Distinct Differentiation Programs Triggered by IL-6 and LPS in Teleost IgM+ B Cells in the Absence of Germinal Centers

Abstract

Although originally identified as a B cell differentiation factor, it is now known that mammalian interleukin-6 (IL-6) only regulates B cells committed to plasma cells in response to T-dependent (TD) antigens within germinal centers (GCs). Even though adaptive immunity is present in teleost fish, these species lack lymph nodes and GCs. Thus, the aim of the present study was to establish the role of trout IL-6 on B cells, comparing its effects to those induced by bacterial lipopolysaccharide (LPS). We demonstrate that the effects of teleost IL-6 on naïve spleen B cells include proliferation, activation of NF-κB, increased IgM secretion, up-regulation of Blimp1 transcription and decreased MHC-II surface expression that point to trout IL-6 as a differentiation factor for IgM antibody-secreting cells (ASCs). However, LPS induced the secretion of IgM without up-regulating Blimp1, driving the cells towards an intermediate activation state in which antigen presenting mechanisms are elicited together with antibody secretion and expression of pro-inflammatory genes. Our results reveal that, in trout, IL-6 is a differentiation factor for B cells, stimulating IgM responses in the absence of follicular structures, and suggest that it was after follicular structures appeared that this cytokine evolved to modulate TD responses within the GC.

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Scientific RepoRts | 6:30004 | DOI: 10.1038/srep30004
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Distinct Dierentiation Programs
Triggered by IL-6 and LPS in Teleost
IgM+ B Cells in The Absence of
Germinal Centers
Beatriz Abós1, Tiehui Wang2, Rosario Castro1, Aitor G. Granja1, Esther Leal1,
Jerey Havixbeck3, Alfonso Luque1, Daniel R. Barreda3, Chris J. Secombes2 & Carolina Tafalla1
Although originally identied as a B cell dierentiation factor, it is now known that mammalian
interleukin-6 (IL-6) only regulates B cells committed to plasma cells in response to T-dependent (TD)
antigens within germinal centers (GCs). Even though adaptive immunity is present in teleost sh,
these species lack lymph nodes and GCs. Thus, the aim of the present study was to establish the role of
trout IL-6 on B cells, comparing its eects to those induced by bacterial lipopolysaccharide (LPS). We
demonstrate that the eects of teleost IL-6 on naïve spleen B cells include proliferation, activation of
NF-κB, increased IgM secretion, up-regulation of Blimp1 transcription and decreased MHC-II surface
expression that point to trout IL-6 as a dierentiation factor for IgM antibody-secreting cells (ASCs).
However, LPS induced the secretion of IgM without up-regulating Blimp1, driving the cells towards
an intermediate activation state in which antigen presenting mechanisms are elicited together with
antibody secretion and expression of pro-inammatory genes. Our results reveal that, in trout, IL-6
is a dierentiation factor for B cells, stimulating IgM responses in the absence of follicular structures,
and suggest that it was after follicular structures appeared that this cytokine evolved to modulate TD
responses within the GC.
e immune system comprises both innate and adaptive immune responses. While the innate immune system
is genetically programmed to detect invariant features of invading microbes, the cells of the adaptive immune
system, such as conventional B cells (B2) and T cells, detect specic epitopes through somatically recombined
receptors. However, it is now recognized that both branches of immunity are highly interconnected and B cells
also possess a certain capacity to directly sense and respond to pathogens though the expression of certain pattern
recognition receptors (PRRs) or through the action of cytokines produced by cells of the innate immune system1.
In general, conventional B cells are activated in response to T-dependent (TD) antigens within the lymphoid
follicles and trigger the formation of germinal centers (GCs). ese sites promote the close collaboration between
proliferating antigen-specic B cells, T follicular helper cells, and the specialized follicular dendritic cells (DCs)
that constitutively occupy the central follicular zones of secondary lymphoid organs. In this environment,
B cells divide in response to antigens and acquire the capacity to dierentiate into antibody-secreting cells (ASCs),
reaching a terminal state of plasma cells or memory B cells, both of them with the capacity to secrete high anity
antibodies. is TD pathway provides a strong long-lived immunological memory, but is relative slow to occur.
us, it must be integrated with additional T-independent (TI) pathways that mainly involve other B cell subsets
such as B1 cells or marginal zone (MZ) B cells. ese TI responses do not require cooperation from T cells, but
instead are much more responsive to products secreted by cells of the innate immune system and have a greater
capacity to directly recognize pathogens1.
