1. The Effects of Noradrenaline on B Cells in vitro
and their Potential Role in the Pathogenesis of
Stroke-Associated Infection
Matriculation No. s0900885
Word Count: 3000/3000

2. 1
Declaration of Support Received and Acknowledgements
I would like to take this opportunity to extend my gratitude to Dr Laura McCulloch for her
patient tuition in a number of lab-based techniques and for her invaluable advice and support
over the course of this project, which could not have existed without her.
I express special thanks to Dr Sara Clohisey and Dr Kenneth Baillie for assisting in the acquisition
of healthy human blood samples and to Dr Anna Raper for her instruction and continuous
assistance in the operating of the flow cytometer.
I sincerely thank Dr Barry McColl for overseeing this work and am grateful to all members of the
McColl Lab for their warm welcome and instrumental guidance throughout my time at the
Roslin Institute.

3. 2
Abstract
Introduction
Stroke is the second leading cause of death worldwide and infection is a common post-stroke
complication that carries a significant degree of morbidity and mortality. There is increasing
recognition that cerebral ischaemia suppresses systemic immunity and that catecholamines
may play an important role in this process. Marginal Zone (MZ) B-cells are innate-like
lymphocytes that constitute part of the initial defence against bacterial infection. This study
investigates the effects of noradrenaline on MZ cells and other B-cells to explore whether these
may be implicated in the pathogenesis of post-stroke infection.
Methods
Peripheral venous blood was extracted from healthy volunteers and white blood cells were
isolated and cultured in varying concentrations of noradrenaline. Antibody labelling and flow
cytometry were used to identify B-cell subpopulations and measure cell number and viability.
Results
The highest concentration of noradrenaline was associated with an increase in number but
decrease in viability of total B-cells. Noradrenaline was also linked to a significant decline in MZ
B-cell numbers at 48 hours.
Conclusion
This study shows that noradrenaline can reduce the viability of B220+ cells and the number of
MZ B cells in vitro and proposes that further research is warranted into the potential role this
plays in post-stroke immunosuppression.
(Word Count: 198/200)

4. 3
Abbreviations
β2-AR β2-Adrenergic Receptor
BCR B Cell Receptor
CNS Central Nervous System
FACS Flow-Assisted Cell Sorting (tubes/buffer)
FCS Foetal Calf Serum
FSC Forward Scatter (of laser light in flow cytometry)
HPA Hypothalamus-Pituitary-Adrenal Axis
Ig_ Immunoglobulin (A/D/E/G/M)
ILC Innate Lymphoid Cells
LPS Lipopolysaccharide
MCAO Middle Cerebral Artery Occlusion
MZ Marginal Zone (B Cells)
NA/NE Noradrenaline/Norepinephrine
PBl Pacific Blue™ (fluorophore)
PI Propidium Iodide
PS Phosphatidyl Serine
RPMI Roswell Park Memorial Institute (Medium)
SD Standard Deviation
SSC Side Scatter (of laser light in flow cytometry)
Th1 Type 1 Helper (T Cells)
TLR Toll-like Receptor

