Cyanobacterial harmful bloom lipopolysaccharides: pro-inflammatory effects on epithelial and immune cells in vitro

Cyanobacterial harmful blooms (CyanoHABs) pose a global ecological problem, and their lipopolysaccharides (LPS) are among the bioactive compounds they release. Previous studies on CyanoHAB-LPS from single cyanobacterial species have shown varying bioactivities in different in vitro cell models. In this study, we isolated LPS from 19 CyanoHAB samples collected at 18 water bodies in the Czech Republic over two consecutive seasons. The proportions of cyanobacteria, Gram-negative bacteria (G-), and other bacteria in the biomass were determined by qPCR, while the cyanobacterial genera were identified using light microscopy. In vitro models of keratinocytes (HaCaT), the intestinal epithelium (co-culture of differentiated Caco-2 cells and peripheral blood mononuclear cells — PBMC), and PBMC alone were treated with isolated LPS at concentrations of 50, 100, and 1 µg/ml, respectively. The endotoxin activities of these concentrations were within the range measured in the aquatic environment. Approximately 85–90% of the samples displayed biological activity. However, the potency of individual LPS effects and response patterns varied across the different in vitro models. Furthermore, the observed activities did not exhibit a clear correlation with the taxonomic composition of the phytoplankton community, the relative share of microbial groups in the biomass, endotoxin activity of the LPS, or LPS migration and staining pattern in SDS-PAGE. These findings suggest that the effects of CyanoHAB-LPS depend on the specific composition and abundance of various LPS structures within the complex environmental sample and their interactions with cellular receptors. Supplementary Information The online version contains supplementary material available at 10.1007/s00204-023-03644-8.

(14.8%), algae (6.8%) Cyanobacterial genera and/or species with more than 5% biovolume share in the concentrated biomass are listed.The only exception is the sample B, where all the cyanobacterial species are listed due to a very low biovolume share of cyanobacteria in this mixture.The sum of biovolume share of all eukaryotic algae is also provided.n.d.= not detected.
Tab. S3 LPS yield from the CyanoHAB biomasses and endotoxin activity.The endotoxin activity of the isolated CyanoHAB-LPS and the water from the site of the collection was assessed by Pyrogene TM assay.The endotoxin activity of the final concentration used for the treatment in the in vitro experiments was calculated.The green color denotes biomasses, blue water in situ, and yellow culture media.

DETAILED METHODOLOGY
LPS isolation 1 g of lyophilized cyanobacterial biomass was mixed with 50 ml of MilliQ water and sonicated using an ultrasonic bath (10 min).The suspension was then heated to 68 °C, mixed with 50 ml of pre-warmed 90% phenol, and stirred for 20 min at 68 °C.After cooling to 4 °C, the mixture was centrifuged (5 630 g, 30 min, 4 °C), and the supernatant (aqueous phase with LPS) was collected.The phenol layer was re-extracted with 50 ml of MilliQ water, and pooled supernatants were purified by dialysis (48 h) using cellulose membranes (33x21 mm, Sigma-Aldrich, USA, D9652) against MilliQ water (1 l) containing toluene (10 μl/l) to prevent bacterial contamination.After 24 h of dialysis, the water was changed and dialysis continued for another 24 h.The dialyzed extracts were centrifuged (5 630 g, 30 min, 4 °C), collected as supernatants and lyophilized.The resulting semi-purified freeze-dried extract of LPS was resuspended in 3.75 ml of 0.1M Tris-HCl buffer (pH 7.4) containing 25 µg/ml ribonuclease A (RNase, Sigma-Aldrich, R4642) and incubated for 16 h at 37 °C.Next, 3.75 ml of 90% phenol in 0.1M Tris-HCl was added, vortexed, and incubated for 4 min at room temperature (RT).The solution was centrifuged (18 410 g, 15 min, RT), the aqueous phase was separated and purified for 48 h by dialysis for a second time (see above) and then lyophilized.The purified LPS (freeze-dried powder) was weighed to assess the content of LPS in the biomass and kept at -20 °C.Isolated LPS were dissolved (at a concentration of 10 mg/ml) in MilliQ water for characterization by SDS-PAGE or agarose electrophoresis, or in PBS with 0.1% BSA (v/v, Sigma-Aldrich) for PyroGene TM rFC or in vitro cell assays.

