Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Jun 5.
Published in final edited form as: Toxicol Lett. 2017 May 9;275:101–107. doi: 10.1016/j.toxlet.2017.05.013

Effects of cyanobacteria Oscillatoria sp. lipopolysaccharide on B cell activation and Toll-like receptor 4 signaling

Michelle Swanson-Mungerson a,*, Ryan Incrocci a, Vijay Subramaniam b, Philip Williams c, Mary L Hall d, Alejandro MS Mayer d
PMCID: PMC5510639  NIHMSID: NIHMS876468  PMID: 28499610

Abstract

Cyanobacteria (“blue-green algae”), such as Oscillatoria sp., are a ubiquitous group of bacteria found in freshwater systems worldwide that are linked to illness and in some cases, death among humans and animals. Exposure to cyanobacteria occurs via ingestion of contaminated water or food-products. Exposure of the gut to these bacteria also exposes their toxins, such as lipopolysaccharide (LPS), to B cells in the gut associated lymphoid tissue. However, the effect of Oscillatoria sp. LPS on B cell activation is unknown. To test the hypothesis that Oscillatoria sp. LPS exposure to murine B cells would result in B cell activation, murine B cells were incubated in the absence or presence of Oscillatoria sp. LPS or E. coli LPS as a positive control. The data indicate that Oscillatoria sp. LPS induces B cells to proliferate, upregulate MHC II and CD86, enhance antigen uptake and induce IgM production at low levels. Additional studies demonstrate that this low level of stimulation may be due to incomplete TLR4 signaling induced by Oscillatoria sp. LPS, since IRF-3 is not induced in B cells after stimulation with Oscillatoria sp. LPS. These findings have important implications for the mechanisms of toxicity of cyanobacteria in both humans and animals.

Keywords: Cyanobacteria, Oscillatoria sp, Lipopolysaccharide (LPS), B cells, Toll-like receptor 4 signaling

1. Introduction

Cyanobacteria (“blue-green algae”) are a ubiquitous group of Gram-negative bacteria found in freshwater systems worldwide (Kagalou et al., 2008; Palus et al., 2007; Stewart et al., 2006) that negatively impact human, animal, and environmental health. In particular, marine and freshwater strains belonging to the genera Anabaena, Aphanizome-non, Cylindrospermopsis, Lyngbya, Microcystis, Nodularia, Oscillatoria, and Planktothrix are linked to illness and, in some cases, death among humans and other animals (i.e. livestock, wildlife) (Bernardova et al., 2008; Mayer et al., 2011; Notch et al., 2011; Stewart et al., 2006). Exposure to cyanobacteria is via absorption through skin, inhalation, ingestion of drinking water, and food-products contaminated through bioaccumulation (Lang-Yona et al., 2014; Stewart et al., 2006). Exposure of the gut to these bacteria also exposes their toxins to the gut associated lymphoid tissue (GALT). Within the GALT resides a significant proportion of the body’s B cells (Pabst et al., 2008) of the immune system that respond to pathogen invasion by proliferating, serving to activate other cells of the immune system, and producing antibodies that neutralize pathogen invasion and toxins (LeBien and Tedder, 2008). Lipopolysaccharides (LPS), widely recognized as toxins in Gram-negative bacteria, are implicated in the toxicity of cyanobac-teria (Stewart et al., 2006). Cyanobacteria LPS is suggested to cause illnesses in humans, ranging from headache, fever, allergy, respiratory disease, and gastro-intestinal illness (Carmichael, 1993; Codd et al., 1999, 2005). Cyanobacteria LPS is thought to contribute to these symptoms through innate immune cells such as monocytes and macrophages (Ohkouchi et al., 2015). However, B cells of the adaptive immune system are known to become activated after exposure to LPS through the stimulation of Toll Like Receptor 4 (TLR4) (Mikheyskaya et al., 1977; Minguet et al., 2008) and despite the exposure of GALT B cells to cyanobacteria LPS, the ability of cyanobacteria to modulate their activation is unknown.

Previous studies have assessed the ability of Oscillatoria cyanobac-teria LPS to activate cells of the immune system (Mayer et al., 2011, 2016; Ohkouchi et al., 2015). One strain of Oscillatoria, Oscillatoria planktothrix FP1, produces an LPS-like molecule (CyP) that acts as a TLR4 antagonist that blocks the toxicity associated with other Gram-negative bacteria (Carillo et al., 2014; Jemmett et al., 2008). Additional studies using CyP indicate that CyP also antagonizes microglial cytokine release in vitro (De Paola et al., 2012). In contrast, a different strain of Oscillatoria, Oscillatoria sp., produces an LPS molecule that activates rat microglia to produce cytokines and chemokines in vitro (Mayer et al., 2016). However, none of the previous studies have addressed the ability of Oscillatoria LPS to activate B cells, which are likely exposed to Oscillatoria LPS after ingestion of contaminated water or food.