5. 4
Introduction
Stroke is reported to be the second leading cause of death worldwide and is responsible for a
significant degree of morbidity and disability in those who survive the initial neurological
insult1. One of the most common and immediate post-stroke complications is infection,
affecting approximately 30% of patients and worsening their clinical outcomes2. Bacterial
infections of the respiratory and urinary tracts are particularly prevalent and mortality rates are
three times higher amongst those who develop pneumonia2,3.
Although functional impairments such as immobility4 and dysphagia with subsequent
aspiration5,6 can partly explain this phenomenon, an increasing volume of research in both
human patients and mouse models is revealing a systemic immunodepressed state induced by
cerebral ischaemia7,8,9
. Profound splenic and thymic atrophy has been observed following
experimental middle cerebral artery occlusion (MCAO) in mice accompanied by an 80%
reduction in the number of circulating B-lymphocytes10. In humans, peripheral inflammatory
markers are detectable in the circulation early after stroke11 and are followed by a dramatic loss
of T-lymphocytes12 and functional deactivation of both monocytes and Th1 cells9. It is therefore
likely that stroke itself results in a complex, multi-faceted depression of systemic immunity and
that this is responsible for the increased risk of infection.
Approximately 75% of post-stroke infections occur within 3 days of the event2. Innate immune-
mediated mechanisms respond rapidly to pathogens whilst elements of adaptive immunity
usually require days-weeks13. Hence, this time course implies that impaired functioning of the
innate immune system is one possible mechanism contributing to this phenomenon. Classically,
B and T-lymphocytes have been considered members of the adaptive immune system.
However, recent investigations into the functioning of these cells as well as the discovery of
Innate Lymphoid Cells (ILCs), B1 Cells and Marginal Zone B-Cells has blurred this boundary and
led to the recognition that many lymphocytes do indeed possess important innate-like
functions14,15.

6. 5
Human Marginal Zone (MZ) B-cells are so named because of their primary site of residence in
the marginal zone of the spleen although they are also found within lymph nodes, tonsils,
Peyer’s patches and the peripheral bloodstream, where they constitute approximately 5% of
circulating B-cells16. Their positioning at these interfaces between lymphoid-tissue and the
circulation enable MZ B-cells to act as first-line sentinels, responding rapidly to blood-borne
antigens by differentiating into short-lived plasma cells and secreting antibodies15,17. MZ B-cells
accomplish this independently of T-cells, with the high speed and low specificity that is
characteristic of the innate immune system, primarily through the activation of polyreactive
BCR and TLR receptors15
. Dual-stimulation of these receptors by LPS (an endotoxin found in the
outer membrane of encapsulated bacteria such as Streptococcus pneumoniae and Haemophilus
influenzae) results in a rapid mobilisation of MZ B-cells and subsequent differentiation into IgM,
IgG and IgA-secreting plasma cells15,16,18. Interestingly, encapsulated bacteria are commonly
implicated in pneumonia leading to the speculation that stroke-induced changes to MZ B-cells
could be one plausible explanation for the increased risk of pneumonia in stroke patients. In
support of this theory is the observation that hyposplenic or asplenic individuals are similarly
vulnerable to life-threatening infections with encapsulated bacteria and that this susceptibility
correlates with a reduced number of circulating MZ B-cells18,19
.
Complex, bi-directional interactions exist between the CNS and the immune system, mediated
by neural and humoral pathways which include the vagus nerve, the sympathetic nervous
system and the HPA axis20. Lymphoid organs, including the spleen, are highly innervated by
sympathetic nerve fibres and β2-AR receptors have been identified on several populations of B
and T-cells21. This has led some to propose that CNS injury might modulate the immune
response via sympathetic signalling to the spleen7. Blockade of the sympathetic nervous system
reduces the incidence of bacterial infection following model stroke in mice whilst blocking the
HPA axis does not8. Additionally, administration of the β-adrenoceptor blocker propranolol to
these mice significantly reduces post-stroke infection and mortality rates8.

7. 6
In conjunction, the above findings provide compelling evidence that ischaemic stroke prompts
adrenergic signalling to the spleen resulting in a systemic state of immunosuppression which
predisposes the individual to subsequent bacterial infection. The time course and typical
pathogens involved suggest that MZ B-cells and other innate-like lymphocytes may play an
important role in this process.
This study forms part of a larger body of work seeking to investigate the mechanisms
underlying post-stroke immunosuppression with a particular emphasis on innate-like B cells and
adrenergic signalling. The present report aims to determine the effects that noradrenaline
elicits on peripherally-circulating, human MZ B-cells and other B-cell subpopulations via in vitro
cell culture. As noradrenaline is the primary neurotransmitter utilised by the sympathetic
nervous system, the results of this research could provide important insights into how CNS
ischaemia can modulate some aspects of immunity via adrenergic signalling and why this might
result in an increased risk of post-stroke infection.

