Up-Regulation of the Nrf2/HO-1 Antioxidant Pathway in Macrophages by an Extract from a New Halophilic Archaea Isolated in Odiel Saltworks

Citation: Ávila-Román, J.; Gómez- Villegas, P.; de Carvalho, C.C.C.R.; Vigara, J.; Motilva, V.; León, R.; Talero, E. Up-Regulation of the Nrf2/HO-1 Antioxidant Pathway in Macrophages by an Extract from a New Halophilic Archaea Isolated in Odiel Saltworks. Antioxidants 2023, 12, 1080. https://doi.org/10.3390/ antiox12051080 Academic Editors: Susana M. Cardoso, Dulcineia Ferreira Wessel and Marcelo D. Catarino Received: 30 March 2023 Revised: 4 May 2023 Accepted: 8 May 2023 Published: 11 May 2023 Copyright: © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). antioxidants Article Up-Regulation of the Nrf2/HO-1 Antioxidant Pathway in Macrophages by an Extract from a New Halophilic Archaea Isolated in Odiel Saltworks Javier Ávila-Román1,*,† , Patricia Gómez-Villegas 2,3,† , Carla C. C. R. de Carvalho 3,4 , Javier Vigara 2, Virginia Motilva 1, Rosa León2and Elena Talero 1 1 Department of Pharmacology, Faculty of Pharmacy, Universidad de Sevilla, Profesor García González Street, 41012 Seville, Spain 2 Laboratory of Biochemistry, Center for Natural Resources, Health, and Environment, Universidad de Huelva, Avda. de las Fuerzas Armadas s/n, 21071 Huelva, Spain 3 iBB—Institute for Bioengineering and Biosciences, Department of Bioengineering, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal 4Associate Laboratory i4HB—Institute for Health and Bioeconomy, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049-001 Lisbon, Portugal *Correspondence: [email protected]; Tel.: +34-954559880 † These authors contributed equally to this work. Abstract: The production of reactive oxygen species (ROS) plays an important role in the progression of many inflammatory diseases. The search for antioxidants with the ability for scavenging free radicals from the body cells that reduce oxidative damage is essential to prevent and treat these pathologies. Haloarchaea are extremely halophilic microorganisms that inhabit hypersaline environments, such as saltworks or salt lakes, where they have to tolerate high salinity, and elevated ultraviolet (UV) and infrared radiations. To cope with these extreme conditions, haloarchaea have developed singular mechanisms to maintain an osmotic balance with the medium, and are endowed with unique compounds, not found in other species, with bioactive properties that have not been fully explored. This study aims to assess the potential of haloarchaea as a new source of natural antioxidant and anti-inflammatory agents. A carotenoid-producing haloarchaea was isolated from Odiel Saltworks (OS) and identified on the basis of its 16S rRNA coding gene sequence as a new strain belonging to the genus Haloarcula. The Haloarcula sp. OS acetone extract (HAE) obtained from the biomass contained bacterioruberin and mainly C18 fatty acids, and showed potent antioxidant capacity using ABTS assay. This study further demonstrates, for the first time, that pretreatment with HAE of lipopolysaccharide (LPS)-stimulated macrophages results in a reduction in ROS production, a decrease in the pro-inflammatory cytokines TNF- α and IL-6 levels, and up-regulation of the factor Nrf2 and its target gene heme oxygenase-1 (HO-1), supporting the potential of the HAE as a therapeutic agent in the treatment of oxidative stress-related inflammatory diseases. Keywords: bacterioruberin; carotenoids; fatty acids; haloarchaea; inflammation; macrophages; Odiel Saltworks; oxidative stress 1. Introduction Many pathologies such as inflammatory bowel disease, psoriasis, and rheumatoid arthritis have an inflammatory base, triggered in part by a deficient antioxidant system that leads to ROS overproduction and chronic inflammation. Impairment of the antioxidant defense systems causes the overaccumulation of these oxidative species, which produces oxidative damage in lipids, proteins, and DNA, inducing cell death [ 1 ]. For this reason, new sources of antioxidants are desirable, not only to treat chronic diseases or promote health but also to be used as additives for food and pharmaceutical products, in order to prevent their deterioration and extend their shelf-life. Most natural antioxidants ingested by humans come from plants; however, microorganisms can also be a source of Antioxidants 2023,12, 1080. https://doi.org/10.3390/antiox12051080 https://www.mdpi.com/journal/antioxidants Antioxidants 2023,12, 1080 2 of 15 these compounds to substitute undesirable artificial ones [ 2 , 3 ]. In this regard, extremely halophilic microorganisms that inhabit hypersaline environments, such as saltworks or salt lakes, are particularly interesting due to their ability to cope with extreme conditions. High salinity is the main hindrance found in these habitats, but other extreme conditions, including high ultraviolet (UV) and infrared radiations from the sun, must also be endured. Halophilic archaea are usually the main representatives of the microbiota at the highest salinity ( ≥ 5 M NaCl), exceeding Bacteria and Eukarya [ 4 , 5 ]. Haloarchaea possess specific strategies to proliferate in such harsh environments, which include the accumulation of isomolar concentrations of KCl to balance the osmotic pressure with the surrounding medium, and an adapted cellular machinery able to work at high salt concentrations [ 6 ]. In addition, Archaea count with unique cell membranes, which contain