Sample collection and identification

The polar microalgae samples KSF0003, KSF0015, KSF0037, and KSF0041 were collected in 2006 and KSF0134 was collected in 2014 from freshwater in Byers Peninsula, Livingston Island in Antarctica (S 62 40’09.81” W 61 06’26.61”). KNM0029C was obtained from seawater in the vicinity of Dasan Station in Ny-Ålesund, Spitsbergen, Norway (78°55′N, 11°56′E) in 2008. All microalgae samples were deposited at the Korea Polar Research Institute located in the Republic of Korea and stored at 2–3 °C under a white light source from light-emitting diodes (35 µmol·m− 2·s− 1). Based on small subunit ribosomal DNA sequence similarities within the NCBI GenBank database, KSF0015 and KSF0041 were identified as belonging to the genus Micractinium sp., KNM0029C, KSF0037, and KSF0134 were determined to be of the genus Chlamydomonas sp., and KSF0003 was found to be of the genus Chlorococcum sp. As control strains, Micractinium sp. (LIMS-PS-1788), Chlamydomonas sp. (LIMS-PS-0082), and Chlorococcum sp. (LIMS-PS-0085), isolated from moderate-temperature regions, were obtained from the Library of Marine Samples (LIMS) of the Korea Institute of Ocean Science and Technology.

Microalgae culture

KSF0015 and KSF0041 were cultured on Bold’s Basal Medium, and KSF0003, KSF0037, KSF0134, and KNM0029C were cultured on Tris-acetate-phosphate (TAP) medium [48]. All strains were grown at 11–12 °C for 4 weeks under controlled conditions in a 15-L photo-bioreactor with white LED lights at 80 µmol photon m− 2s− 1 on a 16:8 h light-dark cycle and supplied with filtered air at a rate of 5 L/min [49]. Micractinium sp. (LIMS-PS-1788) was cultured in BG-11 medium, and Chlamydomonas sp. (LIMS-PS-0082) and Chlorococcum sp. (LIMS-PS-0085) were cultured in 1 L of TAP medium with the same light conditions at 24 °C and supplied with filtered air at a rate of 0.5 L/min. Biomass was harvested through centrifugation (15 °C, 30 min, 12,000 rpm) and dried in a freeze-dryer to remove moisture.

Extract preparation

Microalgae extract was prepared following a previously described method with minor modification [12]. Dried microalgae material (10 g) was suspended in 40 mL of 80% methanol in 50-mL conical tubes. Conical tubes were incubated on a reciprocating shaker for 24 h with continuous agitation at 150 rev/min to dissolve bioactive compounds. The conical tubes were then centrifuged (4 °C, 15 min, 4000 rpm) and supernatants were filtered through Whatman no. 1 filter paper. A rotary vacuum evaporator with a water bath temperature of 37 °C was used to remove the solvents. Residues were collected and used for the experiments.

Gas chromatography-mass spectrometry (GC-MS) analysis

GC-MS analysis was performed using an Agilent 7890 B gas chromatograph equipped with a 5977B mass selective detector (GC/MSD) (Agilent Technologies, Santa Clara, CA, USA). Chromatographic separation was achieved using a Rxi-5HT capillary column (30 m × 0.25 mm I.D., 0.25 μm film thickness; RESTEK, Bellefonte, PA, USA). An autosampler (Agilent Technologies; 7683B) was used for all experiments. The temperature of the injector was 280 °C. Using split mode, 1 µL of each extract was injected at a ratio of 1/50. The carrier gas was helium C-60 at a constant flow rate of 1 mL/min. The GC oven temperature was initially 60 °C for 1 min, increased to 160 °C in 3 °C/min increments, and then increased to 320 °C in 10 °C/min increments, followed by a hold for 10 min. The mass spectrometer used the electron ionization mode at 70 eV with the ion source temperature set at 250 °C. The running time was 30 min. Scan mode was used in the range of 30–600 m/z with a scan rate of 2.6 scan/s. Agilent Mass Hunter Qualitative Analysis B.04.00 software was used for data analysis. Single compounds were identified by comparing mass spectra with NIST mass spectral libraries (National Institute of Standards, 2020 version) and the Wiley Registry 12th Edition.