8. 7
Materials and Methods
Blood Samples
40ml of venous blood was extracted via peripheral venepuncture from healthy volunteers
under ethical approval granted by the Lothian Research Ethics Committee (11/AL/0168).
Samples were immediately centrifuged at 1400rpm for 10 minutes at room temperature and
the resultant plasma supernatant discarded. Erythrocytes were then lysed using an erythrocyte
lysis buffer (BioLegend, Cat.#420301) and centrifuged to produce a pellet of white cells which
were subsequently re-suspended in the appropriate medium, outlined below.
Viable white cells were counted on a haemocytometer under light-microscopy following
staining with 0.4% Trypan Blue (ThermoFisher Scientific, Cat.#15250-061). Cell concentrations
were then adjusted to 5x106cells/ml via the addition of medium and 100µl of cell suspension
added per well on a 96-well plate.
Cell Culture Media
Two separate, liquid media were compared during the initial stages of investigation. AIM-V®
Medium (ThermoFisher Scientific, Cat.#12055-091) was purchased pre-synthesised and is
claimed to be a highly effective medium for human lymphocyte cultures. Another medium
optimised for B-cell survival was created by mixing RPMI with 1% L-Glutamate, 10% Heat-
inactivated FCS, 1% Penicillin-Streptomycin, 1% Sodium Pyruvate, 1% Non-Essential Amino
Acids and 0.1% β-mercaptoethanol.
Noradrenaline Preparations
1mM aliquots of Noradrenaline were produced from crystalline (-)-Norepinephrine (Sigma-
Aldrich Cat.#51-41-2). This was further diluted with medium to give final concentrations of
1µM, 10µM, 100µM and 500µM per well.
Cells with varying concentrations of noradrenaline, or medium alone, were then cultured for 4,
24 or 48 hours in a cell incubator at 37oC with 0.5% CO2.

9. 8
Antibody Labels
Following culture, all plated cells were centrifuged at 1400rpm for 10 minutes at 4o
C and the
resultant supernatant discarded. Cells were re-suspended in 50µl of 1/500 Human TruStain
FcX™ (Biolegend, Cat.#422301) and incubated on a plate shaker for 15 minutes at room
temperature. This ensures that the Fc receptors on white cells are saturated and limits false
antibody labelling.
50µl of an antibody cocktail was added to each well with the purpose of identifying particular B-
cell subsets. The cocktail contained the following antibodies: 1/100 APC Anti-Human/Mouse
CD45R(B220)(RA3-6B2) (TONBO Biosciences Cat.#20-0452-U100) to identify all B-lymphocytes;
1/50 PerCP/Cy5.5 anti-human CD23 (BioLegend Cat.#338518) to exclude mature B-cells, T-cells,
monocytes, macrophages, Langerhans cells and eosinophils; 1/20 Pacific Blue™ anti-human
CD1c (Biolegend Cat.#331507) to partially identify MZ B-cells; and 1/200 APC/Cy7 anti-human
IgM (BioLegend Cat.#314519) to identify IgM positive B-cells. Cells were incubated in the above
cocktail for 30 minutes on a plate shaker at room temperature prior to centrifuge and disposal
of the supernatant.
Analysis of Cell Viability
Cell viability was assessed using the Alexa Fluor®-488 Annexin-V/Dead Cell Apoptosis Kit
(ThermoFisher Scientific Cat.#V-13245) which contains Annexin-V Alexa Fluor™ 488, Propidium
Iodide (PI) and Annexin Binding Buffer.
Once cells had been washed in PBS, they were re-suspended in 100µl of Annexin Binding Buffer.
1µl of 1/10 PI and 5µl of Annexin-V-488 were added to each well and cells incubated for 15
minutes at room temperature. In apoptotic cells, Phosphatidyl Serine (PS) is translocated to the
outer surface of the plasma membrane. Annexin-V binds to PS, thus labelling apoptotic cells. PI
does not permeate the plasma membrane of live or apoptotic cells and thus will only label
necrotic cells. Viable cells will remain unlabelled and exhibit no fluorescence.