SDS-PAGE and agarose electrophoresis of CyanoHAB-LPS
Isolated LPS samples (10 µg) were diluted with MilliQ water and 4× Laemmli loading buffer (Bio-Rad, Hercules, CA, USA, 161-0747) containing 2-mercaptoethanol (final concentration 357 mM), and heated for 10 min at 65 °C.Commercially available LPS purified from Escherichia coli serotype O111:B4 (Sigma-Aldrich, L2630) was used as a positive control (1 or 10 µg per lane).The 4% (w/v) acrylamide stacking gels were used for SDS-PAGE in combination with 10-20% (w/v) gradient separating gels (1 mm thickness) prepared from 40% (w/v) acrylamide/bis-acrylamide solution, 37.5:1 (Serva, Heidelberg, Germany) using a Mini-Protean gel casting and electrophoresis system (Bio-Rad).Electrophoresis was conducted at 130 V, and the gels were then washed with deionized water, and stacking gels were removed.For the silver staining of LPS, the washed gels were fixed with 40% (v/v) ethanol-5% (v/v) acetic acid overnight at RT.Then, the fixed gels were oxidized with 0.7% (w/v) periodic acid in 40% (v/v) ethanol-5% (v/v) acetic acid for 5 min, and washed with deionized water (2×5 min and 1×10 min).The silver staining solution was freshly prepared by mixing 28 ml of 0.1M sodium hydroxide, 2.3 ml of 24% (w/w) ammonium hydroxide, 5 ml of 20% (w/v) silver nitrate (Sigma-Aldrich) and filled up to 150 ml with deionized water.The gels were incubated in the staining solution for 10 min and then washed with deionized water (2×5 min and 1×10 min).The developing process was carried out with 50 µg/ml citric acid in 0.05% (v/v) formalin for approximately 2-3 min, followed by washing with deionized water to achieve suitable levels of background staining.Finally, the gels were fixed with 5% (v/v) acetic acid for 1 min, washed, and documented under white light (Alliance Q9 Advanced system, Uvitec Cambridge, UK).LPS were stained with the Pro-Q TM Emerald 300 Lipopolysaccharide Gel Stain Kit (ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturer's protocol.Washed gels were fixed with 50% (v/v) methanol-5% (v/v) acetic acid for 45 min at RT and then washed with 3% (v/v) acetic acid for 2×15 min.LPS were oxidized for 30 min with a periodic acid-based oxidizing solution supplied with the kit.The gels were washed again with 3% (v/v) acetic acid (3×15 min) and then incubated for 90 min with 1× Pro-Q TM Emerald 300 staining buffer.Before UV-light visualization and documentation (Alliance Q9 Advanced), the gels were washed with 3% acetic acid for 2×20 min.To visualize protein contamination of LPS, the gels were restained overnight with SYPRO® Ruby (ThermoFisher Scientific), washed with 10% methanol with 7% acetic acid (v/v) for 30 min and finally with deionized water for 2×5 min, prior documentation (Alliance Q9 Advanced).Agarose gel electrophoresis of LPS and nucleic acids was conducted as reported previously (Sychrová et al., 2022, https://doi.org/10.1016/j.etap.2022.103869),using 1% gel, TAE buffer, and Bioline HyperLadder™ 50 bp molecular weight marker (MWM).The gels were stained using a 1% solution of ethidium bromide and documented with the Alliance Q9 Advanced system (Cambridge, UK).LPS from Escherichia coli serotype O111:B4 (Sigma-Aldrich) and its mixture with DNA or RNA from a fish cell line NKA-1 (isolated as reported previously in Součková et al., 2023, https://doi.org/10.1016/j.aquatox.2023.106517)were used as positive controls and to create a calibration curve for the RNA amount in the mixture with LPS.Densitometric analysis was conducted using ImageJ software (Schneider et al., 2012(Schneider et al., , https://doi.org/10.1038(Schneider et al., /nmeth.2089)).