Although evolutionarily jawed sh constitute the rst group of animals in which adaptive immunity based on
Ig receptors is present2, many structural immune peculiarities predict important functional dierences between
sh and mammalian B cells. e teleost spleen constitutes the main secondary immune organ in the absence of
lymph nodes. However, the splenic white pulp is poorly developed in teleosts in comparison to mammals and
1Centro de Investigación en Sanidad Animal (CISA-INIA), Madrid, Spain. 2Scottish Fish Immunology Research Centre,
University of Aberdeen, Aberdeen, UK. 3Department of Biological Sciences, University of Alberta, Alberta, Canada.
Correspondence and requests for materials should be addressed to C.T. (email: tafalla@inia.es)
Received: 27 April 2016
Accepted: 28 June 2016
Published: 02 August 2016
OPEN

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no GCs are apparent3. Regarding mucosal immunity, although sh B cells have been reported in surfaces such as
gills, skin, digestive tract and nasal cavities4,5, they are scattered throughout the mucosa in disorganized lymphoid
structures6. Additionally, sh contain only three immunoglobulin classes IgM, IgD and IgT (designated as IgZ
in some species). IgT is a teleost sh-specic Ig that seems specialized in mucosal immunity7,8 and IgT+ B cells
constitute a distinct linage7, thus no class switch recombination has ever been reported in sh. As a result, the
lack of teleost follicular structures already anticipates that sh B cell responses best resemble mammalian extr-
afollicular responses. Consequently, teleost B cells share many features of mammalian B1 cells, as for example a
high phagocytic capacity9,10, constitutive expression of many PRRs4,11 or expression of B1-specic cell markers12.
Interleukin 6 (IL-6) is a multi-functional cytokine produced by a wide range of cell types in the early stages
of infection. IL-6 modulates a plethora of immune functions through a receptor composed of the restricted IL-6
receptor chain (IL-6R) and a common signal transducer, gp13013. Although initially described as a B cell dif-
ferentiation factor14, it was later demonstrated that IL-6 is a potent growth and maturation factor only for cells
that have already initiated a dierentiation process towards plasma cells, but has minimal capacity to directly
induce plasma cell dierentiation15. Besides, IL-6 enhances antibody production of ASCs but only those that are
antigen-specic, whereas non-specic ASCs are unresponsive to IL-616. Interestingly, the normal development of
GCs is signicantly altered in the absence of IL-617. Consequently, IL-6 deciency signicantly impairs early TD
IgG production, but has no eect on IgM TI responses17,18. us, it has been postulated that IL-6 is predominantly
involved in the maturation of TD plasma cells in the early stages of GC formation19. At a mucosal level, IL-6 also
seems implicated in promoting IgA TD responses20. Strikingly, the number of B1-derived IgA-secreting cells
signicantly increases in the absence of IL-620, demonstrating that B1 cells are not regulated by this cytokine and
can even be negatively aected by it. Despite this, IL-6 in combination with an anti-IgM antibody induces the
expression of surface CD5 on conventional B2 cells that acquire phenotypic characteristics of B1 cells21.
Taking into account the main role of IL-6 on TD responses in mammals and the unique attributes of sh
B cells, it is important to determine the eect of IL-6 on sh B cells, which we do here using the rainbow trout
(Oncorhynchus mykiss) as a model. We compare the eects elicited by IL-6 to those triggered by lipopolysaccha-
ride (LPS). LPS, a protein-free endotoxin from the cell wall of Gram-negative bacteria, is the most extensively
studied TI antigen in mammals and is directly recognized by B cells through low anity B cell receptors (BCRs)
and PRRs such as Toll like receptors (TLRs)22. LPS is not a mitogen for human B cells, since human B cells express
neither TLR4 nor CD14, the two canonical ligands for Gram (− ) bacterial LPS23,24. In contrast, mice B cells
express TLR4 and are polyclonally activated by it25. Interestingly, although TLR4 is thought to be absent from the
genome of salmonid sh26, some eects of LPS on salmonid B cells have been reported27.