In the current study, we tested whether Oscillatoria sp. LPS directly activates B cells. Our data indicate that purified murine B cells proliferate, upregulate activation markers, increase antigen uptake and produce IgM in response to Oscillatoria sp. LPS at low levels. Additional studies demonstrate that this low level of stimulation may be due to incomplete TLR4 signaling induced by Oscillatoria sp. LPS, as IRF-3 is not induced in B cells after stimulation with Oscillatoria sp. LPS. Understanding the potential of LPS from different species of Gram-negative bacteria to activate immune cells will help us to understand the mechanism by which these bacteria induce disease in both animals and humans.

2. Materials and methods

2.1. B cell isolation and culture conditions

Murine B cells were purified from the spleens of forty 6–8 week old male and female c57Bl/6 mice using Stemcell “EasySep™” (STEMCELL Technologies Inc., Vancouver, Canada) murine B cell isolation kit following the manufacturer’s instructions. B cells purification reached > 97% purity as determined by flow cytometry and yielded approximately 20 × 106 B cells per spleen (data not shown). Purified B cells were then cultured in the absence or presence of increasing concentrations of Oscillatoria sp. LPS or E. coli LPS Stain 0111:B4 (Sigma) in cRPMI (10% Fetal Calf Serum (FCS), penicillin/streptomycin, and L-glutamine) at 37 °C, 5% CO2. Oscillatoria sp. LPS was prepared from Oscillatoria sp. strain HCC-1097 by hot phenol/water extraction followed by further extraction to remove RNA and protein impurities as described previously (Mayer et al., 2016). The Oscillatoria sp. LPS endotoxin activity was quantified using Toxinsensor chromo-genic endotoxin assay kit (Genscript, Piscataway, NJ), according to the manufacturer’s instructions, which uses E. coli LPS as a reference standard. E. coli LPS purchased from Sigma is also purified by phenol/water extraction.

2.2. Proliferation assay

Purified B cells (5 × 105) were incubated in quadruplicate in 96 well plates as described above for 72 h before proliferation was assessed using an MTT assay. Briefly, MTT 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (Sigma, St. Louis, MO) was added at a final concentration of 2.5 mg/ml to each well and incubated for 1 h at 37 °C in 5% CO2. The absorbance was determined at 562 nm using a Multiskan FC microplate reader (Thermo Fisher Scientific, Waltham, MA). The average of the individual wells from each experiment was used to combine three independent experiments to calculate the level of proliferation and perform statistical analysis.

2.3. Immunofluorescence staining for B cell activation markers

Purified B cells (5 × 105) were incubated in individual wells of a 12 well plate as described above for 48 h. Cells were harvested, washed three times in FACS buffer (PBS/1% FCS) and stained with either PE-conjugated rat anti-mouse MHC II (IAb) or PE-conjugated rat anti-mouse CD86 (Biolegend, Sand Diego, CA) for 30 min in the dark on ice. The cells were subsequently washed three times in PBS and analyzed using a FACS Calibur followed by Cell Quest Software (BD Biosciences, San Jose, CA). The Mean Fluorescence Intensity (MFI) for each individual culture from each experiment was used to combine three independent experiments to calculate the mean fold change in MFI and perform statistical analysis.

2.4. Antigen internalization assay

Purified B cells (2 × 106) were incubated as described above in individual wells of a 12 well plate and harvested 48 h later. The cells were washed three times in cRPMI before plating 5 × 105 B cells with FITC-Dextran (Thermo Fisher Scientific, Waltham, MA) for 1 h at 37 °C in 5% CO2, as described previously (Xu et al., 2008). Alternatively, cells were incubated with FITC-Dextran at 4 °C for 1 h to serve as a basis for non-specific binding. All cells were then incubated for five minutes with Trypan Blue to quench any autofluorescence, washed, and analyzed using the Amnis Flowsight (EMD Millipore, Billerica, MA) to determine intracellular mean fluorescence. Not only were cells compared to controls incubated with FITC-Dextran at 4 °C, but the membrane of cells analyzed were also “masked” using the Amnis Flowsight IDEAS software to remove any membrane-specific fluorescence (Wang et al., 2009). The Mean Fluorescence Intensity for each individual culture from each experiment was used to combine three independent experiments to calculate the level of antigen internalization (MFI) and perform statistical analysis.

2.5. IgM ELISA

Purified B cells (1 × 105) were incubated in quadruplicate in 200 ul of cRPMI in 96 well plates as described above for 5 days before 100 ul of supernatants were harvested and analyzed for IgM production using the Mouse IgM Ready Set Go ®ELISA kit (eBioscience Inc., San Diego, CA), following the manufacturer’s instructions. The average of the individual wells from each experiment was used to combine three independent experiments to calculate the level of IgM and perform statistical analysis.