DNA extraction
Briefly, freeze-dried biomass (4-10 mg) was resuspended in 0.5 ml of 0.15 M NaCl/0.1 M EDTA solution and homogenized using three freeze-thaw cycles with liquid nitrogen.Samples were then centrifuged (7 200 g, 10 min), the supernatant was discarded, and the pellet was resuspended in 0.5 ml of TE buffer.Then, 1 μl of RNase (10 mg/ml) was added and the samples were incubated at 37 °C for 1 h.Afterwards, 100 μl of lysozyme (50 mg/ml) was added, and the samples were incubated at 37 °C for 30 min.After incubation, 10 μl of protein kinase K (50 mg/ml) and 2% (final concentration) sodium dodecyl sulfate (SDS) were added, and samples were incubated at 55 °C for 1 h.Then, selective precipitation was performed by adding 150 μl of 5M NaCl to the tubes followed by 0.1 volumes (of total volume) of 10% cetyltrimethylammonium bromide stock solution, mixed by inversion, and incubated at 65 °C for 10 min.For purification (to allow for protein precipitation), 1 volume of chloroform was added, and tubes were incubated on ice for 30 min.Then, were centrifuged (7 200 g, 10 min, 4 °C), and the supernatant was transferred to fresh tubes, mixed with 0.6 volumes of isopropanol, and incubated overnight at 4 °C.Following this, samples were centrifuged (16 000 g, 30 min, 4 °C), the isopropanol supernatant was discarded, and the pellet was washed twice with 1 ml of 70% ethanol.Finally, samples were centrifuged (16 000 g, 30 min, 4 °C), the supernatant discarded, the pellet air-dried, and then resuspended in 30 μl of TE buffer.The concentration and quality of the extracted DNA were checked via NanoDrop (Thermo Scientific) and agarose gel electrophoresis.The isolated DNA underwent further processing through qPCR.

Treatment
Confluent HaCaT cells were treated with LPS at a final concentration of 50 μg/ml.Positive control was treated with E. coli O111:B4 LPS (Sigma-Aldrich) diluted in 0.1% BSA in PBS, while the negative control cells received no treatment.After 1 day of exposure, the medium was collected, and the cells were washed with ice-cold PBS and prepared for western blot.Co-culture was assembled by putting an insert with differentiated Caco-2 cells into the well with PBMC.LPS was applied to the apical part of the insert (on Caco-2 cells) at a final concentration of 100 μg/ml.The positive control consisted of a co-culture treated with E. coli LPS, while the negative control group was a co-culture without any treatment.Additional controls included an insert with Caco-2 cells without PBMC, either untreated or treated with E. coli LPS, and untreated PBMC without the insert.After 4 days of exposure, the media were collected for further analyses, the Caco-2 cells were washed with ice-cold PBS and prepared for western blot, PBMC were collected and prepared for flow cytometry.PBMC were treated with LPS at a final concentration of 1 μg/ml.Positive control was treated with E. coli LPS, and negative control was untreated.After 4 days of exposure, the medium was collected for further analyses, the cells were collected and prepared for flow cytometry.
Lactate dehydrogenase assay 80 μl of the sample was mixed with the reaction mixture in a ratio of 1:1 and incubated RT.The absorbance was measured at 492 nm using a SPECTRA Sunrise microplate reader (Tecan, Mannedorf, Switzerland).Untreated cells lysed by supplier-provided lysis buffer were used as a positive control.

Flow cytometry
The medium from PBMC was collected and centrifuged to capture all floating cells.PBMC in wells were incubated with 500 μl of 10 mM EDTA/PBS (10 min, 37 °C).Detached cells were moved to a flow cytometry tube.Another 500 μl of 10 mM EDTA/PBS was added to the wells, and any remaining cells were scraped with a cell scraper and moved to the tube with the rest of the respective sample.Cells were washed with PBS and blocked with 150 μl of PBS with 10% human serum (15 min, 4 °C).Human serum was a heat-inactivated pool of sera from 4 healthy donors prepared in our lab.Blocked cells were stained with antibodies against CD14 (APC anti-human CD14, 2109040) and CD16 (FITC anti-human CD16, 2110030, both SONY Biotechnology), 3 μl of each per sample (20 min, 4 °C).Stained cells were washed with cold PBS, the cells were re-suspended in 150 μl of cold PBS and kept on ice until measured on a flow cytometer (BD FACSVerse, BD Biosciences, USA).Immediately before measurement, 2 μl of propidium iodide (1 mg/ml) was added to the samples as a viability marker.

ELISA
Kits from Invitrogen and RaD Systems were used for the analyses.Invitrogen: Human CCL2 Uncoated ELISA Kit, Human IL-6 Uncoated ELISA Kit, Human IL-8 Uncoated ELISA Kit, Human IL-10 Uncoated ELISA Kit, Human TNFα Uncoated ELISA Kit; RaD Systems: Human CCL20 DuoSet ELISA.The absorbance was measured using a SPECTRA Sunrise microplate reader.