Our ndings demonstrate that, in contrast to what occurs in mammals, B cells obtained from healthy unstim-
ulated trout respond to IL-6. e eects that IL-6 exerted on these IgM+ B cells included proliferation, activa-
tion of NF-κ B, increased IgM secretion, up-regulation of Blimp1 transcription, increased size and decreased
MHC-II surface expression, all pointing to trout IL-6 as a dierentiation factor for naïve IgM B cells towards
ASCs. Interestingly, LPS also increased IgM secretion but the activation prole was very dierent to that elicited
by IL-6, driving the IgM+ B cells towards a pro-inammatory state. Additionally, IL-6 stimulated B cells mobilized
more intracellular calcium in response to BCR cross-linking, demonstrating that IgM ASCs retain a functional
BCR on the cell surface. us, our results demonstrate that in trout, where follicular structures have not yet been
developed, IL-6 regulates IgM B cell responses. is suggests that the emergence of follicular structures marked a
critical point in IL-6 evolution, acquiring a novel capacity to specically regulate TD antibody responses.
Results
The IL-6 receptor complex is transcribed in IgM+ B cells from naïve sh that are activated by
IL-6 stimulation. IL-6 exerts its biological activities through two molecules: the IL-6 receptor, IL-6Rα (also
known as gp80 and CD130) and the associated signal transducer glycoprotein gp130. While IL-6Rα is important
for ligand binding, gp130 is required for adequate intracellular signaling28. Prior to studying the eects of IL-6
on naïve B cells in sh, we determined if these two molecules were constitutively transcribed in sorted naïve
IgM+ B cells from spleen, blood and kidney, being this last organ the main hematopoietic tissue in sh. We found
that IgM+ B cells from all three tissues constitutively transcribed IL-6Rα and gp130 at similar expression levels
(Supplemental Figure S1A), suggesting that naïve B cells have the capacity to respond to IL-6. ese results con-
trast those of mammals, where only activated B cells express IL-6R16,28.
To conrm that unstimulated B cells from trout are responsive to IL-6, we rst examined the expression of
SOCS3 and STAT3, two genes involved in the signaling of IL-6, in sorted IL-6-stimulated trout IgM+ B cells. Both
SOCS3 and STAT3 were signicantly up-regulated in IgM+ B cells in response to IL-6 stimulation (Supplemental
Figure S1B), as previously reported in other IL-6-responsive cell types in trout29. LPS, on the other hand, induced
the transcription of STAT3 but had no eect on SOCS3 mRNA levels (Supplemental Figure S1B). To further
conrm these results and demonstrate a direct eect of IL-6 on IgM+ B cells, we sorted IgM+ cells and aerwards
incubated them with IL-6 or LPS. Aer 1 h of incubation, the phosphorylation of STAT3 was conrmed through
Western blot in response to IL-6 and LPS (Supplemental Figure S1C). ese results conrm a direct eect of IL-6
on IgM+ B cells.
IL-6 induces IgM+ cell proliferation. As already mentioned, mammalian IL-6 has no eect on naïve B
cells, but has been shown to induce the proliferation of pre-B cells30 as well as plasmablasts31. In contrast, LPS is
a potent inducer of B cell proliferation in mice and sh27,32. us, we compared the lymphoproliferative eects
of LPS to those of IL-6 in trout splenocytes. Our results show that trout spleen IgM+ B cells signicantly prolif-
erated in response to IL-6 (Fig.1a,c), although at levels signicantly lower than those elicited by LPS (Fig.1a,c).
When the percentage of IgM+ B cells was evaluated in these cultures without taking into account BrdU uptake,
the percentage of IgM+ cells was quite similar in LPS- and IL-6-treated cultures, suggesting an additional positive

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eect of IL-6 on B cell survival (Fig.1b). e proliferative and survival eects observed were specic to IL-6 since
another cytokine produced and tested in parallel, IL-4/13A, was unable to increase the number of trout IgM+ cells
during the in vitro culture33.
Since in mammals IL-6 only activates B cells previously stimulated with TD antigens17,18, we also evaluated
the potential synergistic eect of IL-6 on IgM+ B cell proliferation induced by a TD antigen (TNP-KLH) or a
TI antigen (TNP-LPS). We performed this experiment because despite their lack of follicular structures, sh
can orchestrate responses to TD antigens34, possibly through a mechanism that resembles rare extrafollicular
TD responses reported in mammals35–37. For this, we incubated splenocytes with TNP-KLH or TNP-LPS in the
presence or absence of IL-6 and evaluated the percentage of BrdU+/IgM+ B cells aer 4 days. We observed that
the addition of IL-6 could further enhance the proliferative eects provoked by TNP-LPS or TNP-KLH (Fig.1d),
indicating that, in trout, IL-6 can have regulatory eects on both TD and TI responses.
