pylori infection depend on the complex relationship between host and bacterium [8,9]. The bacterium virulence, the host response, together with environmental factors contribute to the ability of H. pylori to colonize the harsh gastric environment over a long period, thus promoting long-term inflammation, a key feature for the development of severe gastric or extra-gastric diseases [10].
Inflammation is a defensive response triggered by the host innate immune system, in order to survive during an infection or an injury, favoring a return to homeostasis [11][12][13].
However, when prolonged, inflammation may cause more damage to the host than the pathogen [14], inducing intracellular metabolic changes and epigenetics modifications [15]. The immune system coordinates the inflammatory response through innate receptors, also known as pattern recognition receptors (PRRs), able to recognize highly conserved microbial structures [16].
Formyl peptide receptors (FPRs) are pattern recognition receptors (PRRs) belonging to the family of G i -protein coupled receptors, comprising FPR1, FPR2 and FPR3 [17,18]. Even though the nature of the evolutionary process responsible for FPRs' differentiation is poorly understood, it is clear that it originates from a common ancestor and that they acquire functional differences through gene duplication and natural selection [19]. As PRRs, FPRs trigger cellular defense mechanisms, by sensing pathogen associated molecular patterns (PAMPs) [20]. Consequently, they play a critical role in host defense as well as in the regulation of inflammatory processes by participating in the pathogenesis of inflammatory disorders [21,22]. FPRs can detect both bacteria and host mitochondria-derived formylated peptides. In addition, FPRs, and specifically FPR2, can respond to a large variety of structurally different ligands, including not-formylated peptides.
The recent return to traditional medicine and natural drugs has increased the interest in marine natural products with potential pharmacological activity. Moreover, the current problem of marine biological invasion and their resulting biological, economic and social impact [28] have prompted the investigation of the biological activities of the natural products present in some of the most invasive species. In particular, Caulerpa cylindracea, a green macroalga native to South Western Australia and invasive in the Mediterranean Sea, has been found to contain high levels of the bis-indole alkaloid Caulerpin (Cau), showing anti-inflammatory and antioxidant activity [29][30][31]. The indole nucleus is a promising scaffold for the discovery of new anti-inflammatory and antinociceptive drugs, as it provides suitable ligands for G-protein coupled receptors [32,33].
We investigated, for the first time, whether Cau could exert an anti-inflammatory role in the context of H. pylori infection. We found that Cau inhibits FPR2, thus reverting the Hp(2-20)-induced signaling cascade. Our findings suggest a prospective therapeutic application of Cau as an adjuvant to control H. pylori-associated chronic inflammation and to prevent its related adverse effects. Nevertheless, based on the key role of FPRs in inflammatory disorders and of indole nucleus as "privileged structure" in drug discovery, Cau could represent a potential competitive alternative to classical anti-inflammatory approaches by preventing long-term inflammatory damage.

The Effect of Cau on Cell Viability
First, we evaluated the effect of Cau on the metabolic activity of AGS gastric adenocarcinoma epithelial cells, using an MTT (3-4,5-dimethylthiazol 2,5-diphenyltetrazolium bromide solution) assay. AGS cells were cultured with different concentrations of Cau (spanning from 1.6 µM to 405 µM) for 24 h. As shown in Figure 1, Cau was not toxic up to  45 µM, since cell viability was higher than 80% compared to the untreated control. More specifically, we determined CC 50 and observed that the concentration of Cau that caused a 50% decrease of cell viability was 61.20 µM.

Cau Prevents Hp(2-20)-Induced Inflammatory Response in Macrophages
Monocyte-derived macrophage recruitment is an important hallmark during inflammation [36]. According to different stimuli, macrophages change their polarization from M1 (classical activated macrophages) to M2 (alternatively activated macrophages) and vice versa, exhibiting either a pro-inflammatory or anti-inflammatory phenotype, respectively [37]. Previous results (Figures 2A and 3) demonstrated that Hp(2-20)-stimulated cells up-regulated the expression of CCL2, CCL3 and TNF-α genes, which are known to induce the macrophage pro-inflammatory phenotype [38,39]. Therefore, we evaluated the production of IL-1β and TNF-α-pro-inflammatory cytokines released by classically activated monocyte-derived macrophages-in THP-1 macrophages, cultured with the conditioned medium from AGS cells stimulated with Hp(2-20) either treated with Cau or left untreated. The THP-1 macrophages were incubated with the AGS conditioned medium for up to 24 h and their medium was collected at different time points, in order to measure the concentration of IL-1β and TNF-α using an ELISA assay, which normalized to the concentration of IL-1β and TNF-α contained in the conditioned medium of AGS cells that were differently stimulated. Figure 4