2.6. Western blot analysis

After incubation with either E. coli or Oscillatoria sp. LPS, murine B cells were washed twice with PBS and resuspended in RIPA buffer (Thermo Fisher Scientific, Waltham, MA) at a concentration of 10 × 106 cells/50 uL along with HaltTM Protease and Phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA). Protein lysates were boiled with LDS Sample Buffer and Reducing Agent (Thermo Fisher Scientific, Waltham, MA) at 95 °C for 5 min. Samples were then loaded into 4–12% Bolt Bis-Tris Plus Gels (Thermo Fisher Scientific, Waltham, MA) and run for 20 min at 180 V. The gel was then transferred to a PVDF membrane using Bolt Transfer (Thermo Fisher Scientific, Waltham, MA) and run for 1 h at 20 V. The membrane was coated with blocking buffer containing milk and rocked for 1 h at room temperature. Antibodies were then added against Phospho-IκBα (Ser32/36), Total-IκBα, Phospho-p38 MAPK (Thr180/Tyr182), or Total-p38 MAPK, (Cell Signaling Technology, Danvers, MA) in blocking buffer containing milk and rocked at 4 °C overnight. After washing the blot in TBS-T, secondary anti-rabbit IgG-HRP (Pierce) or anti-mouse IgG-HRP (Cell Signaling Technology, Danvers, MA) conjugated antibodies were diluted in blocking buffer and added to membrane to rock for 1 h at room temperature. Following additional washes with TBS-T, blots were incubated for 5 min at room temperature with Amersham™ ECL Select™ (GE Healthcare, Buckinghamshire, UK) and chemiluminescent signals were measured using a Bio-Rad imager. Image analysis and quantification was done using ImageJ software (National Institutes of Health, Bethesda, MD). Each experiment was performed three times independently with similar results.

2.7. Statistics

Experiments with three or more groups were initially analyzed using a one-way analysis of variance (ANOVA) to determine if there were statistically significant differences within the experiment. Subsequently, a Bonferroni post-hoc test was used to compare individual groups. A finding is determined to be significant when the p value was equal to or less than 0.05. Data were analyzed with Prism Software Version 7.00.

3. Results

The ability of cyanobacteria LPS to activate human and animal B cells is unknown. LPS from other Gram-negative bacteria, such as E. coli, induces B cell proliferation, activation, and differentiation into plasma cells that produce high levels of antibody. To investigate if LPS from cyanobacteria Oscillatoria sp. induces the proliferation of murine B cells, purified B cells were incubated with increasing concentrations of either Oscillatoria sp. LPS or E. coli LPS (as a positive control) for 72 h. As shown in Fig. 1, E. coli LPS at concentrations of 1000 ng/ml–100,000 ng/ml induces murine B cells to proliferate 10- to 12-fold greater when compared to B cells not exposed to any LPS (p < 0.05). In contrast, Oscillatoria sp. LPS induces a 3-fold increase in proliferation of B cells at 100,000 ng/ml when compared to unexposed B cells (Fig. 1, p < 0.05). These data indicate that Oscillatoria sp. LPS has agonistic effects on B cells, but only at high concentrations.

Fig. 1.

Fig. 1

Open in a new tab

E. coli or Oscillatoria LPS activate purified murine B cells to proliferate. Murine B cells were isolated from c57Bl/6 mice and incubated with increasing concentrations of LPS for 72 h. Proliferation was assessed using the MTT assay as described in the Materials and methods. The data are a combination of three experiments. The error bars represent the standard error of the mean (SEM). * Indicates p < 0.05 from B cells not exposed to any LPS. The F value for the data in Fig. 1A is 29 with 5 degrees of freedom. The F value for Fig. 1B is 19 with 5 degrees of freedom.