Figure 1. IL-6 and LPS elicit B cell proliferation and increased survival. Spleen leukocytes were incubated
with IL-6 (200 ng/ml) or LPS (100 μ g/ml) for 3 days at 20 °C. Aer this time, BrdU was added to the cultures and
incubated for a further 24 h. e percentage of proliferating (BrdU+) IgM+ B cells was determined as described in
Material and Methods. (a) Percentage (mean + standard deviation) of proliferating IgM+ B cells (BrdU+/IgM+)
aer treatment with IL-6 and/or LPS (n = 6). (b) IgM+ B cell survival estimated as percentage of IgM+ cells
(proliferating and non-proliferating cells) in cultures (mean + standard deviation) (n = 6). (c) Representative
dot plots are shown. (d) Spleen leukocytes were stimulated with TNP-KLH (5 μ g/ml) or TNP-LPS (5 μ g/ml) in
the presence or absence of IL-6 (200 ng/ml). e percentage of proliferating IgM+ cells was assessed as described
above. Data are shown as the mean fold change relative to the control value for unstimulated controls + standard
deviation (n = 5). Asterisks denote signicant dierences between cells treated with IL-6 or LPS and their
corresponding controls and between IL-6 and LPS treated cells when indicated. * P < 0.05, * * P < 0.01.

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IL-6 up-regulates IgM secretion in naïve B cells. In mammals, IL-6 is unable to induce dierentiation
of naïve B cells towards plasmablasts on its own15. In contrast, the in vitro incubation of naïve splenic B cells with
IL-6 was sucient to induce the secretion of IgM, as veried in an ELISPOT assay (Fig.2a,b). When this experi-
ment was carried out in previously immunized sh, IL-6 also increased the secretion of IgM, at levels signicantly
higher than those observed in non-immunized healthy trout (Supplemental Figure S2). As previously reported27,
LPS also induced the secretion of IgM by trout B cells, however, our results suggest that the mechanisms through
which IgM secretion is induced dier between IL-6 and LPS-stimulated B cells. At the transcript level, only IL-6
up-regulated the transcription of Blimp1, a protein required for the development of ASCs and the maintenance of
long-lived plasma cells38, while LPS had no eect (Fig.2b). Interestingly, IL-6 also up-regulated the transcription
of ACKR2, an atypical chemokine receptor specic for innate B cells39, while LPS showed no eect (Fig.2b). us,
Figure 2. IL-6 and LPS activate IgM secretion in naïve B cells. (a) ELISPOT analysis of IgM-secreting cells
in splenocyte cultures treated with IL-6 (200 ng/ml), LPS (100 μ g/ml) or non-stimulated. Splenocytes were
cultured for 3 days in ELISPOT plates previously coated with anti-trout IgM mAb (2 μ g/ml) in the presence or
absence of the dierent stimuli. Aer incubation, cells were washed away and a biotinylated anti-trout IgM mAb
(1 μ g/ml) was used to detect numbers of spot forming cells. Duplicates from a representative experiment (le)
and quantication of spot forming cells (right) from 5 independent experiments are shown (mean + standard
deviation). (b) Spleen leukocytes were incubated with media containing IL-6, LPS or control media alone for 24 h
at 20 °C. Aer that time, IgM+ B cells were sorted using an anti-trout IgM mAb and RNA was extracted. Relative
transcript expression of Blimp-1 and ACKR2 is shown (mean + standard deviation, n = 6). (c) Membrane IgM
and total IgM expression of IgM+ cells aer incubation with IL-6, LPS or control media for 24, 48 and 72 h. Mean
uorescence intensity (MFI) + standard deviation is shown (n = 9). (d) Dot plots and histograms showing the
Forward scatter (FSC) from IgM+ B cells and BrdU+/IgM+ B cells incubated in the presence or absence of LPS
or IL-6, from one representative experiment. Graphs showing FSC MFI values from 8 independent experiments
(mean + standard deviation) are included next to the histograms for stimulated cultures. * P < 0.05, * * P < 0.01,
* * * P < 0.001.

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our results demonstrate that, unlike mammalian IL-6, sh IL-6 is a dierentiation factor for naïve B cells towards
an ASC prole. On the other hand, LPS seems to induce the secretion of IgM in a Blimp1-independent fashion.