isoprenoids ether linked to sn-glycerol-1-phosphate, contrarily to bacterial and eukaryotic cells where the fatty acid chains are ester linked to the sn-glycerol-3-phosphate backbone. This lipid structure is totally different from that found in the membrane of bacteria or eukaryotes and is one of the structural characteristics that supported the definition of Archaea as an independent domain of life [ 7 ]. Previous studies have shown that several archaeal species contain fatty acids (FAs) and phospholipid FAs [ 8 – 11 ]; nonetheless, there is practically no information about the physiological role and the composition of haloarchaeal FAs. Most members of the class Halobacteria are pink or red pigmented due to the presence of carotenoids and retinal proteins in their membranes [ 12 ]. Carotenoids are one of the most abundant bioactive compounds in the marine environment and are highly demanded products with many applications in cosmetics, pharmaceutics, nutraceuticals, foods, and beverages [ 13 ]. These pigments are usually obtained from plants, algae, or yeasts, while prokaryotic microbial sources are less exploited. Interestingly, carotenoids obtained from different species of haloarchaea have been reported to exhibit health-promoting properties such as antimicrobial, antihemolytic, neuroprotective, or antiviral, among others [ 14 – 17 ]. The main carotenoid found in haloarchaea is bacterioruberin, a 50-carbon carotenoid with 13 conjugated double bonds and 4 terminal hydroxyl groups, which give the cells exceptional properties against oxidative stressors, acting as a radical scavenger in hypersaline environments [ 18 ]. Bacterioruberin protects the haloarchaea against UV light and could act as a membrane stabilizer, connecting the inner and outer leaflets of the membrane bilayer by the interaction of its hydroxyl groups through hydrogen bonds with the hydrophilic head groups of polar lipids [19]. In the present study, a new strain of haloarchaea belonging to the genus Haloarcula was isolated from Odiel Saltworks, and the antioxidant and anti-inflammatory properties of an acetone extract from this microorganism, containing carotenoids and FA, were evaluated by both in vitro and cellular assays, to explore the molecular mechanisms underlying these activities. 2. Materials and Methods 2.1. Sample Collection and Strain Isolation Haloarchaeal strains were isolated from water samples from a crystallizer pond in Odiel Saltworks (37.255025, − 6.972945), located in the Odiel River Marshlands in Huelva (SW, Spain). Water samples (20 µ L) were plated in agar plates and incubated at 37 ◦ C for 30 days. The culture medium used for the isolation contained per liter: 10 g glucose, 156 g NaCl, 13 g MgCl 2· 6H 2 O, 20 g MgSO 4· 7H 2 O, 1 g CaCl 2· 6H 2 O, 4 g KCl, 0.2 g NaHCO 3 , and 5 g yeast extract. The colony with the most intense red color was purified by five streaking rounds on fresh agar plates and preserved in 20% glycerol (w/v) at − 80 ◦ C for further use. 2.2. Molecular Identification The selected microorganism was identified by 16S ribosomal RNA gene sequencing. Genome DNA was extracted by using the Gene JET Genomic Purification kit (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer’s instructions. The Antioxidants 2023,12, 1080 3 of 15 full length of the 16S rRNA coding gene was amplified with specific primers for archaea 21F (5 0 -TTCCGGTTGATCCTGCCGGA-3 0 ) and 1492R (5 0 -GGTTACCTTGTTACGACTT- 3 0 ). Polymerase chain reaction (PCR) was carried out in a total volume of 25 µ L, using an Eppendorf thermo-cycler. The reaction mixture contained 1 µ L of genomic DNA, 0.2 U RED Taq ® DNA polymerase (Sigma Aldrich, St. Louis, MO, USA), and 2.5 µ L of its specific 10 × buffer that contained 10 pM of each primer, 0.2 mM dNTPs, and 2.5 mM MgCl 2 . The thermal profile was set to 0.5 min at 96 ◦ C, 0.5 min at 56 ◦ C, and 1 min at 72 ◦ C for 30 cycles, followed by 10 min of final extension. The PCR products were checked by electrophoresis, on a 1% agarose gel, and sent to Stabvida (Lisbon, Portugal) for Sanger sequencing. The 1.4 kb 16S rRNA gene sequences obtained were compared to those available at the GenBank and the European Molecular Biology Laboratory (EMBL) databases, using the Basic Local Alignment Search Tool (BLASTn) at the National Center for Biotechnology Information (NCBI) [20]. 2.3. Culture Medium and Extract Preparation The selected microorganism was grown in the liquid medium previously detailed for the strain isolation at 37 ◦ C and 150 rpm for 10 days, and the growth was monitored according to the optical density at 580 nm. When the culture reached the stationary phase of growth, 1 L was collected by centrifugation at 19,800 × gfor 15 min at 4 ◦ C. The cellular pellet was treated overnight with 10 mL of cold acetone ( − 20 ◦ C) per gram of fresh biomass in the dark, and vortex several times until a colorless pellet was obtained. Then, the sample was centrifuged at 4 ◦ C and 19,800 × gfor 30 min, and the supernatant, which contains carotenoids and other lipophilic compounds, was dried in a rotary vacuum evaporator and freeze dried. 