Cell culture

HaCaT cells were cultured at 37 °C in a 5% CO2 atmosphere in Dulbecco′s Modified Eagle′s Medium (DMEM)-high glucose, supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin-streptomycin, 1 M hydroxyethyl piperazineethanesulfonic acid, 100 mM sodium pyruvate, 50 mM 2-mercaptoethanol, 10 mM non-essential amino acids, and L-glutamine. For inflammatory stimuli, HaCaT cells were irradiated with 25 mJ/cm2 UVB and then cultured for 4 h to induce epithelial damage. For the psoriatic keratinocyte model, HaCaT cells were stimulated with a final concentration of 2.5 ng/mL recombinant IL-17 A, IL-22, oncostatin M, IL-1α, and TNF- α (Peprotech, Rocky Hill, NJ, USA) for 3 h, as previously described [25, 26].

Radical-scavenging assay

Microalgae extracts (0.1 µg/mL or 1 µg/mL) and 50 µM 2′,7′-dichlorofluorescein diacetate (DCHF‐DA) (Sigma–Aldrich, St. Louis, MO, USA) were incubated with 10 mM hydrogen peroxide (H2O2) (Sigma–Aldrich) or 60 mM H2O2 and 0.75 mM FeCl2 in pH 7.4 phosphate-buffered saline (PBS) at 37℃ for 10 min [50]. All experiments were performed in a dark room to prevent oxidation of the DCHF-DA. Fluorescence quenching was measured using a spectrofluorometer (VICTOR3, PerkinElmer, Waltham, MA, USA) with excitation and emission wavelengths of 488 and 530 nm, respectively.

Cell-based ROS scavenging assay

Cell-based ROS scavenging assay was performed using a previously reported method with minor modification [50]. HaCaT cells were plated in 96-well black plates at a density of 1 × 104 cells/well in 100 µL MEM and incubated for 24 h. The antioxidant activity of the microalgae extracts was assessed by adding 250 mM H2O2 after the cells were first overnight incubated with the polar microalgae extracts (1 µg/mL). Cells were then washed with pH 7.4 PBS and incubated for 30 min with pH 7.4 PBS containing 10 µM DCHF-DA. Fluorescence was measured using a spectrofluorometer (VICTOR3) with excitation and emission wavelengths of 485 and 535 nm, respectively.

Cell viability assay

The effect of microalgae extracts on the viability of HaCaT cells was determined using a water-soluble tetrazolium salt assay kit according to the manufacturer’s instructions (EZ Cytox cell viability assay kit; DoGEN Bio, Seoul, Korea). Absorbance was measured at 450 nm (VICTOR3).

Fluorescein isothiocyanate (FITC)-dextran permeability assay

HaCaT cells were seeded on tissue culture polycarbonate membrane filters (pore size, 0.4 μm) in 24-well transwell plates (SPL Life Sciences, Gyeonggi-do, Korea) at a seeding density of 1.0 × 105 cells/cm2. Epithelial permeability across HaCaT monolayers was assessed by measuring the flux of 4-kDa FITC-labeled dextran (2 mg/mL) (Sigma–Aldrich) from the apical chamber to the basolateral chamber of the transwells [9]. FITC-dextran was added to the apical chamber and incubated for 2 h at 37 °C. Fluorescence in the basolateral compartment was then measured at an excitation of 485 nm and emission of 535 nm (VICTOR X4, PerkinElmer).

Real-time polymerase chain reaction (PCR) analysis

Total RNA was extracted using QIAzol® lysis reagent (Qiagen, Hilden, Germany) and subsequently column purified using an RNeasy® Mini Kit (Qiagen). The RNA (500 ng) was then treated with DNase I (New England Biolabs, Ipswich, MA, USA), and cDNA was synthesized using an iScript™ cDNA synthesis kit (Bio-Rad Laboratories, Hercules, CA, USA). Real-time PCR was performed using iQ SYBR® Green Supermix (Bio-Rad Laboratories) on a CFX Connect™ real-time PCR detection system (Bio-Rad Laboratories). Primers are listed in Table S1.

RNA extraction, library construction, and sequencing

RNA was extracted from HaCaT cells using QIAzol® and purified using an RNeasy® mini kit (Qiagen). Purified RNA was processed with DNase I (New England Biolabs) to remove genomic DNA. Total RNA concentrations were calculated using Quant-IT RiboGreen (Invitrogen, Waltham, MA, USA). To assess the integrity of the total RNA, samples were run on a TapeStation RNA ScreenTape (Agilent). Only high-quality RNA preparations with RNA integrity number greater than 7.0 were used for RNA library construction. Libraries were independently prepared with 1 µg of total RNA for each sample using an Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA, USA). The libraries were quantified using KAPA Library Quantification kits for Illumina Sequencing platforms according to the qPCR Quantification Protocol Guide (Kapa Biosystems, Wilmington, MA, USA) and qualified using a TapeStation D1000 ScreenTape. Indexed libraries were then submitted to an Illumina NovaSeq, and paired-end (2 × 100 bp) sequencing was performed by Macrogen (Seoul, Korea).