In mammals, the dierentiation of B cells to plasma cells provokes a down-regulation of membrane Igs that
completely loose the BCR when terminally dierentiated40. However, recent reports have demonstrated that this
seems to only be true for IgG plasma cells, since fully dierentiated IgM or IgA-secreting plasma cells retain a
functional BCR in the cell membrane41. In our studies, we observed that both IL-6 and LPS increased the levels of
expression of total IgM in B cells (Fig.2c), along with increased membrane IgM levels (Fig.2c). erefore, these
IL-6 or LPS-induced plasmablasts retain and even up-regulate IgM levels in the cell surface while increasing their
secretion of IgM, similar to mammalian IgM plasma cells. e secretion of IgM in response to LPS or IL-6 was
associated with an increase in the size of IgM+ cells, as shown by increased forward side scatter (FSC) in stimu-
lated cells in comparison to control cells. is increase in size was signicant when proliferating cells were ana-
lyzed (Fig.2d) demonstrating that LPS and IL-6 induce the proliferation of IgM+ B cells and part of the progeny
dierentiate into ASCs that retain IgM on the cell surface.
IL-6 activates NF-κB in spleen IgM+ B cells. Aer binding to its receptor, IL-6 activates gene expression
mainly through the STAT3 pathway28,42 and, in line with its pro-inammatory nature, it has been shown to acti-
vate NF-κ B in intestinal epithelial cells43. Despite this, whether IL-6 activates NF-κ B in mammalian B cells has
not been established. In mice, LPS induces a dual BCR/TLR-signaling by engaging TLR4 through the lipid A moi-
ety and the BCR through the polysaccharidic moiety44. As a result, both the canonical and non-canonical NF-κ B
signaling pathways are activated in B cells in response to LPS. To determine whether IL-6 and LPS activate the
NF-κ B signaling pathway in our system, we analyzed the translocation of the NF-κ B p65 subunit to the nucleus
of IgM+ B cells using a quantitative imaging ow cytometry-based approach which we have used previously11. We
observed a signicant increase in the percentage of total splenocytes and IgM+ B cells in which p65 translocated
to the nucleus in response to IL-6 and LPS in comparison with control cells, indicating that both stimuli activate
the canonical NF-κ B pathway (Fig.3a).
However, when the transcriptional activity of NF-κ B-dependent genes such as IL-1β 1, IL-8 or TNF-α 3 was
studied, only LPS was capable of inducing a signicant up-regulation of their mRNA levels (Fig.3b). Similarly,
dierential eects of LPS and IL-6 in the transcriptional activation of antimicrobial peptides was also observed.
While LPS up-regulated cathelicidin 1 (CATH1) mRNA levels (Fig.3b) CATH2 was signicantly up-regulated
by both LPS and IL-6, although the eect of IL-6 was much stronger (Fig.3b). Taken together, these results show
that although IL-6 and LPS activate NF-κ B in IgM+ B cells, the downstream eects of this activation are dierent.
IL-6 and LPS-stimulated IgM+ B cells are predisposed for BCR-mediated calcium mobili-
zation. Anti-IgM stimulation of B cells mimics the recognition of a high affinity antigen by the BCR,
Figure 3. Eect of IL-6 and LPS on NF-κB activation and transcription of pro-inammatory and
antimicrobial genes in B cells. (a) Translocation of NF-κ B to the nucleus in IgM+ splenocytes following
stimulation with LPS (100 μ g/ml) or IL-6 (200 ng/ml) for 24 h. e mean percentages of NF-κ B translocation in
total cells (upper panel) and IgM+ B cells (lower panel) from six independent experiments are shown. * * P < 0.01,
* * * P < 0.001. (b) Spleen leukocytes were incubated with IL-6, LPS or control media alone for 24 h at 20 °C. e
eect of IL-6 and LPS on the transcription of IL-1β 1, IL-8, TNF-α 3, cathelicidin-1 (CATH1), CATH2 and hepcidin
was then studied in sorted IgM+ B cells from these cultures. e relative transcript expression (mean + standard
deviation) of 6 independent experiments is shown. * P < 0.05, * * P < 0.01, * * * P < 0.001.

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consequently leading to a rapid increase of intracellular calcium45. is calcium mobilization plays an important
role in B cell activation and is required for correct downstream signaling of the BCR45. us, we also studied the
eect of anti-IgM cross-linking of the BCR in splenocyte cultures that had been previously stimulated with IL-6
or LPS and compared them to those in non-stimulated control cells, given the fact that IL-6 or LPS-stimulated
cells seem to retain their BCR. As expected, anti-IgM induced a rapid mobilization of intracellular calcium, and
furthermore, the levels of calcium released upon anti-IgM stimulation were signicantly higher in cells that had
been previously stimulated with IL-6 or LPS (Fig.4a,b). is stimulatory eect was observed aer 24, 48 or 72 h of
stimulation. us, our results reveal that IL-6 renders the cells more responsive to BCR engagement.