10. 9
Flow Cytometry
Cells were transferred to FACS tubes containing Annexin Binding Buffer and underwent flow
cytometry on a Becton Dickinson LSR Fortessa utilising the following lasers: R670 to detect
B220-APC; B710 to detect CD23-PerCP/Cy5.5; V450 to detect CD1c-PBl; R780 to detect IgM-
APC/Cy7; YG586 to detect PI; and B530 to detect Annexin-V-488.
Forward Scatter (FSC) and Side Scatter (SSC) were used to draw a gate around putative
lymphocytes and cytometry continued until 10,000 cells within this gate were recorded for each
sample, unless otherwise stated. Flow cytometry results were further analysed using Summit-
v4.3 software.
Unlabelled and single antibody-labelled controls were used to calibrate the positive gating-
threshold for each fluorophore. Some emission spectra bled through into nearby channels and
had to be digitally compensated to limit false labelling. Once all gates and compensation levels
had been set, these remained unchanged throughout the experiments to ensure consistency
between replicates.
Statistical Analysis
Due to large differences in the recorded number of cell populations between each of the
biological replicates, experiments had to be analysed individually for statistical significance.
Only significant findings which were common to all biological samples (n=4) are presented,
using 3 technical replicates unless otherwise stated. Graphs and figures are drawn from one
sample for demonstrative purposes showing mean values ± SD between technical replicates.
For normally distributed data, differences were analysed using Student’s t-test for 2 groups or
1-way ANOVA with Dunnett’s multiple comparison test to compare multiple groups with a
single control. All data were analysed using the statistics package built in to GraphPad Prism-
v6.07 and P-values <0.05 were accepted as statistically significant throughout.

11. 10
Results
Comparison of AIM-V® Medium and Murine B-cell Medium
An appropriate medium for cell culture had to be ascertained prior to further investigation. Two
potential candidates were assessed and compared during the initial phase of the study, namely
AIM-V® medium and a B-cell medium optimised for murine B-cell cultures.
No significant differences in the total number of B220+ cells were seen between the two media
at either 4, 24 or 48 hours. However, the proportion of B220+ cells which were viable was
considerably higher in the AIM-V® medium at all three time points [Fig. 1]. This difference was
observed to be greatest at 48 hours (27.6 ± 3.1% viable in AIM-V® medium compared to 1.8 ±
0.8% viable in B-cell medium).
Despite this finding, the decision was made to continue using the B-cell medium for the
remaining experimental cultures to ensure comparability with experiments already utilising this
medium to culture murine splenocytes. It is also worth noting that this experiment was
performed early in the author’s training and shows lower mean viability than later experiments
once various lab skills had been improved.
Figure 1: Comparison of Cell Culture Media. The percentage of viable B220+
cells was greater in the AIM-V®
medium at all measured time points though the total number of B220+
cells did not vary significantly. Results
shown are for one biological sample with three technical replicates. Statistical significance was determined using
multiple student’s t-tests with Holm-Šídák method. Columns show mean values with SD error bars. ***P<0.001;
****P<0.0001.

12. 11
The Effects of Noradrenaline on B220+ cells
Next, the effects of noradrenaline (NA) on B-lymphocytes were assessed by counting the total
number of B220+ cells within each culture and measuring the proportions of viable, apoptotic
and dead cells. Although B220 is a B-cell marker, it is present on some other white cells and
thus, it cannot be confidently asserted that B-cells are solely responsible for the trends outlined
below.
No significant differences were seen in the number of B220+ cells at 4 hours, though there was
a paradoxical rise in their number in the highest concentration of NA at the 24 and 48-hour
time points [Fig. 2a]. This increase in B220+ cell number did not translate into an increase in the
number of live cells however as it was coupled with a dramatic decline in the proportion of
viable B220+
cells [Fig. 2b].
Figure 2: The Effects of Noradrenaline on B220+
cell Number and Viability. (a) – The number of B220+
cells was
significantly higher in culture wells with 500µM NA compared to medium alone at 24 and 48 hours. (b) – The % of
viable B220+
cells was markedly lower in 500µM NA across all three time points. Results shown are for 1 biological
sample with 3 technical replicates but trend was significant across 4 biological samples (n=4). Statistical
significance was determined using one-way ANOVA with Dunnett’s multiple comparison test. Columns show mean
values with SD error bars. ***P<0.001; ****P<0.0001.