In addition to inducing B cell proliferation, LPS enhances the ability of B cells to serve as antigen presenting cells to activate T cells by increasing MHC Class II and CD86 expression (Krieger et al., 1985; Snyder et al., 2002; Van Gool et al., 1996). Therefore, we tested whether Oscillatoria sp. induced the expression of these two critical molecules in antigen presentation. Purified B cells were exposed to either Oscillatoria sp. or E. coli LPS for 48 h. As shown in Fig. 2A, the addition of E. coli or Oscillatoria sp. LPS resulted in an increase in the mean fluorescence intensity (MFI) for MHC Class II as indicated by a shift of the shaded histogram to the right when compared to the non-shaded histogram. When the fold change in MFI of MHC Class II expression was combined from three independent experiments, it was determined that Oscillatoria sp. LPS could only significantly increase the level of MHC Class II expression at the highest concentration of LPS used (100,000 ng/ml) (ΔMFIOsc. sp. = 1.45-fold increase, p < 0.05 when compared to non-LPS exposed B cells) (Fig. 2B). In contrast, E. coli LPS significantly increased MHC Class II expression at 10,000–100,000 ng/ml (ΔMFIE.coli = 2.2–2.4-fold increase, respectively. p < 0.05 when compared to non-LPS exposed B cells) (Fig. 2B). Similarly, LPS from both strains of gram-negative bacteria significantly increase the level of CD86 expression (Fig. 2C–D: ΔMFIE.coli = 2.1–8.5-fold increase at 100–100,000 ng/ml; ΔMFIOsc. sp = 2.0-fold increase at 100,000 ng/ml, p < 0.05 when compared to non-LPS exposed B cells for both groups), suggesting that Oscillatoria sp. LPS at high concentrations is capable of increasing cell surface markers critical for T cell activation.

Fig. 2.

Fig. 2

Open in a new tab

E. coli or Oscillatoria LPS increases MHC II expression and CD86 expression. B cells were isolated from c57Bl/6 mice and incubated in the absence or presence of E. coli or Oscillatoria LPS (105 ng/ml) for 48 h. B cells were then washed and stained either with a (A–B) PE-rat anti-mouse anti-MHC II antibody (IAb) (top panels) or (C–D) PE-rat anti-mouse CD86 antibody before analysis by flow cytometry. The data in (A) and (C) are representative of three experiments with similar results. Unshaded histograms represent unstimulated B cells, while the shaded histograms represent LPS-stimulated B cells. The data in (B) and (D) are a combination of the MFI of each group from three individual experiments and are expressed as a fold change in MFI. The error bars represent the standard error of the mean (SEM). *Indicates p < 0.05 from B cells not exposed to any LPS. The F value for the data in Fig. 2B is 17.4 with 2 degrees of freedom. The F value for Fig. 2D is 6.1 with 2 degrees of freedom.

However, for B cells to present peptide in the context of MHC II, the B cells must first uptake antigen. Previous data indicate that LPS from E. coli increases the ability of B cells to uptake antigen through pinocytosis (Xu et al., 2008). To determine if Oscillatoria sp. LPS increases antigen uptake, purified murine B cells were incubated in the absence or presence of Oscillatoria sp. LPS or E. coli. LPS for 48 h. At the end of this incubation, the cells were washed to remove any residual LPS and incubated with fluoresceinated-dextran (FITC-Dextran-MW 40,000) particles. Antigen internalization was visualized manually and automatically by the use of the Flowsight flow cytometer. As shown in Fig. 3A, unstimulated B cells do not demonstrate any detectable fluorescence, which is in contrast to murine B cells exposed to either E. coli or Oscillatoria sp. LPS. When the fluorescence intensity from multiple independent experiments are combined, both E. coli and Oscillatoria sp. LPS induce significant increase in MFI when compared to unstimulated B cells (ΔMFIE.coli = 2340; ΔMFIOsc. sp. = 787, p < 0.05 when compared to non-LPS exposed B cells for both groups), indicating that Oscillatoria sp. LPS increases antigen internalization by B cells.

Fig. 3.

Fig. 3

Open in a new tab

E. coli and Oscillatoria sp. LPS increases B cell antigen internalization. B cells were isolated from c57Bl/6 mice and incubated in the absence or presence of E. coli or Oscillatoria LPS (105 ng/ml) for 48 h. B cells were then washed and incubated with FITC-dextran for 1 h before analysis by flow cytometry using the Amnis Flowsight flow cytometer. (A) Flowsight analysis of antigen internalization of FITC-dextran by individual cells as described in the Material and Methods. The numbers next to the bright field image refers to the sample number of 10,000 cells that were analyzed. (B) The increase in MFI as an indication of antigen internalization of FITC-Dextran was calculated from three independent experiments and combined. The error bars represent the standard error of the mean (SEM). *Indicates p < 0.05 when compared to B cells not exposed to any LPS (–LPS). The F value for the data in Fig. 2B is 6.1 with 2 degrees of freedom.

Another outcome of LPS engagement on B cells is the production of IgM (Lu and Munford, 2016). To test whether Oscillatoria sp. LPS activates B cells to produce IgM, murine B cells were activated in the absence or presence of increasing concentrations of Oscillatoria sp. or E. coli LPS for 5 days and analyzed by ELISA for IgM production. As shown in Fig. 4, at concentrations of E. coli LPS of 10,000 ng/ml–100,000 ng/ml, murine B cells produce significantly more IgM (14- to 33-fold increase) when compared to B cells not exposed to any LPS (p < 0.05). In contrast, Oscillatoria sp. LPS induces a 7-fold increase in IgM production by B cells at 100,000 ng/ml when compared to unexposed B cells (Fig. 4, p < 0.05). These data indicate that Oscillatoria sp. LPS is agonistic to induce IgM production by murine B cells at high concentrations.