IL-6 down-regulates the surface expression of MHC-II in naïve trout B cells. e response of
B cells against an antigen not only requires antigen binding to and signaling through the BCR but also the
processing and presentation of the BCR-bound antigen to helper T cells in the context of MHC-II46. us, we
also determined the eect of IL-6 or LPS stimulation on the levels of expression of surface MHC-II of naïve
B cells using a specic anti-trout MHC-II antibody. We observed that IL-6 provoked a signicant decrease of
surface MHC-II expression that was detectable from 24 h post-stimulation up to 72 h post-stimulation (Fig.5a).
In contrast, LPS-stimulated B cells showed increased surface MHC-II levels at all time points studied (Fig.5a).
Along with this increase in MHC II levels, LPS also up-regulated the levels of transcription of two co-stimulatory
molecules: CD80/86, a molecule with similar homologies to mammalian CD80 and CD8647, and CD83 (Fig.5b).
IL-6, on the other hand, had no eect on their transcription levels (Fig.5b).
IL-6 has no eect on the phagocytic activity of IgM+ B cells. Since trout B cells have a potent phago-
cytic activity9, we also investigated whether IL-6 or LPS pre-stimulation of naïve IgM+ B cells could have an
eect on their phagocytic activity. For this, splenocytes were incubated with IL-6 or LPS for dierent incubation
periods, or le-unstimulated in the same conditions. ereaer, 1 μ m polystyrene-based uorescent beads were
added to the cultures and aer 3 h of incubation the phagocytic activity of IgM+ cells was determined in ow
cytometry. We observed that while pre-stimulation of B cells with LPS signicantly increased the percentage of
phagocytic IgM+ B cells in spleen, pre-stimulation with IL-6 produced no eect (Fig.6a,b).
IL-6 has a synergistic eect on antigen-specic IgM responses in vivo. Given the stimulatory
eects observed for IL-6 on IgM+ B cells in vitro, we next investigated whether this stimulatory eect on naïve
IgM+ B cells could also condition IgM responses elicited in vivo in response to a specic antigen. For this, we
immunized sh with inactivated infectious pancreatic necrosis virus (IPNV) either alone or in combination with
IL-6. Control groups treated with IL-6 alone or PBS were also included. e administration of IL-6 alone sig-
nicantly increased the number of IgM-secreting cells in the spleen at day 2, but no eect was observed in the
following days (Fig.7a). IPNV on its own was unable to increase the number of IgM-secreting cells in spleen or
kidney when compared to controls, however when IPNV was combined with IL-6, the number of IgM-secreting
cells signicantly increased in spleen at day 6 post-immunization and in the kidney at day 2 post-immunization,
when compared to controls or sh injected with IPNV alone (Fig.7a). ese positive eects of IL-6 on the num-
ber of ASCs elicited by IPNV, correlated with signicantly increased IPNV-specic IgM titers in sera of sh
injected with IPNV and IL-6 when compared to titers in sh injected with IPNV alone (Fig.7b). Surprisingly,
this increase in IPNV-specic IgM had no eect on total IgM titers (Fig.7b) at the time point sampled (day 15
post-immunization).
To get additional information on the antibodies present in the sera we also analyzed the titers of natural anti-
bodies against four classic antigens, BSA, galactosidase, phosphorylcholine and dsRNA (poly I:C) in the same
Figure 4. IL-6 and LPS predispose IgM+ B cells to calcium mobilization aer BCR cross-liking. Spleen
leukocytes were incubated with IL-6 (200 ng/ml), LPS (100 μ g/ml) or control media alone for 24, 48 and 72 h
at 20 °C. Aer the dierent incubation periods, cells were loaded with Fluo-3 AM (5 μ M nal concentration),
and its baseline emission was measured by ow cytometry for 30 s, and then stimulated with 0.5 ug/ml
of Alexa 647 conjugated anti-IgM mAb. Fluorescence was then measured for a further 180 s. (a) Mean
uorescence intensity (MFI) plus standard deviation of intracellular Ca2+ levels (Fluo-3) in IgM+ B cells is
shown (n = 6). * P < 0.05, * * P < 0.01, * * * P < 0.001. (b) Scatter plot showing Fluo-3 MFI levels in spleen
IgM+ B cells aer 48 h of incubation with the appropriate stimuli.