Cau Acts as FPR2 Inhibitor
To confirm whether Cau acts on FPR2, inhibiting Hp(2-20) downstream signaling, we first examined FPR2 gene expression in response to Cau by performing quantitative real time PCR (qPCR). Figure 7A shows a similar trend in mRNA levels of FPR2 by WRW4 and Cau. Specifically, neither WRW4 nor Cau were found to activate FPR2. Interestingly, All samples were normalized to GAPDH as reference housekeeping gene. Furthermore, relative gene expression was normalized to basal activity (untreated control), in order to obtain relative fold expression. Graphs report the results of at least three independent experiments, represented as means ± SD. Statistical analysis was performed by GraphPad Prisma software, using one-way ANOVA followed by Bonferroni post hoc for multi-comparison (more than two groups) or student's t test for single-comparison (two groups). ** p < 0.001; *** p < 0.0001.

Cau-FPR2 Interaction: Predictive Computational Studies
Finally, predictive molecular modeling studies were performed to investigate the interaction between Cau and FPR2. As shown in Figure 8A, Cau fits well with FPR2 ligand binding domain, occupying a small area of the receptor binding pocket. In addition, a partial overlap between WKYMV (FPR2 agonist) and Cau was predicted ( Figure 8B). Cau was found to form hydrophobic interactions with FPR2 amino acids involved in hydrophobic interactions with WKYMV ( Figure 8C). Specifically, Cau interacts with the amino acids (Phe257, Asp281, Asn285 and Arg201, Arg205) showing a critical role in ligand binding and formation of hydrogen bounds for FPR2 [48]. Despite the limits of this predictive approach, these data provide further evidence of the direct binding of Cau with FPR2.

Cau: A More Potent Anti-Inflammatory Molecule Than Indomethacin
Indomethacin (Indo) is a member of the non-steroidal anti-inflammatory drugs (NSADs) class, used to treat inflammation and pain. Alongside the essential role of indomethacin in inhibiting prostaglandins synthesis, additional mechanisms could explain its potency. Both indomethacin and Cau possess the indole scaffold in their structure. More specifically, Cau presents an additional indole ring to indomethacin ( Figure 9A). The structural similarity and the capability of indomethacin in suppressing formyl-peptides induced cell migration [49] led us to examine the potential interaction between indomethacin and FPRs. Indomethacin pretreatment, indeed, was found to modulate FPR2 induction triggered by Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), as well as in cells pretreated with Cau ( Figure 9B). These data provide evidence about the role of indomethacin on FPRs. We also observed a more potent anti-inflammatory effect for Cau than indomethacin. Figure 9C, shows a higher decrease in cytokines (MIP-1β, IL-8 and TNF-α) by Cau than indomethacin in cells stimulated with Hpcf.

Cau: A More Potent Anti-Inflammatory Molecule than Indomethacin
Indomethacin (Indo) is a member of the non-steroidal anti-inflammatory drugs (NSADs) class, used to treat inflammation and pain. Alongside the essential role of indomethacin in inhibiting prostaglandins synthesis, additional mechanisms could explain its potency. Both indomethacin and Cau possess the indole scaffold in their structure. More specifically, Cau presents an additional indole ring to indomethacin ( Figure 9A). The structural similarity and the capability of indomethacin in suppressing formyl-peptides induced cell migration [49] led us to examine the potential interaction between indomethacin and FPRs. Indomethacin pretreatment, indeed, was found to modulate FPR2 induction triggered by Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20), as well as in cells pretreated with Cau ( Figure 9B). These data provide evidence about the role of indomethacin on FPRs. We also observed a more potent anti-inflammatory effect for Cau than indomethacin. Figure 9C, shows a higher decrease in cytokines (MIP-1β, IL-8 and TNF-α) by Cau than indomethacin in cells stimulated with Hpcf.