13. 12
Identification of B-cell Subpopulations
All B220+ cells which were also found to be positive for CD23 were excluded as this is a negative
marker for MZ B-cells. The remaining B220+ cells were plotted on a graph of V450xR780
fluorescence which determines the expression of CD1c and IgM respectively. Three distinct cell
populations were observed and can be classified as: B220+CD23-CD1chiIgMhi cells; B220+CD23-
CD1cloIgMlo cells; and B220+CD23-CD1c-IgM- cells [Fig. 3]. The first of these populations closely
resembles the description of human MZ B-cells found within the literature16,17,22. All three B-cell
subpopulations were investigated further to determine any effects that noradrenaline exerted
upon them.
Figure 3: Flow Cytometry Identification of B cell Subpopulations. B220+
CD23-
cells were plotted on the dot-plot
diagram above. The X-axis displays fluorescence detected after stimulation with a V450 laser and indicates CD1c
expression. The Y-axis displays fluorescence detected after stimulation with an R780 laser and indicates IgM
expression (the “Comp” refers to digital compensation which limits cross-channel bleed-through). Note that both
axes are logarithmic. Three distinct populations are observable. (i) – A population of B220+
CD23-
CD1chi
IgMhi
cells
which resemble MZ B cells. (ii) – A population of B220+
CD23-
CD1clo
IgMlo
cells. (iii) – A population of B220+
CD23-
CD1c-
IgM-
cells.

14. 13
The Effects of Noradrenaline on MZ B cells and other B cell Subpopulations
To assess the effects of noradrenaline on MZ B-cells, the total number of B220+CD23-
CD1chiIgMhi cells were counted within each culture [Fig. 3(i)]. The proportions of viable,
apoptotic and necrotic cells were also assessed.
Within the 48-hour cultures, there was a significant decline in the number of MZ B-cells in the
highest concentration of noradrenaline compared to medium alone [Fig. 4]. A similar trend
seemed to be apparent at 24 hours but did not achieve statistical significance across all
samples.
The proportion of viable MZ B-cells did not vary significantly between different noradrenaline
concentrations at any time point. However, these cells did prove uniquely vulnerable to cell
death during culture with only 54.2 ± 21.3% of cells remaining viable at 4 hours in medium
alone (n=4).
Finally, two other B-cell subpopulations [Fig. 3(ii-iii)] were examined, however no significant
trends were observed in either population at any time point that were common to all biological
samples.
Figure 4: The effects of Noradrenaline on MZ B cells. The number of B220+
CD23-
CD1chi
IgMhi
cells (MZ B) cells are
displayed for each noradrenaline concentration following 48 hour culture. A significant decline in cell number is
seen in the 500µM culture well compared to medium alone. Results shown are for 1 biological sample with 3
technical replicates but trend was significant across 4 biological samples (n=4). Statistical significance was
determined using one-way ANOVA with Dunnett’s multiple comparison test. Columns show mean values with SD
error bars. ***P<0.001.