Fig. 4.

Fig. 4

Open in a new tab

E. coli or Oscillatoria sp. LPS induces IgM production. B cells were isolated from c57Bl/6 mice and incubated in the absence or presence of increasing concentrations of E. coli or Oscillatoria LPS for 5 days. Supernatants were harvested before analysis by ELISA. The data are a representative of three experiments with similar results. The error bars represent the standard error of the mean (SEM). *Indicates p < 0.05 from B cells not exposed to any LPS. The F value for the data in Fig. 4A is 5.5 with 5 degrees of freedom. The F value for Fig. 4B is 3.6 with 5 degrees of freedom.

A striking finding among all of the data is that Oscillatoria sp. LPS activates murine B cells, but only at high concentrations. This low level of Oscillatoria sp. LPS-induced B cell activation could be due to alterations in the level of TLR4 engagement after exposure to Oscillatoria sp. LPS vs E. coli LPS. LPS-mediated activation of TLR4 induces the MYD88-dependent activation of both p38 and NF-kB, through phosphorylation of IKKβ (Carpenter and O’Neill, 2009; Carter et al., 1999; Kawai and Akira, 2007). To test if Oscillatoria sp. LPS induced TLR4 signaling, murine B cells were incubated in the absence or presence of either Oscillatoria sp. or E. coli LPS (as a positive control) for 10–30 min and the levels of IKKβ phosphorylation were analyzed by Western blot analysis. As shown in Fig. 5A, the data confirm that exposure of murine B cells to E. coli LPS results in a significant increase in phosphorylation of IKKβ at 10 min, with the levels decreasing at 30 min. This decrease at 30 min in the E. coli LPS-treated murine B cells is in part due to the degradation of IKKβ, as indicated by decreased levels of total IKKβ (Fig. 5A). This decrease in total IKKβ is not due to changes in protein levels loaded into each well, since the levels of immunoglobulin light chain were consistent between all groups within the experiment. When the levels of IKKβ-phosphor-ylation are analyzed after exposure to Oscillatoria sp. LPS, there is a slight increase in phosphorylated IKKβ at 10 min with levels returning to normal by 30 min (Fig. 5A). Additionally, E. coli LPS induced strong and sustained phosphorylation of p38K at both 10 and 30 min (Fig. 5B), which is in contrast to Oscillatoria sp. LPS in which p38K phosphoryla-tion is not detected until after 30 min of exposure. Finally, TLR4 signaling activates IRF-3 in addition to the MYD88 pathway (Kawai and Akira, 2006; McCoy et al., 2008). When the levels of phosphory-lated IRF-3 were analyzed in B cells exposed to either E. coli or Oscillatoria sp. LPS, E. coli LPS induced significant phosphorylation of IRF-3 by 60 min, which increased through 180 min. In contrast, in multiple experiments, Oscillatoria sp. LPS did not significantly enhance IRF-3 phosphorylation through 180 min (Fig. 5C). Taken together, these data suggest that the low level of B cell activation induced by Oscillatoria sp. LPS, as seen in Figs. 1–5, are due to incomplete signaling through TLR4.

Fig. 5.

Fig. 5

Open in a new tab

E. coli or Oscillatoria LPS induces altered Toll like receptor 4 (TLR4) signaling. B cells were isolated from c57Bl/6 mice and incubated in the absence or presence of E. coli or Oscillatoria LPS (105 ng/ml) for 10 or 30 min. Protein lysates were obtained and analyzed by Western blot analysis for (A) phospho-IKKβ, total IKKβ, or mouse light chain (as a control for protein loading), (B) phospho-p38K and total p38K, (C) phospho-IRF-3 and total IRF-3. Data are representative of 3 experiments with similar results.

4. Discussion

Multiple studies have analyzed the ability of cyanobacteria LPS to influence the function of innate immune cells (Jemmett et al., 2008; Macagno et al., 2006; Mayer et al., 2016). However, LPS also has significant effects on B cells of the adaptive immune system. Since it is highly likely that the B cells found within the GALT will be exposed to LPS from cyanobacteria after the ingestion of contaminated food and/or water, the current study investigated whether cyanobacteria Oscillatoria LPS could activate murine B cells. Our findings indicate that Oscillatoria sp. LPS significantly activates B cells to proliferate, serve as an antigen-presenting cells and produce antibody, albeit at low levels.