Discussion
Helicobacter pylori is one of the most common human-colonizing bacteria, and its resultant infection can promote chronic inflammation. It is usually acquired during early childhood [50], remaining asymptomatic for long time. The significant capability of the bacterium in evading the host immune system and developing strategies to resist the common antimicrobial therapy means H. pylori is able to persist for decades in the harsh gastric environment, establishing lifelong chronic inflammation, which leads to severe clinical outcomes [1,9]. Strategies to control chronic inflammation, by modulating the immune system, may represent a promising approach to improve H. pylori clinical outcomes and counteract chronic diseases.
In the present study, we focused our attention on FPRs, proposing FPRs as a novel target to ameliorate the detrimental effects derived from H. pylori-induced chronic Values were normalized to basal activity (CTR) and represent mean ± SD of at least three independent experiments, each performed in triplicate. Statistical analysis was performed by GraphPad Prisma software, using one-way ANOVA followed by Bonferroni post hoc correction. ** p < 0.001; *** p < 0.0001.

Discussion
Helicobacter pylori is one of the most common human-colonizing bacteria, and its resultant infection can promote chronic inflammation. It is usually acquired during early childhood [50], remaining asymptomatic for long time. The significant capability of the bacterium in evading the host immune system and developing strategies to resist the common antimicrobial therapy means H. pylori is able to persist for decades in the harsh gastric environment, establishing lifelong chronic inflammation, which leads to severe clinical outcomes [1,9]. Strategies to control chronic inflammation, by modulating the immune system, may represent a promising approach to improve H. pylori clinical outcomes and counteract chronic diseases.
In the present study, we focused our attention on FPRs, proposing FPRs as a novel target to ameliorate the detrimental effects derived from H. pylori-induced chronic inflammation. In particular, we investigated the potential capability of Cau to act as an attractive target for FPRs, inhibiting Hp(2-20) signaling pathway. The choice to use Cau in this study was based on its particular chemical structure, characterized by two indole nuclei. Indole, in fact, has been considered the most privileged scaffold in drug discovery [51,52] because of its anti-inflammatory, anti-cancer, antioxidant, anti-diabetic, antimicrobial, antiviral and anti-hypertensive roles [32,51,53]. In addition, the presence of two indole nuclei makes Cau similar to W-rich peptides, such as WRW 4 , which was found to interact with FPR2, exerting antagonistic effects [54].
For the first time, our results show the potential capability of Cau in antagonizing FPR2, as well as WRW 4 . Cau was tested as a probable target for all FPRs. However, it was found to distinguish between the three FPRs, interacting selectively with FPR2. The unique characteristic of indole rings in transferring electrons and favoring amino acids receptor reaction makes them effective components of the molecule. Specifically, Cau was observed to occupy the FPR2 binding pocket, forming a hydrophobic environment that could contribute to the stabilization of the receptor interaction, potentially competing with other ligands. This view was supported by the finding that Cau limited Hp(2-20)-induced cellular responses, via the inhibiting oxidative burst, chemotaxis and pro-inflammatory cytokines release.
Finally, we compared Cau with indomethacin, a traditional anti-inflammatory drug containing one indole nucleus. Extraordinarily, our data revealed that indomethacin may modulate the expression of FPR2, thus elucidating its potential pharmacological effect, which is still poorly understood. Nevertheless, Cau displayed a greater inhibitory effect against FPR2 than indomethacin and, at the same time, a more effective anti-inflammatory role. These results highlight the association between FPRs and inflammatory conditions and the importance of indole as a scaffold for anti-inflammatory drugs, by targeting FPRs.

Cau Extraction and Purification
Caulerpa cylindracea was collected in Italy in the Gulf of Pozzuoli and exhaustively extracted with acetone at room temperature, as reported by Magliozzi et al. [75]. Briefly, the acetone extract was evaporated at a reduced pressure and the residual water was extracted with diethyl ether. The diethyl ether extract was first fractionated on Sephadex column (CHCl 3 /MeOH; 1:1) and the obtained fraction was further purified by silica-gel column chromatography (gradient of light petroleum ether/Et 2 O, as eluent) to produce pure Cau, identified by comparing 1 H-and 13 C-NMR spectroscopic data with the literature [76,77]. Size-exclusion chromatography was achieved using Sephadex LH-20 column, whereas silica-gel column chromatography was performed using Merck Kieselgel 60 powder. NMR data were recorded on a Bruker Avance-400 spectrometer using an inverse probe fitted with a gradient along the z axis.