15. 14
Discussion
Interpretation of Results
The number of total B220+ cells appears to significantly increase in cultures with high
concentrations of noradrenaline at 24 hours and 48 hours. This could reflect an increase in B-
cell number and indeed some previous studies have shown that B-cell β2-AR activation can
induce proliferation and increased antibody secretion21. However, B220 can be found on a
number of other white cells which were neither labelled nor assessed as part of this study.
Noradrenaline has been observed to decrease proliferation rates and induce apoptosis in T-cell
populations both in vitro and in vivo21,23 and thus, it is unlikely that T-cell expansion is
responsible for the observed trend. However, it cannot be excluded that other white cells (e.g.
monocytes and macrophages) are at least partially responsible for this increase in B220+ cell
number.
The above finding was coupled with a profound decline in B220+ cell viability. It is unclear
whether this was a direct effect of noradrenaline stimulating B2-AR receptors or an indirect
effect mediated by another cell within the culture. It could be possible that this decline in
viability is a consequence of a greater concentration of cells competing for limited resources
within the medium24. However, this is unlikely as viability is seen to be compromised as early as
4 hours, prior to an increase in cell count. One could argue that the increase in cell number is
spurious and reflects apoptotic B220+ cell-fragments being counted as whole cells by the flow
cytometer but this explanation is also improbable due to steps of the analysis comparing the
height and area of all events to exclude cell-clusters and debris.
A population of B220+CD23-CD1chiIgMhi MZ B-cells was identified which comprised
approximately 5-10% of the total circulating B-cell population, matching similar figures from
previous authors16,17. Numbers of MZ B-cells were found to be lowest in cultures with the
highest concentration of noradrenaline at 48 hours. This time course fits with the peak
incidence of stroke-associated bacterial infection occurring at 3 days2 and thus, it is one
plausible explanation for this phenomenon.

16. 15
Observed levels of apoptosis and necrosis among MZ B-cells did not show statistically
significant variations in the present study. However, it is still uncertain whether the decline in
MZ B-cell number is due to apoptosis, necrosis, or differentiation and further experimentation
is required to elucidate this. Although this trend appeared unique to MZ B-cells and was not
significant among other measured B-cell subpopulations, further biological replicates are
required to confirm or refute this finding.
All cells analysed within this report were from peripheral circulating blood and any elicited
effects may not be representative of how these cells would behave within the spleen. It is
possible that human splenocytes behave differently when exposed to noradrenaline and this
requires further study.
Limitations of Study
There were a number of limitations within the current study which could be partially resolved
through the modification of future experiments.
Firstly, due to time constraints and the lengthy nature of cell cultures, only four biological
samples could be analysed for the bulk of experiments. This had an impact on the statistical
power of the results and, because of large variations in cell numbers between samples,
experiments had to be analysed individually and common trends identified.
Inter-experimental inconsistencies arose which were partly the result of increasing skill in
performing lab-based techniques and partly due to unforeseen circumstances. For example,
low levels of antibody during one experiment necessitated that only two technical replicates be
performed for each culture whilst three replicates were used elsewhere. Some variations were
unavoidable, such as donor white cell counts and disparity in the initial proportions of
lymphocytes and B-cell subpopulations.

17. 16
Future Directions
The finding that AIM-V® medium is associated with a greater degree of B220+ cell viability
warrants its use in future B-cell culture studies.
Repeating the experimental protocol outlined above with a greater number of biological
samples would maximise statistical significance and hopefully allow results to be analysed as
mean values across all biological replicates. Apparent trends within this current study which
failed to achieve statistical significance could also be more confidently asserted or refuted.
Proposed modifications to the above protocol would include the use of greater concentrations
of noradrenaline as concentrations below 500µM did not appear to elicit any measurable
effects on cells. Additionally, culturing the cells for longer time periods and adding Propranolol
to certain culture wells may yield additional interesting results. Finally, culturing B-cells from
human spleen samples could explore potential differences in the effects of noradrenaline
between splenocytes and circulating B-cells.
Conclusion
In conclusion, the present study reveals that culturing white cells in noradrenaline may result in
an increase in B220+ cell number but a decrease in cell viability. It further demonstrates that
noradrenaline exposure may decrease the number of MZ B-cells over a period of 48 hours with
relative sparing of other B-cell subpopulations. Combined with current research, this suggests
that a noradrenaline-mediated decline in MZ B-cell number is one plausible mechanism
underlying the observed increased risk of bacterial infection following stroke and that this
warrants further investigation.

18. 17
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