LPS typically acts as a B cell mitogen that induces the proliferation and differentiation of B cells into plasma cells that produce high levels of antibody, irrespective of their specificity (Lu and Munford, 2016). Our data indicate that at high concentrations of Oscillatoria sp. LPS, B cells proliferate and differentiate into IgM-producing plasma cells. The mucosal lymphoid tissue that surrounds the gut has an extremely high concentration of B cells (Pabst et al., 2008), including B cells with specificities that result in pathology (Arnaboldi et al., 2005; Cardoso et al., 2008). The inappropriate expansion of B cells increases the likelihood the Oscillatoria sp. LPS-mediated activation could contribute to disease, such as allergic responses seen after Oscillatoria sp. exposure (Stewart et al., 2006). Future experiments that analyze the ability of cytokines to promote B cell switch to IgE in the presence of Oscillatoria sp. LPS may provide insight into a possible mechanism of allergic responses seen after Oscillatoria exposure.

One of the most striking findings is that Oscillatoria sp. LPS only induced B cell activation at the highest concentrations of Oscillatoria sp. LPS and with incomplete TLR4 signal transduction. There are multiple reasons that could be responsible for this finding. First, LPS must first interact with LPS binding protein (LBP) and subsequently with CD14. This complex can then interact with TLR4 to activate B cells (Maeshima and Fernandez, 2013). It is possible that Oscillatoria sp. LPS binds to these subunits and/or TLR4 with low affinity to generate a weak and incomplete TLR4 signal. While we have not determined the affinity of Oscillatoria sp. LPS for TLR4, our data indicate that TLR4 engagement by Oscillatoria sp. LPS does not signal in a conventional manner (Fig. 5).

Alternatively, differences in the Lipid A structure between E. coli and Oscillatoria sp. LPS could yield the findings in this study. The number of lipid chains directly impacts the functional activity of LPS (Maeshima and Fernandez, 2013). For example, lipid A variants with six lipid chains results in full TLR stimulation and activation, while lipid A variants with five lipid side chains have approximately 100-fold less activity (Rhee, 2014). Unfortunately, the composition of Oscillatoria sp. LPS is not known and therefore, it is impossible to currently compare the structure between the LPS of these two species. However, preliminary studies suggest Oscillatoria sp. LPS is comprised of different lipids than E. coli LPS (Philip Williams, unpublished data). Additional experiments analyzing the lipid content and structural aspects of Oscillatoria sp. LPS are currently under investigation.

It is likely that there are structural differences between not only Oscillatoria sp. LPS and E. coli LPS, but also with the LPS-like molecule, CyP from Oscillatoria planktothrix FP1. CyP acts as an MD2-TLR4 antagonist, and inhibits the ability of LPS from other Gram-negative bacteria to induce cytokine production by innate immune cells (Jemmett et al., 2008; Macagno et al., 2006; Maeshima and Fernandez, 2013). Structural information regarding CyP was recently determined and it identified differences in the lipid A portion and a much higher molecular mass of the O-chain of LPS, when compared to another cyanobacteria LPS from Synechococcus (Carillo et al., 2014). Therefore, it seems as if differences in the LPS structure among species of cyanobacterium may dictate their ability to induce disease through activation or antagonism of TLR4.

The data presented here are the first to show that Oscillatoria sp. LPS acts as an agonist to stimulate B cell activation and differentiation into antibody-producing cells, which may contribute to cyanobacterial disease. Future studies that analyze the effects of Oscillatoria sp. LPS on other TLR4-expessing immune cells, such as monocytes, will help us to understand the interplay of immune cells that is induced in vivo after exposure to Oscillatoria sp. LPS. Furthermore, the determination of the structure of Oscillatoria sp. LPS and its interaction with TLR4 may give further insight into the mechanisms by which this cyanobacteria contributes to disease in humans and animals.

Acknowledgments

Funding

This research was supported in part by the Office of Research at Midwestern University One Health Intramural Grant, The Biomedical Sciences Program in the College of Health Sciences, Midwestern University (M.S.M. and A.M.S.M.) and the National Institute for Aging [1R01AG039468] (P.W.).

The authors would like to thank Dr. Bryan Bjork and Dr. Sophie Lasalle for providing spleen cells for B cell isolation.

Footnotes

Conflict of interest

None.