2.4. Lipid Analysis The FAs were extracted and simultaneously methylated to fatty acid methyl esters (FAMEs) using the Instant FAME TM kit from MIDI, Inc. (Newark, DE, USA), after acetone removal at 40 ◦ C and 100 mBar on a vacuum evaporation system (RapidVap from Labconco, Kansas City, MO, USA). At least two samples of extract were used per lipid extraction. For lipid quantification, the FA C19:0 (Sigma-Aldrich, Darmstadt, Germany), was added as an internal standard prior to transesterification. The base-catalyzed kit allows the methylation of FA to FAMEs and their extraction to an organic solvent for analysis. The FAMEs were analyzed by gas chromatography on a 6890N gas chromatograph, equipped with a flame ionization detector (FID) and a 7683B series injector, all from Agilent Technologies (Santa Clara, CA, USA). The gas chromatograph was equipped with a 25 m long Agilent J&W Ultra 2 capillary column and was programmed and controlled by the Sherlock software package, version 6.2 (from MIDI, Inc.). Peak identification was carried out by Sherlock Software by comparison with standards. Their identification was confirmed by injection of the same samples on an Agilent 7820A gas chromatograph equipped with a 7693A autoinjector and a 5977E quadrupole mass spectrometer (MS; Agilent Technologies, Santa Clara, CA, USA). A 25 m long Agilent J&W Ultra 2 capillary column was used, and the carrier gas was helium at 1.5 mL/min. 2.5. Carotenoid Analysis The content of total carotenoids in the acetone extract was calculated by the absorbance of the sample at 496 nm in a cuvette with a 1 cm path length and using the specific absorption coefficient A 1% = 2540 (100 mL g −1 cm −1 ) [ 21 ]. The chromatographic analysis of the carotenoids present in the extract was performed in a Merck-Hitachi LaChrom Elite (Prague, The Czech Republic) HPLC equipped with a DAD (L-2455) detector. Separation of carotenoids was performed using an RP-18 column (5 µ m × 25 cm × 4.6 mm id), with an equilibration time of 20 min, and a flow rate of 1 mL min −1 . Solvents were (A) a mixture of acetonitrile/water (9:1 v/v) and (B) pure ethyl acetate. The gradient elution program was as follows: 0–16 min 0–60% A; 16–30 min 60% A; 30–35 min 100% A. The column temperature Antioxidants 2023,12, 1080 4 of 15 was kept at 25 ◦ C, and the injection volume was 100 µ L. Chromatograms were recorded in the range of 450 nm and the EZ ChromeElite program was used for data processing. The compounds were identified on the basis of their retention time and UV–Vis spectra. 2.6. Antioxidant Capacity Assessment The antioxidant activity of the compounds as a scavenger was performed by the ABTS colorimetric method [ 22 ]. The ABTS · + radical was generated with 3.84 mg/mL of ABTS (Applichem, Darmstadt) and 6.6 mg/mL of potassium persulfate (Sigma-Aldrich Química, SA, Spain) dissolved in water and incubated for at least 16 h in the dark. The compounds were prepared into 96-well plates at different concentrations (0–200 µ g/mL) and ABTS · + solution, equivalent to an absorbance of 0.7 ± 0.02, was incubated with samples for 6 min at room temperature. Then, the absorbance was measured at 734 nm by using a Synergy HT multimode microplate reader (BioTek Instruments, Winooski, VT, USA). Trolox was used as the standard antioxidant in the same concentration range. The effective concentration 50% (EC 50 ) was calculated, and results were expressed as Trolox Equivalent Antioxidant Capacity, TEAC (EC50 sample/EC50 Trolox). 2.7. Cell Cultures Human acute monocytic leukemia cell line, THP-1, was purchased from the American Type Culture Collection (TIB-202, ATCC, Manassas, VA, USA) and cultured in RPMI 1640 media (GIBCO ® , Life Technologies, New York, NY, USA) containing 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (PAA®, Pasching, Austria), in a humidified atmosphere containing 5% CO2at 37 ◦C. 2.8. Cell Viability Assay The in vitro cytotoxicity assessment of HAE was determined by the 3-(4,5-dimethylthiazol- 2-yl)-2,5 diphenyltetrazolium bromide (MTT, Calbiochem, Darmstadt, Hesse, Germany) method [ 23 ] by using THP-1 cells transformed into macrophages. Briefly, for differentiation into macrophages, THP-1 cells were seeded into 96-well plates (100 µ L/well) at a density of 10 5 cells/mL in the presence of 8 nM phorbol 12-myristate 13-acetate (PMA, Sigma-Aldrich Química, S.A., Madrid, Spain) and, incubated in a humidified atmosphere of 5% CO 2 at 37 ◦ C for 72 h. Afterward, the medium was removed, and cells were washed twice with ice-cold phosphate saline buffer (PBS, 4 ◦ C). Then, the macrophages were treated with different concentrations of the acetone extract (0, 6.25, 12.5, 25, 50, and 100 µ g/mL) for 24 h prepared in DMSO stock solutions at a concentration of 50 mg/mL and diluted to desired concentration directly in the culture medium. Controls were incubated in a fresh medium containing DMSO (0.2% v/v), which did not affect cell viability. Then, the cells were washed (PBS, 4 ◦ C) prior to the addition of 100 µ L of 0.25 mg/mL MTT solution into each well and were incubated for 4 h. The formazan crystals were dissolved with DMSO (200 µ L) and 0.1 M glycine buffer pH 10.5 (25 µ L). Finally, the absorbance was measured at 550 nm by using a Multiskan EX microplate reader (Labsystems, Thermo Scientific, Helsinki, Finland) and the 50% inhibitory concentration (IC50) was calculated. 