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groups. We observed a signicant decrease in the titers of antibodies that bind these four antigens in all the groups
treated with IL-6 or/and IPNV. Interestingly, at the same time that IL-6 signicantly increased IPNV-specic IgM
titers when compared to those elicited by IPNV alone (Fig.7b), it decreased the amount of natural antibodies
reactive against BSA or galactosidase in serum (Fig.7b). Taken together, our results strongly suggest that when
combined with an antigen in vivo, IL-6 preferentially activates antigen-specic B cells as occurs in mammals.
Discussion
Although mammalian IL-6 exclusively signals in antigen-experienced cells15, thus only aecting the outcome
of switched antibody isotypes such as IgG or IgA18,19,48; a recent study reported that fugu (Takifugu rubripes)
unstimulated B cells transcribe both IL-6Rα and gp130 and that the in vitro stimulation of blood leukocytes with
IL-6 up-regulates IgM transcription levels42. ese results suggest that sh B cells from healthy unstimulated sh
could be responsive to IL-6. Hence, we decided to investigate further the capacity of IL-6 to regulate B cell activity
in sh, using the rainbow trout as a model. Once we had veried that rainbow trout unstimulated IgM+ B cells
also transcribe IL-6Rα and gp130, we examined the eects of IL-6 on dierent relevant functions of sh B cells.
We compared the eects provoked by IL-6 to those elicited by LPS, since there is still some controversy about the
degree to which trout cells respond to LPS. Although LPS stimulation was reported to increase IgM secretion and
lymphocyte proliferation in trout27, TLR4 seems absent from salmonid genomes26. In this context, our studies
would help to establish how, in the absence of follicular structures, sh IL-6 regulates the activity of IgM+ B cells.
Furthermore, our results would provide additional evidence as to how teleost B cells respond to LPS.
LPS is a potent mitogen of B cells and the in vitro responses of these cells to LPS are very robust both in mice32
and in sh49. In our experiments, LPS and IL-6 were both mitogenic for trout IgM+ cells. Although the prolifera-
tion levels in response to IL-6 were signicantly lower than those observed for LPS, the percentage of viable IgM+
B cells in the cultures was similar in response to the dierent stimuli and signicantly higher than the percentage
observed in control cultures, suggesting that non-proliferating B cells have an increased survival in IL-6-treated
Figure 5. Dierential eects of IL-6 and LPS on antigen presenting properties of IgM+ B cells. Spleen
leukocytes were incubated with IL-6 (200 ng/ml), LPS (100 μ g/ml) or control media alone for 24, 48 and 72 h at
20 °C. e levels of MHC-II expression on the surface of IgM+ B cells were then measured via ow cytometry
using a specic mAb against trout MHC-II. (a) MFI + standard deviation from 6 independent experiments.
* P < 0.05, * * P < 0.01, * * * P < 0.001. Representative histogram from one experiment shown below. (b) Spleen
leukocytes were incubated with IL-6, LPS or control media alone for 24 h at 20 °C. e eect of IL-6 and LPS on
the transcription of CD80/86 and CD83 co-stimulatory molecules was then studied in sorted IgM+ cells from
these cultures. e relative transcript expression (mean + standard deviation) of 5 independent experiments is
shown. * P < 0.05, * * P < 0.01.

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cultures, even higher than that observed in LPS-treated cultures. In mammals, while some authors have reported
a proliferative eect of IL-6 on pre-activated B cells50, other researchers have described stimulation of Ig secretion
in the absence of proliferative responses51,52. However, even in those cases where proliferating eects have been
reported, they seem to be associated only with previously activated cells and are never seen in unstimulated B
cells. Although it could be possible that some of the B cells in our cultures have been previously exposed to an
antigen, it seems unlikely that all the B cells responding in our experiments are pre-activated cells given the fact
that these cells express IgD on the cell membrane and transcribe surface IgM and IgD at high levels (data not
shown). On the other hand, even though we stimulated B cells in cultures in which other cells were present dur-
ing the incubation period, we have demonstrated that STAT3 is phosphorylated in IgM+ B cells incubated with
IL-6 in the absence of other cell types. Likewise, it should be noted that there was no signicant proliferation of
IgM− cells in these cultures (Fig.1c) and that even if IgM+ B cells already accounted for approximately 30% of the
leukocyte population in the spleen, this percentage was increased to approximately 50% in IL-6-treated cultures
aer 4 days. All the evidence points to a preferential eect of IL-6 on a quite abundant population of B cells within
the spleen of unstimulated sh. In addition to these experiments in which leukocytes were treated with IL-6
alone, we also determined the eect of IL-6 on the B cell proliferation induced by antigen encounter, and again
observed stimulatory eects. ese synergistic eects were visualized in response to either TD (TNP-KLH) or TI
(TNP-LPS) antigens, unlike the situation in mammals where IL-6 only seems to promote TD responses17,18. us,
trout IL-6 has positive eects on proliferation and survival of B cells stimulated with TD antigens, and in contrast
to mammalian IL-6, it also has mitogenic eects on trout naïve B cells and cells activated with TI antigens.