References

  1. Arnaboldi PM, Behr MJ, Metzger DW. Mucosal B cell deficiency in IgA−/− mice abrogates the development of allergic lung inflammation. J Immunol. 2005;175(2):1276–1285. doi: 10.4049/jimmunol.175.2.1276. [DOI] [PubMed] [Google Scholar]
  2. Bernardova K, Babica P, Marsalek B, Blaha L. Isolation and endotoxin activities of lipopolysaccharides from cyanobacterial cultures and complex water blooms and comparison with the effects of heterotrophic bacteria and green alga. J Appl Toxicol. 2008;28(1):72–77. doi: 10.1002/jat.1257. [DOI] [PubMed] [Google Scholar]
  3. Cardoso CR, Provinciatto PR, Godoi DF, Vieira TS, Ferreira BR, Teixeira G, Rossi MA, Cunha FQ, Silva JS. B cells are involved in the modulation of pathogenic gut immune response in food-allergic enteropathy. Clin Exp Immunol. 2008;154(2):153–161. doi: 10.1111/j.1365-2249.2008.03748.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carillo S, Pieretti G, Bedini E, Parrilli M, Lanzetta R, Corsaro MM. Structural investigation of the antagonist LPS from the cyanobacterium Oscillatoria planktothrix FP1. Carbohydr Res. 2014;388:73–80. doi: 10.1016/j.carres.2013.10.008. [DOI] [PubMed] [Google Scholar]
  5. Carmichael WaFI. In: Algal toxins in seafood and drinking water. Falconer I, editor. Academic Press; London: 1993. [Google Scholar]
  6. Carpenter S, O’Neill LA. Recent insights into the structure of Toll-like receptors and post-translational modifications of their associated signalling proteins. Biochem J. 2009;422(1):1–10. doi: 10.1042/BJ20090616. [DOI] [PubMed] [Google Scholar]
  7. Carter AB, Monick MM, Hunninghake GW. Both Erk and p38 kinases are necessary for cytokine gene transcription. Am J Respir Cell Mol Biol. 1999;20(4):751–758. doi: 10.1165/ajrcmb.20.4.3420. [DOI] [PubMed] [Google Scholar]
  8. Codd G, Bell S, Kaya K, Ward C, Beattie K, Metcalf J. Cyanobacterial toxins, exposure routes, and human health. Eur J Phycol. 1999;34:405–415. [Google Scholar]
  9. Codd GA, Morrison LF, Metcalf JS. Cyanobacterial toxins: risk management for health protection. Toxicol Appl Pharmacol. 2005;203(3):264–272. doi: 10.1016/j.taap.2004.02.016. [DOI] [PubMed] [Google Scholar]
  10. De Paola M, Mariani A, Bigini P, Peviani M, Ferrara G, Molteni M, Gemma S, Veglianese P, Castellaneta V, Boldrin V, Rossetti C, Chiabrando C, Forloni G, Mennini T, Fanelli R. Neuroprotective effects of toll-like receptor 4 antagonism in spinal cord cultures and in a mouse model of motor neuron degeneration. Mol Med. 2012;18:971–981. doi: 10.2119/molmed.2012.00020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jemmett K, Macagno A, Molteni M, Heckels JE, Rossetti C, Christodoulides M. A cyanobacterial lipopolysaccharide antagonist inhibits cytokine production induced by Neisseria meningitidis in a human whole-blood model of septicemia. Infect Immun. 2008;76(7):3156–3163. doi: 10.1128/IAI.00110-08. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kawai T, Akira S. TLR signaling. Cell Death Differ. 2006;13(5):816–825. doi: 10.1038/sj.cdd.4401850. [DOI] [PubMed] [Google Scholar]
  13. Kawai T, Akira S. Signaling to NF-kappaB by Toll-like receptors. Trends Mol Med. 2007;13(11):460–469. doi: 10.1016/j.molmed.2007.09.002. [DOI] [PubMed] [Google Scholar]
  14. Krieger JI, Grammer SF, Grey HM, Chesnut RW. Antigen presentation by splenic B cells: resting B cells are ineffective, whereas activated B cells are effective accessory cells for T cell responses. J Immunol. 1985;135(5):2937–2945. [PubMed] [Google Scholar]
  15. Lang-Yona N, Lehahn Y, Herut B, Burshtein N, Rudich Y. Marine aerosol as a possible source for endotoxins in coastal areas. Sci Total Environ. 2014;499:311–318. doi: 10.1016/j.scitotenv.2014.08.054. [DOI] [PubMed] [Google Scholar]
  16. LeBien TW, Tedder TF. B lymphocytes: how they develop and function. Blood. 2008;112(5):1570–1580. doi: 10.1182/blood-2008-02-078071. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lu M, Munford R. LPS stimulates IgM production in vivo without help from non-B cells. Innate Immun. 2016;22(5):307–315. doi: 10.1177/1753425916644675. [DOI] [PubMed] [Google Scholar]