2.9. Intracellular ROS Production The production of intracellular ROS was determined by using DCF-DA dye assay (ab113851 Abcam, Cambridge, UK) in THP-1 macrophages. Briefly, THP-1 cells were seeded into a 96-well black plate (100 µ L/well) at a density of 10 5 cells/mL with 8 nM PMA as described above. Then, the medium was removed, and cells were washed (PBS, 4 ◦ C). Subsequently, a pretreatment with the HAE (5, 25, and 50 µ g/mL), and dexamethasone (Dex) as control (1 µ M) was carried out for 1 h. Then, the intracellular ROS production was induced by the addition of lipopolysaccharide from E. coli (LPS, 1 µ g/mL) for 24 h. Control groups, unstimulated (Control) and stimulated (LPS) were incubated with a medium containing DMSO (0.2% v/v). Then, the supernatants were removed, and cells were washed (PBS, 4 ◦ C) supplemented with 20 µ M DCF-DA (100 µ L/well) and incubated for Antioxidants 2023,12, 1080 5 of 15 45 min, according to the manufacturer’s instructions. Fluorescence was measured by using a fluorescence plate reader (Sinergy HT, Biotek ® , Bad Friedrichshall, Germany) at 485 nm for excitation and 535 nm for emission. 2.10. Determination of TNF-αand IL-6 Levels The production of pro-inflammatory cytokines was studied in human THP-1 macrophages. Briefly, THP-1 were seeded into a 96-well plate (100 µ L/well) at a density of 10 5 cells/mL in the presence of 8 nM PMA as described previously. After differentiation, the medium was removed, and cells were washed twice (PBS, 4 ◦ C). Subsequently, a pretreatment with the HAE at different concentrations (5, 25, and 50 µ g/mL) and Dex (1 µ M) as positive reference compound was performed for 1 h. Then, the inflammatory response was induced by the addition of LPS (1 µ g/mL) for 24 h. Control groups were incubated with a growth medium containing DMSO (0.2% v/v). Afterward, the supernatants were collected and stored at − 80 ◦ C until cytokines determination by ELISA (Diaclone GENPROBE, Besançon, France), according to the manufacturer’s protocol. The absorbance was measured with a microplate reader (Labsystems Multiskan EX, Thermo Scientific, Helsinki, Finland) at 450 nm. 2.11. Assessment of Antioxidant Proteins Expression The expression of the antioxidant proteins Nrf2 and HO-1 was determined by Western blotting in THP-1 macrophages. Briefly, THP-1 were seeded into 6-well plates (2000 µ L/well) at a density of 4 × 10 5 cells/mL in the presence of 8 nM PMA as previously described. After the incubation period, the medium was removed, the cells were washed twice (PBS, 4 ◦ C), and incubated with different concentrations of the HAE (5, 25, and 50 µ g/mL) for 1 h. The inflammatory response was induced by the addition of LPS (1 µ g/mL) for 24 h. The Control group was incubated with a growth medium containing DMSO (0.2% v/v). Subsequently, cell pellets were mixed with cold lysis buffer containing a cocktail of protease inhibitors (50 mM Tris-HCl pH 7.5, 8 mM MgCl 2 , 5 mM ethylene glycol bis (2-aminoethyl ether)-N,N,N’N’-tetraacetic acid, 0.5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 0.01 mg/mL leupeptin, 0.01 mg/mL pepstatin, 0.01 mg/mL aprotinin, and 250 mM NaCl (40 µ L per sample) and incubated on ice for 30 min. After that, cell homogenates were centrifugated (12,000 × g, 5 min, 4 ◦ C) to discard cell debris and DNA. The protein cytoplasmic content in the supernatants was determined using the Bradford method. Aliquots of supernatants containing equal protein (50 µ g) were separated on 10% acrylamide gel by SDS-polyacrylamide-gel electrophoresis. Then, the proteins were electrophoretically transferred onto a nitrocellulose membrane and incubated with specific primary antibodies as follows: rabbit anti-Nrf2 (1:1000) and rabbit anti-HO-1 (1:1000) (Cell Signaling, Danvers, MA, USA). Each membrane was washed three times for 10 min and incubated with the secondary horseradish peroxidase-linked anti-rabbit (Pierce Chemical, Rockford, IL, USA). To prove equal loading, the blots were analyzed for β -actin expression by using an antiβ - actin antibody (Sigma-Aldrich, St. Louis, MO, USA). Immunodetection was performed by using a chemiluminescence light-detecting kit (Super-Signal West Pico Chemiluminescent Substrate, Pierce, IL, USA). Densitometric data were analyzed following normalization to the housekeeping gene ( β -actin), and the signals were quantified with Scientific Imaging Systems (Biophotonics Image J Analysis Software; National Institute of Mental Health, Bethesda, MD, USA). 2.12. Statistical Analysis All values in the figures, tables, and text are expressed as arithmetic means ± standard error of the mean (S.E.M.). Data were evaluated by using the GraphPad Prism Version 6.00 software (GraphPad Software, Inc., San Diego, CA, USA). The Kolmogorov– Smirnov test was used to verify the normality of the data. pvalues < 0.05 were considered statistically significant. The statistical test used for individual analyses is provided in the figure legends. Antioxidants 2023,12, 1080 6 of 15 3. Results 3.1. Selection and Identification of a Carotenoid-Rich Haloarchaea A new strain of haloarchaea was isolated from a crystallizer pond in Odiel Saltworks with 32% salinity at the moment of sampling. This microorganism was selected for its fast growth ( µ = 1.02 days −1 ), as shown in Supplementary Materials (Figure S1), and intense red color. The identification of the isolate was carried out based on the amplification and sequencing of the complete 16S rRNA encoding gene (Supplementary Materials, Figure S2). The obtained sequence was compared to those available in the NCBI database using the BLASTn tool, showing around 97.5% sequence identity with other Haloarcula species, such as H. hispanica,H. japonica, or H. marismortui. In this regard, multiple alignments were generated by Multiple Sequence Comparison by Log-Expectation (MUSCLE). Thus, it