Sequence annotation and statistical analysis of gene expression

Raw reads were preprocessed from the sequencer to remove low-quality and adapter sequences before analysis; processed reads were aligned with Homo sapiens (GRCh38) using HISAT v2.1.0 [51]. The relative gene abundance was measured in Read Count using StringTie [52, 53]. Genes with one more than the zeroed Read Count value in the samples were excluded. To facilitate log2 transformation, 1 was added to each Read Count value of each filtered gene. Filtered data were log2-transformed and subjected to relative log expression normalization. Statistical significance of the differential expression data was determined using the nbinomWaldTest using DESeq2 and fold change in which the null hypothesis was that no difference existed among groups. The false discovery rate (FDR) was controlled by adjusting the p-value using the Benjamini-Hochberg algorithm.

Multidimensional scaling (MDS)

To visualize the similarities among samples, MDS was used, which converts the structure in a similarity matrix to a simple geometrical picture as scatter plots. The larger the dissimilarity between two samples, the further apart the points representing the experiments in the picture should be. Euclidean distance was applied as the measure of dissimilarity.

Gene set enrichment analysis (GSEA)

GSEA was performed using GSEA v4.2.3 software provided by the Broad Institute (Cambridge, MA, USA) as previously described [54]. Enrichment analysis was performed using hallmark gene sets of the MsigDB database. To determine the enrichment of ontology gene sets (C5.all.v2022.1), mouse gene symbols were remapped to human orthologs. Selected gene sets with p < 0.05 and FDR < 0.25 are shown.

Mice

Six-week-old, female, specific pathogen-free C57BL/6 mice were purchased from Orient Bio (Gyeonggi-do, Korea). Female mice were selected because of their robust inflammatory response against psoriatic inflammation induced by TLR7 application [55, 56]. Mice were maintained under standard temperature and humidity in specific pathogen-free conditions. All procedures involving mice were reviewed and approved by the Center of Animal Care and Use of the Lee Gil Ya Cancer and Diabetes Institute, Gachon University (Number: LCDI-2023-0017).

Animal model of psoriasis-like skin inflammation and KSF0041 extract treatment

Mice received five sequential daily topical doses of 62.5 mg of imiquimod cream (5%) (Aldara™; 3 M Pharmaceuticals, Maplewood, MN, USA) or Vaseline cream (Unilever, Rotterdam, Netherlands) on their shaved back, as described [28]. Mice were assessed for body weight. TEWL was measured on the back skin using a Tewameter TM 210 (Courage + Khazaka GmbH, Cologne, Germany). The composition of KSF0041 cream (with 1% KSF0041 extract) was as follows: 50 mg KSF0041 extract, 0.9 g glycerin, 3 g olive oil, and 1.2-2.4 g olive oil wax. The mixture was incubated overnight at room temperature and stored at 4 °C. A daily topical dose of 62.5 mg KSF0041 cream or vehicle cream (without KSF0041) was applied daily during the imiquimod application period. The mice were euthanized by carbon dioxide asphyxiation 5 days after induction of psoriasis-like skin inflammation.

Histology

Skin specimens were fixed in 10% neutral buffered formalin (BBC Biochemical, Mount Vernon, WA, USA) and embedded in paraffin. Six 4-µm sections per experimental group were stained with hematoxylin and eosin and visualized using a DM6 B microscope with a DFC7000T camera (Leica, Wetzlar, Germany). Epidermal thickness was quantified by analyzing and averaging images from three distinct regions of each section. Additionally, each section was analyzed to quantify dermal cell infiltration using i-SOLUTION™ (IMT i-Solution Inc., Vancouver, BC, Canada).

Statistical analysis

All experiments were performed in duplicate. Differences between groups were examined for statistical significance using one-way ANOVA with the Tukey post hoc test. Multiple-comparison tests were performed using two-way ANOVA with Bonferroni’s post hoc test. A p-value < 0.05 was considered statistically significant. GraphPad Prism 9 (GraphPad, San Diego, CA, USA) was used for data analysis. There were no studies in which the investigators were blinded.