Cell Viability Assay
The effects of Cau on AGS cells were assessed by performing MTT assay. Briefly, AGS cells were seeded at a density of 2 × 10 3 per well in a 96-well plate and incubated at 37 • C in a 5% CO 2 atmosphere overnight. After cell attachment, the medium was replaced with fresh medium containing different concentrations of Cau and cells were incubated for 24 h. Twenty µL of 3-4,5-dimethylthiazol 2,5-diphenyltetrazolium bromide solution (MTT) were added to each well and cells were further incubated at 37 • C in a 5% CO 2 atmosphere for 3 h. Finally, the medium was removed, and the resultant formazan crystals were dissolved in 200 µL of DMSO. Absorbance was recorded at 570 nm using an EnVision 2102 multilabel reader (PerkinElmer, Waltham, MA, USA). Cell viability was calculated as the ratio between the mean absorbance of the sample and the mean absorbance of the untreated cells and expressed in percentage.

In Vitro Scratch Assay
AGS cells migration was tested by performing in vitro scratch assay, as described by de Paulis et al. [61]. Briefly, confluent monolayers of cells were treated with mitomycin for 2 h (2 µg/mL) to inhibit cell growth. Monolayers were then scratched using a pipette tip, in order to create a gap. After scratching, medium and cell debris were removed, and cells were washed with a fresh medium and incubated for 12 h with Hp(2-20) with or without Cau pretreatment.

RNA Extraction and Quantitative Real-Time PCR
AGS cells were seeded at a density of 0.5 × 10 6 per well in 12-well plates to analyze the expression profiling of (1) cytokine and chemokine genes; (2) FPRs genes; (3) mitochondrial superoxide dismutase (SOD2) gene and (4) p53 gene at different times post-inflammatory stimulus, represented by Hp(2-20) (synthesized by Innovagen, Lund, Sweden) or Hpcf, in the presence or absence of the Cau pretreatment. Total RNA was extracted from individual wells by PureLink ® RNA Mini Kit (ThermoFisher Scientific, Waltham, MA, USA), according to the manufacturer instructions. Genomic DNA was removed by digestion with DNase I, Amplification Grade (TermoFisher Scientific). Extracted RNA was quantified and analyzed for purity, using Nanodrop-ND 1000 spectrophotometer (TermoFisher Scientific) and finally reverse-transcribed using the high-capacity cDNA Reverse transcription kit (Applied Biosystem, Bedford, MA, USA). Gene transcript levels were measured using TaqMan PCR master 2× reagent or Power SYBR ® Green PCR Master Mix (Applied Biosystem ® ) on a StepOne ™ Real-Time PCR System (Applied Biosystem ® ), according to the standardmode thermal cycling conditions, according to the manufacturer's protocol. The relative expression level of analyzed genes was determined using probes or primers reported in the Supplementary Material, Table S1. All samples were normalized to GAPDH as the reference housekeeping gene and the relative quantitative expression was determined using the 2 −∆∆Ct method [78][79][80].

Measurement of Cytokines Production in Macrophages
Opportunely differentiated THP-1 cells were seeded at a density of 0.5 × 10 6 per well in 12-well plates and stimulated with AGS-conditioned medium for 24 h. AGS-conditioned medium was prepared by seeding 2 × 10 6 cells per well in 6-well plates. After treatment with the pro-inflammatory stimulus Hp(2-20), preceded or not by Cau pre-treatment, the medium was collected and filtered by passage in a 0.22 µm filter (Sigma Aldrich). Supernatants of THP-1 cells were collected at different times post-stimulation with AGSconditioned medium and stored at −80 • C until use. Secretion of IL-1β and TNF-α was detected by Human ELISA kit (Abcam, Waltham, MA, USA), according to the manufacturer instructions.

Intracellular ROS Measurement
AGS cells were split at 80-90% of confluency, seeded (0.5 × 10 6 ) in 35 mm culture dishes, and incubated at 37 • C in a 5% CO 2 atmosphere overnight. After cell attachment, ROS detection assay was assayed using dihydrorhodamine 123 (DHR; Sigma Aldrich, Missouri, USA), as described by Cuomo et al. [81]. Briefly, cells were preloaded with 10 µM DHR for 20 min and treated as detailed in the figure legend. After treatments, DAPI (Thermo Fischer Scientific) was used as a nuclear counterstain, and cells were analyzed with a Zeiss Axioskop 2 Hal100 fluorescence microscope equipped with a digital camera (Nikon). The excitation and emission wavelengths were 488 and 515 nm, respectively. Images were digitally acquired with exposure times of 100-400 ms and processed for fluorescence determination with ImageJ software version 2.1.0/1.53c.