was designated as Haloarcula sp. OS. A molecular phylogenetic tree was constructed by using the Molecular Evolutionary Genetics Analysis (MEGA X, Version 10, Sudhir Kumar, Pennsylvania State University, US) [ 24 ], including the new isolate Haloarcula sp. OS and a series of reference haloarchaeal species. The bootstrap was settled on 1000 replicates, and the thermophilic archaea Methanococcus vulcanus was set as an outgroup. The 16S rRNA encoding sequence of the new isolate clustered with the corresponding genes of the members of the genus Haloarcula (Figure 1). Antioxidants 2023, 12, x FOR PEER REVIEW 6 of 17 following normalization to the housekeeping gene (β-actin), and the signals were quantified with Scientific Imaging Systems (Biophotonics Image J Analysis Software; National Institute of Mental Health, Bethesda, MD, USA). 2.12. Statistical Analysis All values in the figures, tables, and text are expressed as arithmetic means ± standard error of the mean (S.E.M.). Data were evaluated by using the GraphPad Prism Version 6.00 software (GraphPad Software, Inc., San Diego, CA, USA). The Kolmogorov–Smirnov test was used to verify the normality of the data. p values < 0.05 were considered statistically significant. The statistical test used for individual analyses is provided in the figure legends. 3. Results 3.1. Selection and Identification of a Carotenoid-Rich Haloarchaea A new strain of haloarchaea was isolated from a crystallizer pond in Odiel Saltworks with 32% salinity at the moment of sampling. This microorganism was selected for its fast growth (µ = 1.02 days −1 ), as shown in Supplementary Materials (Figure S1), and intense red color. The identification of the isolate was carried out based on the amplification and sequencing of the complete 16S rRNA encoding gene (Supplementary Materials, Figure S2). The obtained sequence was compared to those available in the NCBI database using the BLASTn tool, showing around 97.5% sequence identity with other Haloarcula species, such as H. hispanica, H. japonica, or H. marismortui. In this regard, multiple alignments were generated by Multiple Sequence Comparison by Log-Expectation (MUSCLE). Thus, it was designated as Haloarcula sp. OS. A molecular phylogenetic tree was constructed by using the Molecular Evolutionary Genetics Analysis (MEGA X, Version 10, Sudhir Kumar, Pennsylvania State University, US) [24], including the new isolate Haloarcula sp. OS and a series of reference haloarchaeal species. The bootstrap was settled on 1000 replicates, and the thermophilic archaea Methanococcus vulcanus was set as an outgroup. The 16S rRNA encoding sequence of the new isolate clustered with the corresponding genes of the members of the genus Haloarcula (Figure 1). Figure 1. Molecular phylogenetic analysis by the maximum likelihood method. The tree represents a comparison within 16S rRNA coding gene sequences, including a series of reference haloarchaeal species and the new isolate, Haloarcula sp. OS, highlighted by an asterisk. The tree was constructed with MEGA X. The numbers at nodes indicate the bootstrap values calculated for 1000 replicates. Figure 1. Molecular phylogenetic analysis by the maximum likelihood method. The tree represents a comparison within 16S rRNA coding gene sequences, including a series of reference haloarchaeal species and the new isolate, Haloarcula sp. OS, highlighted by an asterisk. The tree was constructed with MEGA X. The numbers at nodes indicate the bootstrap values calculated for 1000 replicates. The name and the NCBI access number are indicated for all the reference sequences. Methanococcus vulcanus was used as an outgroup. 3.2. Characterization of the Haloarcula sp. OS Acetone Extract An acetone extract was obtained from Haloarcula sp. OS (HAE) with the aim of testing its potential as an antioxidant and anti-inflammatory agent. The carotenoid and FA content of this extract was analyzed by RP-HPLC-DAD and GC-MS, respectively. The elution profile of the carotenoids revealed three main peaks with identical absorption spectra with absorption maxima at 468, 496, and 532 nm and two cis absorption maxima at lower wavelengths (Figure 2), which correspond to different bacterioruberin isomers. The concentration of total bacterioruberin, the only carotenoid detected in the HAE, was 10 µ g per mg of extract, calculated according to Britton (2004) [ 21 ] and using the specific absorption coefficient A1% = 2540 (100 mL g−1cm−1). Antioxidants 2023,12, 1080 7 of 15 Antioxidants 2023, 12, x FOR PEER REVIEW 8 of 17 3.2. Characterization of the Haloarcula sp. OS Acetone Extract An acetone extract was obtained from Haloarcula sp. OS (HAE) with the aim of testing its potential as an antioxidant and anti-inflammatory agent. The carotenoid and FA content of this extract was analyzed by RP-HPLC-DAD and GC-MS, respectively. The elution profile of the carotenoids revealed three main peaks with identical absorption spectra with absorption maxima at 468, 496, and 532 nm and two cis absorption maxima at lower wavelengths (Figure 2), which correspond to different bacterioruberin isomers. The concentration of total bacterioruberin, the only carotenoid detected in the HAE, was 10 µg per mg of extract, calculated according to Britton (2004) [21] and using the specific absorption coefficient A1% = 2540 (100 mL g−1 cm−1). Figure 2. HPLC elution profile of the Haloarcula sp. OS acetone extract (HAE). The three prominent peaks observed in