Fig. S1
Fig. S1 Cytotoxicity of used LPS concentrations.(a) The concentration of LDH in culture medium after treatment of HaCaT cells with LPS in concentration 50 μg/ml for 1 day, n = 3.Cells lysed by supplier-provided lysis buffer were used as a positive control.(b) Percentage of viable (PI-negative) PBMC after treatment of Caco-2/PBMC co-culture with LPS in concentration 100 μg/ml for 4 days, n = 6.(c) Percentage of viable (PI-negative) cells after treatment of PBMC with LPS in concentration 1 μg/ml for 4 days, n = 5.Data were converted to a percentage of the control (untreated cells) and expressed as the mean ± SD.Data were statistically analyzed by one-sample t-test.* p < 0.05

Fig.
Fig. S3 Composition of CyanoHABs used for LPS isolation as a percentage of biovolume.(a) Biovolume share of the main phytoplankton groups.(b) Biovolume of dominant cyanobacterial genera or species.
Fig. S4 Relationship between phytoplankton biovolume and LPS yield or endotoxin activity in CyanoHABs.(a) Cyanobacteria or (b) eukaryotic algae biovolumes normalized per dry weight (d.w.) of biomass and plotted against LPS yield (left) or endotoxin activity (right) per CyanoHAB biomass d.w.(c) Biovolume of cyanobacteria compared to qPCR-based cyanobacterial cell count estimates (equivalents of Microcystis) per CyanoHAB biomass d.w.Letters (A-U) represent the sample codes, ρ s = Spearman's rank correlation coefficient and two-tailed p value

Cyanobacteria(
Fig. S5 Relationship between qPCR-based estimates of microorganism quantity of microorganisms in CyanoHABs and LPS yield or endotoxin activity.(a) Cyanobacteria, (b) Gram-negative bacterial, and (c) other bacterial (total bacteria without Gram-negative) cell counts (equivalents of Microcystis or E. coli) as estimated by qPCR per CyanoHAB biomass dry weight (d.w.), and plotted against either LPS yield (left) or endotoxin activity (right) per CyanoHAB biomass d.w.Letters (A-U) represent the sample codes, ρ s = Spearman's rank correlation coefficient, and two-tailed p-value.

Fig. S8
Fig. S8 Agarose gel electrophoresis of LPS and nucleic acids followed by ethidium bromide staining.(a) Model DNA or RNA (0.5 µg) was loaded alone or in the mix with 10 µg LPS from E. coli O111:B4 (EC LPS).The presence of LPS was found to affect the migration pattern of RNA bands and cause their smearing.(b) Detection of nucleic acids in the LPS samples isolated from CyanoHABs (samples A-U) loaded at 10 µg per lane.As a control, 10 µg LPS from E. coli O111:B4 (EC LPS) was mixed with 0-2 µg of semipurified RNA.(c) Densitometric evaluation of 0-2 µg RNA + 10 µg E. coli O111:B4 (EC LPS).(d) Estimated amounts of RNA in the LPS samples isolated from CyanoHABs (samples A-U) based on densitometric analysis of the agarose gels, expressed as % of sample weight (w/w).Model DNA and RNA were isolated from a fish cell line NKA-1.

Fig
Fig. S9 Migration potential of keratinocytes.Confluent HaCaT cells were treated with 19 CyanoHAB-LPS from 18 recreational water bodies (A-U) at a final concentration of 50 μg/ml for 4 days.Negative control was untreated, and LPS from E. coli (EC) was used as a positive control.Wound closure of the scratched monolayer was assessed 12 h.Cell proliferation was either (a) unaffected or (b) stopped by mitomycin c pre-treatment (1 μg/ml, 2 h).(c) Selection of representative images.Data are expressed as the mean ± SD.Data were statistically analyzed by unpaired t-test.

Fig
Fig. S10 Caco-2 monolayer integrity.Co-culture of differentiated Caco-2 cells and human PBMC were treated with 19 CyanoHAB-LPS from 18 recreational water bodies (A-U) at a final concentration of 100 μg/ml for 4 days.Negative control was untreated, and LPS from E. coli (EC) was used as a positive control.Simple cultures of differentiated Caco-2 cells were used as a control of the model (control Caco).(a) Trans-epithelial electric resistance (TEER) of the Caco-2 monolayer, n = 4. Expression of tight junction proteins: (b) representative blots and relative concentration of (c) claudin 2, (d) claudin 4, (e) occludin, and (f) zonula occludens 1 expressed as the optical density of the bands, n = 4 -8.Data are expressed as the mean ± SD.Data were statistically analyzed by unpaired t-test.
Tab. S1 Sampling localities, dates and dominants of CyanoHABs used for LPS isolation.