Western Blotting for Protein Studies
AGS cells were split at 80-90% of confluency, seeded (2 × 10 6 per well) in 6-well plates and incubated at 37 • C in a 5% CO 2 atmosphere until cell attachment. Cells were serum-starved 12-16 h prior the stimulation, using serum-free DMEM containing 0.25% BSA and incubated at 37 • C in a CO 2 incubator. After treatments, cells were washed and harvested using RIPA buffer (50 mM Tris-HCl pH 8.8, 150 mM NaCl, 0.1% SDS, 0.5% NP-40, 0.5% DOC; protease and phosphatase inhibitor cocktail, Sigma-Aldrich), incubated for 20 min at 4 • C and centrifugated at 10,000× g for 15 min. Pellets were discarded and cell lysates were stored at −80 • C until use. Proteins (30 µg/lane, in Sample Buffer: 4× Laemmli Sample Buffer #1610747) were separated in 7.5-15% SDS-polyacrylamide gel and then transferred to a nitrocellulose membrane (Amersham™ Nitrocellulose Western blotting membranes 0.2um) by electrotransfer. Briefly, filters were blocked for 1 h at room temperature in 5% (w/v) non-fat milk in Tris-buffered saline Tween-20 (TBST: 0.1% Tween, 150 mM NaCl, 10 mM Tris-HCl, pH 7.5) and probed with antibodies as reported in the Supplementary Material, Table S2. After several washings in TBST, membranes were incubated with the appropriate secondary antibodies (Supplementary Material, Table S2). Finally, immunoreactive proteins were visualized with enhanced chemiluminescence (Amersham International, Buckinghamshire, UK). The blots were stripped using a stripping buffer and then re-probed to detect the total protein of interest. Each Western blot band was quantified using Image Lab software version 6.0 (Bio-Rad, Hercules, CA, USA).

Cytokines Assay
AGS cells were split at 80-90% of confluency, seeded (2 × 10 6 per well) in 6-well plates and incubated at 37 • C in a 5% CO 2 atmosphere until cell attachment. Cells were serumstarved 12-16 h prior the stimulation, using serum-free DMEM containing 0.25% BSA and incubated at 37 • C in a CO 2 incubator. After 24 h of treatment, medium was collected and centrifugated at 10,000× g to remove debris and dead cells and analysed for the concentration of IL-8, G-CSF, TNF-α and MIP-1β, by using the Bioplex Multiplex human cytokine assay (Bio-Rad), as indicated by manufacturer's instructions. As a multiplexed assay, the Bioplex assay can simultaneously detect more analytes in a single sample [82].

Molecular Modeling
To predict and characterize the potential interaction of Cau with FPR2, computational studies were assessed. A 3D structure model of FPR2 was downloaded from Protein Data Bank (RCSB PDB), using the crystal structure of the complex FPR2-WKYMVm (PDBcode: 6LW5) as a model. The WKYMV ligand was successively removed by PyMOL Molecular Graphic System (Version 1.3 Shrodinger, LLC, New York, NY, USA), obtaining the receptor pdb file. The initial 3D conformation of Cau was obtained from PubChem (ncbi library). The receptor and ligand were then adapted for docking with the AutoDock Tools. Docking analysis was carried out with AutoDock Vina (Trott and Olson, 2010), setting the grid box at 26Å × 40Å × 32Å for the receptor. In conclusion, molecular details of Cau recognition by FPR2 were analyzed with PyMOL Molecular Graphic System (Version 1.3 Shrodinger, LLC).

Statistical Analysis
Statistical analysis was performed using GraphPad Prism 8.0 software, San Diego, CA, USA. All data were compared using One-way ANOVA followed by Bonferroni's multiple comparisons test, in order to compare different groups. Experimental data are presented as mean ± SD of at least three independent experiments, each performed in triplicate. Lastly, p values < 0.05 were considered statistically significant.

Conclusions
In conclusion, the current study aimed to provide in vitro proofs on the role of FPRs in the pathogenesis of H. pylori-associated chronic inflammation, by interacting with the H. pylori-released peptide Hp (2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Concurrently, it demonstrated the impressive effects of Cau on health, by targeting FPR2, thus controlling the H. pylori-associated chronic inflammation and related disorders. Taken together, our results suggest the potential clinical application of Cau for the control of numerous inflammatory disorders, which are among the main health problems occurring today. Nevertheless, future studies are required to validate our in vitro findings in vivo.

Data Availability Statement:
The data presented in this study are available within the article.