the chromatogram correspond to bacterioruberin isomers with the same absorption spectrum, which is shown on the insert. This UV–Vis spectrum with absorption maxima at 468, 496, and 532 nm conforms to the typical three fingers spectrum of bacterioruberin. In addition to carotenoids, other lipid compounds, such as unsaturated FAs, have been shown to play an important role as antioxidants against ROS species. When the HAE was analyzed by gas chromatography-mass spectrometry, significant amounts of 16:0, 18:0, and 18:0 10-methyl FA were identified. This led to the characterization of the extract in terms of FAs. The analysis of the FAs content of the total lipids in the HAE showed that the most abundant FA was 18:0 (55.5% of total lipids), while the others accounted for ca. 0.4–14% (Table 1). The amount of FAs in the extract was 1.7 ± 0.3 µg FA/mg CDW, which corresponds to 0.8 ± 0.06 µg FA/µg extract. Table 1. Fatty acid composition of the acetone extract of Haloarcula sp. OS (HAE) (represented in ng of fatty acids per mg of cell wet weight and as a percentage of total fatty acid content). FA ng Lipids/mgCDW % (FA/Total FAs) 12:0 36.1 2.1 14:0 8.1 0.5 14:0 anteiso 8.9 0.5 16:0 101.7 6.0 16:0 DMA 5.6 0.3 16:0 N alcohol 62.5 3.7 17:0 7.2 0.4 18:0 948.2 55.5 18:0 10-methyl 230.7 13.5 18:1 w9c 95.1 5.6 18:2 w6c 16.0 0.9 18:3 w6c 187.4 11.0 Minutes 46 8 10 12 14 16 18 20 22 24 26 28 mAU 0 25 50 75 nm 300 400 500 600 700 mAU 0 25 50 75 Figure 2. HPLC elution profile of the Haloarcula sp. OS acetone extract (HAE). The three prominent peaks observed in the chromatogram correspond to bacterioruberin isomers with the same absorption spectrum, which is shown on the insert. This UV–Vis spectrum with absorption maxima at 468, 496, and 532 nm conforms to the typical three fingers spectrum of bacterioruberin. In addition to carotenoids, other lipid compounds, such as unsaturated FAs, have been shown to play an important role as antioxidants against ROS species. When the HAE was analyzed by gas chromatography-mass spectrometry, significant amounts of 16:0, 18:0, and 18:0 10-methyl FA were identified. This led to the characterization of the extract in terms of FAs. The analysis of the FAs content of the total lipids in the HAE showed that the most abundant FA was 18:0 (55.5% of total lipids), while the others accounted for ca. 0.4–14% (Table 1). The amount of FAs in the extract was 1.7 ± 0.3 µ g FA/mg CDW, which corresponds to 0.8 ±0.06 µg FA/µg extract. Table 1. Fatty acid composition of the acetone extract of Haloarcula sp. OS (HAE) (represented in ng of fatty acids per mg of cell wet weight and as a percentage of total fatty acid content). FA ng Lipids/mgCDW % (FA/Total FAs) 12:0 36.1 2.1 14:0 8.1 0.5 14:0 anteiso 8.9 0.5 16:0 101.7 6.0 16:0 DMA 5.6 0.3 16:0 N alcohol 62.5 3.7 17:0 7.2 0.4 18:0 948.2 55.5 18:0 10-methyl 230.7 13.5 18:1 w9c 95.1 5.6 18:2 w6c 16.0 0.9 18:3 w6c 187.4 11.0 3.3. Antioxidant Capacity of the Acetone Extract The antioxidant capacity of the HAE was tested using the 2,2 0 -azino-bis (3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS) method. Several dilutions of the HAE were assayed (Figure 3A) to obtain the effective concentration of 50% (EC50), which was expressed as Trolox Equivalent Antioxidant Capacity, TEAC (EC50 sample/EC50 Trolox) (Figure 3B). The reference compound Trolox showed an EC50 of 11.3 ± 0.9 µ g/mL. The HAE exhibited a potent antioxidant capacity, displaying an EC50 of 20.5 µ g/mL. The HAE showed 1.8 equivalents of Trolox, which means that it is necessary 1.8-fold HAE than Trolox to get the same effect. Antioxidants 2023,12, 1080 8 of 15 Antioxidants 2023, 12, x FOR PEER REVIEW 9 of 17 3.3. Antioxidant Capacity of the Acetone Extract The antioxidant capacity of the HAE was tested using the 2,2′-azino-bis (3-ethylben- zothiazoline-6-sulfonic acid) (ABTS) method. Several dilutions of the HAE were assayed (Figure 3A) to obtain the effective concentration of 50% (EC50), which was expressed as Trolox Equivalent Antioxidant Capacity, TEAC (EC50 sample/EC50 Trolox) (Figure 3B). The reference compound Trolox showed an EC50 of 11.3 ± 0.9 µg/mL. The HAE exhibited a potent antioxidant capacity, displaying an EC50 of 20.5 µg/mL. The HAE showed 1.8 equivalents of Trolox, which means that it is necessary 1.8-fold HAE than Trolox to get the same effect. (A) (B) Figure 3. Scavenger antioxidant activity of the Haloarcula sp. OS acetone extract (HAE). (A) The antioxidant capacity of the HAE at different concentrations (0–200µg/mL) was determined by ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assay, using TROLOX as a reference control. (B) The half-effective concentration (EC50) was calculated and the results were expressed as equivalent to Trolox (EC50 sample/EC50 Trolox). Results are representative of three independent experiments (n = 3). 