  18. Macagno A, Molteni M, Rinaldi A, Bertoni F, Lanzavecchia A, Rossetti C, Sallusto F. A cyanobacterial LPS antagonist prevents endotoxin shock and blocks sustained TLR4 stimulation required for cytokine expression. J Exp Med. 2006;203(6):1481–1492. doi: 10.1084/jem.20060136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Maeshima N, Fernandez RC. Recognition of lipid A variants by the TLR4-MD-2 receptor complex. Front Cell Infect Microbiol. 2013;3:3. doi: 10.3389/fcimb.2013.00003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mayer AM, Clifford JA, Aldulescu M, Frenkel JA, Holland MA, Hall ML, Glaser KB, Berry J. Cyanobacterial Microcystis aeruginosa lipopolysaccharide elicits release of superoxide anion, thromboxane B(2), cytokines, chemokines, and matrix metalloproteinase-9 by rat microglia. Toxicol Sci. 2011;121(1):63–72. doi: 10.1093/toxsci/kfr045. [DOI] [PubMed] [Google Scholar]
  21. Mayer AM, Murphy J, MacAdam D, Osterbauer C, Baseer I, Hall ML, Feher D, Williams P. Classical and alternative activation of cyanobacterium Oscillatoria sp lipopolysaccharide-treated rat microglia in vitro. Toxicol Sci. 2016;149(2):484–495. doi: 10.1093/toxsci/kfv251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. McCoy CE, Carpenter S, Palsson-McDermott EM, Gearing LJ, O’Neill LA. Glucocorticoids inhibit IRF3 phosphorylation in response to Toll-like receptor-3 and -4 by targeting TBK1 activation. J Biol Chem. 2008;283(21):14277–14285. doi: 10.1074/jbc.M709731200. [DOI] [PubMed] [Google Scholar]
  23. Mikheyskaya LV, Ovodova RG, Ovodov YS. Isolation and characterization of lipopolysaccharides from cell walls of blue-green algae of the genus Phormidium. J Bacteriol. 1977;130(1):1–3. doi: 10.1128/jb.130.1.1-3.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Minguet S, Dopfer EP, Pollmer C, Freudenberg MA, Galanos C, Reth M, Huber M, Schamel WW. Enhanced B-cell activation mediated by TLR4 and BCR crosstalk. Eur J Immunol. 2008;38(9):2475–2487. doi: 10.1002/eji.200738094. [DOI] [PubMed] [Google Scholar]
  25. Notch EG, Miniutti DM, Berry JP, Mayer GD. Cyanobacterial LPS potentiates cadmium toxicity in zebrafish (Danio rerio) embryos. Environ Toxicol. 2011;26(5):498–505. doi: 10.1002/tox.20578. [DOI] [PubMed] [Google Scholar]
  26. Ohkouchi Y, Tajima S, Nomura M, Itoh S. Inflammatory responses and potencies of various lipopolysaccharides from bacteria and cyanobacteria in aquatic environments and water supply systems. Toxicon. 2015;97:23–31. doi: 10.1016/j.toxicon.2015.02.003. [DOI] [PubMed] [Google Scholar]
  27. Pabst R, Russell MW, Brandtzaeg P. Tissue distribution of lymphocytes and plasma cells and the role of the gut. Trends Immunol. 2008;8:209–210. doi: 10.1016/j.it.2008.02.006. [DOI] [PubMed] [Google Scholar]
  28. Rhee SH. Lipopolysaccharide: basic biochemistry, intracellular signaling, and physiological impacts in the gut. Intest Res. 2014;12(2):90–95. doi: 10.5217/ir.2014.12.2.90. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Snyder CM, Zhang X, Wysocki LJ. Negligible class II MHC presentation of B cell receptor-derived peptides by high density resting B cells. J Immunol. 2002;168(8):3865–3873. doi: 10.4049/jimmunol.168.8.3865. [DOI] [PubMed] [Google Scholar]
  30. Stewart I, Schluter PJ, Shaw GR. Cyanobacterial lipopolysaccharides and human health—a review. Environ Health. 2006;5:7. doi: 10.1186/1476-069X-5-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Van Gool SW, Vandenberghe P, de Boer M, Ceuppens JL. CD80, CD86 and CD40 provide accessory signals in a multiple-step T-cell activation model. Immunol Rev. 1996;153:47–83. doi: 10.1111/j.1600-065x.1996.tb00920.x. [DOI] [PubMed] [Google Scholar]
  32. Wang JC, Kobie JJ, Zhang L, Cochran M, Mosmann TR, Ritchlin CT, Quataert SA. An 11-color flow cytometric assay for identifying, phenotyping, and assessing endocytic ability of peripheral blood dendritic cell subsets in a single platform. J Immunol Methods. 2009;341(1–2):106–116. doi: 10.1016/j.jim.2008.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Xu H, Liew LN, Kuo IC, Huang CH, Goh DL, Chua KY. The modulatory effects of lipopolysaccharide-stimulated B cells on differential T-cell polarization. Immunology. 2008;125(2):218–228. doi: 10.1111/j.1365-2567.2008.02832.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES