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Toxicol Lett. Author manuscript; available in PMC 2018 June 05.
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Published in final edited form as:

Toxicol Lett. 2017 June 05; 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-Mungersona,*, Ryan Incroccia, Vijay Subramaniamb, Philip Williamsc,
Mary L. Halld, and Alejandro M.S. Mayerd
aDepartment of Microbiology and Immunology, Chicago College of Osteopathic Medicine,
Midwestern University, 555 31st Street, Downers Grove, IL 60515, United States
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bDepartment of Biomedical Sciences, College of Health Sciences, Midwestern University, 555

31st Street, Downers Grove, IL 60515, United States
cDepartment of Chemistry, University of Hawaii-Manoa, 2545 McCarthy Mall, Honolulu, HI 96822,
United States
dDepartment of Pharmacology, Chicago College of Osteopathic Medicine, Midwestern University,
555 31st Street, Downers Grove, IL 60515, United States

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

*
Corresponding author. mswans@midwestern.edu (M. Swanson-Mungerson).
Conflict of interest
None.
 Swanson-Mungerson et al. Page 2

1. Introduction
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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
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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.
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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.
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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.

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2. Materials and methods

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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.,
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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
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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
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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

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with FITC-Dextran at 4 °C for 1 h to serve as a basis for non-specific binding. All cells were
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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
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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
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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
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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

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 Swanson-Mungerson et al. Page 5

groups. A finding is determined to be significant when the p value was equal to or less than
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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
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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.

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
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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.

However, for B cells to present peptide in the context of MHC II, the B cells must first
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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,

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unstimulated B cells do not demonstrate any detectable fluorescence, which is in contrast to

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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.

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
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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.

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
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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
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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.

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4. Discussion
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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.
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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
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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).
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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.

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It is likely that there are structural differences between not only Oscillatoria sp. LPS and E.
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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
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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.).
Author Manuscript

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

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Fig. 1.
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.
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Fig. 2.
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
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(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

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 Swanson-Mungerson et al. Page 13

value for the data in Fig. 2B is 17.4 with 2 degrees of freedom. The F value for Fig. 2D is
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6.1 with 2 degrees of freedom.

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Fig. 3.
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
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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.
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Fig. 4.
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.
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Fig. 5.
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.
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