3.4. Intracellular ROS Assessment The levels of intracellular ROS produced in LPS-stimulated THP-1 macrophages were measured by using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA) (Figure 4). First, to rule out cytotoxic effects, the HAE was tested at different concentrations (0–100 µg/mL) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method. The results showed 100% viability of the macrophages exposed to HAE at all tested concentrations for 24 h (Table 2). Based on these data, the concentrations 5, 25, and 50 µg/mL of HAE were chosen to carry out the ROS assay. Table 2. Viability of THP-1 human macrophages treated with different concentrations of the Haloarcula sp. OS acetone extract (HAE) at different concentrations. Values are the means ± SEM (%), (N = 3). % Viability THP-1 Macrophages (24 h) (µg/mL) 0 6.25 12.5 25 50 100 HAE 100 ± 2.2 112.5 ± 6.5 111.40 ± 4.70 117.06 ± 5.5 111.60 ± 9.7 114.51 ± 11.5 025 50 75 100 125 150 175 200 0 20 40 60 80 100 TROLOX HAE Concentration (μg/mL) Antioxidant Capacity (%) Figure 3. Scavenger antioxidant activity of the Haloarcula sp. OS acetone extract (HAE). ( A ) The antioxidant capacity of the HAE at different concentrations (0–200 µ g/mL) was determined by ABTS (2,2 0 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)) radical scavenging assay, using TROLOX as a reference control. ( B ) The half-effective concentration (EC 50 ) was calculated and the results were expressed as equivalent to Trolox (EC50 sample/EC50 Trolox). Results are representative of three independent experiments (n = 3). 3.4. Intracellular ROS Assessment The levels of intracellular ROS produced in LPS-stimulated THP-1 macrophages were measured by using 2 0 ,7 0 -dichlorodihydrofluorescein diacetate (DCF-DA) (Figure 4). First, to rule out cytotoxic effects, the HAE was tested at different concentrations (0–100 µ g/mL) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) method. The results showed 100% viability of the macrophages exposed to HAE at all tested concentrations for 24 h (Table 2). Based on these data, the concentrations 5, 25, and 50 µg/mL of HAE were chosen to carry out the ROS assay. Antioxidants 2023, 12, x FOR PEER REVIEW 10 of 17 Figure 4. Effect of the Haloarcula sp. OS acetone extract (HAE) on the ROS production in LPS-stim- ulated THP-1 macrophages. THP-1 monocytes were transformed into macrophages with 8 nM PMA for 72 h. Then, cells were pretreated with the BRE (5, 25, and 50 µg/mL) for 1 h and stimulated with LPS (1 µg/mL) for 24 h. Dexamethasone (Dex, 0.39 µg/mL) was used as an internal reference control. Results are representative of four independent experiments (n = 4). Values are the means with standard errors represented by vertical bars. Mean value was significantly different compared to the Control group (+++ p < 0.001; Student’s t-test). Mean value was significantly different compared to the LPS group (*** p < 0.001; * p < 0.01; Kruskal–Wallis test). ROS production was significantly increased in the LPS group in comparison with the Control group (p < 0.001), which showed 57.3% of ROS basal levels. The glucocorticoid dexamethasone (Dex, 1 µM) reduced ROS production to similar levels to those of the Control group (p < 0.001). The treatment with the HAE caused a significant decrease in ROS production at all tested concentrations (p < 0.05), which achieved a reduction close to 40% in ROS levels. 3.5. Pro-Inflammatory Cytokines Levels The in vitro anti-inflammatory activity of the HAE obtained from Haloarcula sp. OS was evaluated by measuring the production of TNF-α and IL-6 in LPS-stimulated THP-1 macrophages. Our results showed that LPS stimulation induced a marked increase in TNF-α and IL-6 production in THP-1 cells compared to unstimulated control cells (p < 0.001). The anti-inflammatory reference drug dexamethasone significantly reduced the levels of both cytokines (p < 0.001). As shown in Figure 5A, pretreatment of THP-1 cells with the HAE at the concentrations of 25 and 50 µg/mL resulted in a significant reduction in LPS-mediated TNF-α production (p < 0.01 and p < 0.001, respectively). Furthermore, the HAE substantially reduced IL-6 levels at all tested concentrations (p < 0.001) (Figure 5B). Figure 5. Effect of the Haloarcula sp. OS acetone extract (HAE) on the pro-inflammatory cytokines production in LPS-stimulated THP-1 macrophages. THP-1 monocytes were transformed into macrophages with 8 nM PMA for 72 h. Then, cells were pretreated with the HAE (5, 25, and 50 µg/mL) for 1 h and stimulated with LPS (1 µg/mL) for 24 h. Dexamethasone (Dex, 0.39 µg/mL) was used as Control LPS Dex 525 50 0 50 100 150 +++ *** *** HAE ROS production (%) Control LPS Dex 525 50 0 2000 4000 6000 8000 +++ *** *** ** TNF-α production (pg/ml) HAE AB Control LPS Dex 525 50 0 100 200 300 400 500 +++ *** *** *** *** IL-6 production (pg/ml) HAE Figure 4. Effect of the Haloarcula sp. OS acetone extract (HAE) on the ROS production in LPS- stimulated THP-1 macrophages. THP-1 monocytes were transformed into macrophages with 8 nM PMA for 72 h. Then, cells were pretreated with the BRE (5, 25, and 50 µ g/mL) for 1 h and stimulated with LPS (1 µ g/mL) for 24 h. Dexamethasone (Dex, 0.39 µ g/mL) was used as an internal reference control. Results are representative of four independent experiments (n = 4). Values are the means with standard errors represented by vertical bars. Mean value was significantly different compared to the Control group (+++ p< 0.001; Student’s t-test). Mean value was significantly different compared to the LPS group (*** p< 0.001; * p< 0.01; Kruskal–Wallis test). Antioxidants 2023,12, 1080 9 of 15 Table 2. Viability of THP-1 human macrophages treated with different concentrations of the Haloarcula sp. OS acetone extract (HAE) at different concentrations. Values are the means ±SEM (%), (N = 3). % Viability THP-1 Macrophages (24 h) (µg/mL) 0 6.25 12.5 25 50 100 HAE 100 ±2.2 112.5 ±6.5 111.40 ± 4.70 117.06 ± 5.5 111.60 ± 9.7 114.51 ± 11.5 ROS production was significantly increased in the LPS group in comparison with the Control group (p< 0.001), which showed 57.3% of ROS basal levels. The glucocorticoid dexamethasone (Dex, 1 µ M) reduced ROS production to similar levels to those of the Control group (p< 0.001). The treatment with the HAE caused a significant decrease in ROS production at all tested concentrations (p< 0.05), which achieved a reduction close to 40% in ROS levels. 3.5. Pro-Inflammatory Cytokines Levels The in vitro anti-inflammatory activity of the HAE obtained from Haloarcula sp. OS was evaluated by measuring the production of TNF- α and IL-6 in LPS-stimulated THP-1 macrophages. Our results showed that LPS stimulation induced a marked increase in TNF- α and IL-6 production in THP-1 cells compared to unstimulated control cells (p< 0.001). The anti-inflammatory reference drug dexamethasone significantly reduced the levels of both cytokines (p< 0.001). As shown in Figure 5A, pretreatment of THP-1 cells with the HAE at the concentrations of 25 and 50 µ g/mL resulted in a significant reduction in LPS-mediated TNF- α production (p< 0.01 and p< 0.001, respectively). Furthermore, the HAE substantially reduced IL-6 levels at all tested concentrations (p< 0.001) (Figure 5B). Antioxidants 2023, 12, x FOR PEER REVIEW 10 of 17 Figure 4. Effect of the Haloarcula sp. OS acetone extract (HAE) on the ROS production in LPS-stim- ulated THP-1 macrophages. THP-1 monocytes were transformed into macrophages with 8 nM PMA for 72 h. Then, cells were pretreated with the BRE (5, 25, and 50 µg/mL) for 1 h and stimulated with LPS (1 µg/mL) for 24 h. Dexamethasone (Dex, 0.39 µg/mL) was used as an internal reference control. Results are representative of four independent experiments (n = 4). Values are the means with standard errors represented by vertical bars. Mean value was significantly different compared to the Control group (+++ p < 0.001; Student’s t-test). Mean value was significantly different compared to the LPS group (*** p < 0.001; * p < 0.01; Kruskal–Wallis test). ROS production was significantly increased in the LPS group in comparison with the Control group (p < 0.001), which showed 57.3% of ROS basal levels. The glucocorticoid dexamethasone (Dex, 1 µM) reduced ROS production to similar levels to those of the Control group (p < 0.001). The treatment with the HAE caused a significant decrease in ROS production at all tested concentrations (p < 0.05), which achieved a reduction close to 40% in ROS levels. 3.5. Pro-Inflammatory Cytokines Levels The in vitro anti-inflammatory activity of the HAE obtained from Haloarcula sp. OS was evaluated by measuring the production of TNF-α and IL-6 in LPS-stimulated THP-1 macrophages. Our results showed that LPS stimulation induced a marked increase in TNF-α and IL-6 production in THP-1 cells compared to unstimulated control cells (p < 0.001). The anti-inflammatory reference drug dexamethasone significantly reduced the levels of both cytokines (p < 0.001). As shown in Figure 5A, pretreatment of THP-1 cells with the HAE at the concentrations of 25 and 50 µg/mL resulted in a significant reduction in LPS-mediated TNF-α production (p < 0.01 and p < 0.001, respectively). Furthermore, the HAE substantially reduced IL-6 levels at all tested concentrations (p < 0.001) (Figure 5B). Figure 5. Effect of the Haloarcula sp. OS acetone extract (HAE) on the pro-inflammatory cytokines production in LPS-stimulated THP-1 macrophages. THP-1 monocytes were transformed into macrophages with 8 nM PMA for 72 h. Then, cells were pretreated with the HAE (5, 25, and 50 µg/mL) for 1 h and stimulated with LPS (1 µg/mL) for 24 h. Dexamethasone (Dex, 0.39 µg/mL) was used as Control LPS Dex 525 50 0 50 100 150 +++ *** *** HAE ROS production (%) Control LPS Dex 525 50 0 2000 4000 6000 8000 +++ *** *** ** TNF-α production (pg/ml) HAE AB Control LPS Dex 525 50 0 100 200 300 400 500 +++ *** *** *** *** IL-6 production (pg/ml) HAE Figure 5. Effect of the Haloarcula sp. OS acetone extract (HAE) on the pro-inflammatory cytokines production in LPS-stimulated THP-1 macrophages. THP-1 monocytes were transformed into macrophages with 8 nM PMA for 72 h. Then, cells were pretreated with the HAE (5, 25, and 50 µ g/mL) for 1 h and stimulated with LPS (1 µ g/mL) for 24 h. Dexamethasone (Dex, 0.39 µ g/mL) was used as an internal reference control. ( A ) TNF- α production. ( B ) IL-6 production. Results are representative of five independent experiments (n = 5). Values are the means with standard errors represented by vertical bars. Mean value was significantly different compared with the Control group (+++ p< 0.001; Student’s t-test). Mean value was significantly different compared with the LPS group (*** p< 0.001, ** p< 0.01; ANOVA test followed by Tukey’s Multiple Comparison test). 3.6. Assessment of Antioxidant Protein Expression Levels To further explore the basis of the potential anti-inflammatory mechanisms of action of the HAE, the expression of the antioxidant proteins Nrf2 and HO-1 was evaluated by Western blotting. THP-1 macrophages were pretreated with the HAE (5, 25, and 50 µ g/mL) for 1 h and then stimulated with LPS (1 µ g/mL). As presented in Figure 6A, LPS stimulation did not induce significant changes in Nrf2 expression, but a slight non-significant increase in HO-1 expression was observed. However, the pretreatment with HAE resulted in a notable up-regulated expression of the Nrf2 protein at 25 and 50 µ g/mL, but only the highest concentration showed a significant increase (p< 0.05) in LPS-stimulated macrophages