IL-6 was originally reported as a cytokine capable of inducing antibody production in B cell lines, augmenting
the secretion of both IgM and IgG on Epstein-Barr virus transformed B cells51,52. However, later reports demon-
strated a preferential eect of IL-6 on switched antibody isotypes, given the fact that IL-6 only plays a role in
Figure 6. IL-6 has no eect on the phagocytic capacity of IgM+ B cells. Splenocytes were cultured in the
presence of IL-6 (200 ng/ml) or LPS (100 μ g/ml) for 24, 48 or 72 h at 20 °C. Non-stimulated controls were
also included. Aer the dierent incubation periods, cells were exposed to uorescent beads for a further
3 h at 20 °C. Non-ingested beads were removed by centrifugation over a cushion of 3% (w/v) BSA in PBS
supplemented with 4.5 (w/v) D-glucose (Sigma). (a) Data are shown as mean percentage of phagocytic IgM+
B cells (le) or Mean uorescence intensity (MFI) (right) + standard deviation from six independent sh.
* P < 0.05, * * P < 0.01. (b) Representative dot plots for each condition.

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the terminal dierentiation of antigen-experienced B cells15. us, IL-6 over-expression in mice provokes IgG
plasmacytosis, but has no eects on plasma cells of other Ig isotypes53,54. Likewise, IL-6-decient mice showed
reduced antigen-specic IgG1, IgG2a and IgG3 levels aer immunization with a TD antigen, but IgM levels were
unaected17. IL-6 also supports the dierentiation of IgA-secreting plasma cells in dierent mucosal surfaces55,56.
Teleost sh do not express IgG, IgA or IgE and rely on non-switched IgM, IgD and IgT responses to ght infec-
tions. In this context, trout IL-6 was capable of inducing IgM secretion in spleen B cells, at a level comparable
Figure 7. Synergistic eects of IL-6 on antigen-induced in vivo IgM production. Rainbow trout were
injected i.p. with 100 μ l of PBS, 100 μ l of PBS with IL-6 (100 ng/sh), 100 μ l of PBS with 2 × 1010 TCID50/ml
inactivated IPNV, or 100 μ l of PBS containing the same amount of IPNV and IL-6. (a) e number of IgM
secreting cells was evaluated at days 2, 6 and 15 post-injection in spleen and kidney leukocyte cultures by
ELISPOT as described in the Materials and methods section (mean + standard deviation; n = 6). Asterisks
indicate signicant dierences between groups as indicated. * P < 0.05. (b) Total IgM, IPNV-specic and natural
(BSA; galactosidase; PC, phosphorylcholine; and poly I:C-specic) IgM titers were measured in the sera of
sh killed at day 15 post-immunization by ELISA. Results are shown as absorbance at 405 nm (for total IgM)
or absorbance at 492 nm (for specic IgMs) for individual sh. Bars indicate mean values in each group and
asterisks denote signicant dierences between groups as indicated. * P < 0.05.

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to that induced by LPS. is increased IgM secretion in response to IL-6 was evidenced in ELISPOT and ow
cytometry and the fact that these IL-6 stimulated cells increased in size and up-regulated Blimp1 further supports
our observations indicating that trout IL-6 has the capacity to induce the dierentiation of unstimulated B cells to
IgM ASCs. Interestingly, LPS also provoked a size increase and augmented IgM secretion but without induction of
Blimp1, suggesting that either LPS exerts its activity on a dierent B cell population to IL-6 or alternatively it acts
on the same population but drives them towards a dierent activation state. In mammals, B2 cells dierentiate
into ASCs in response to LPS along with Blimp1 up-regulation, however, there is some controversy as to whether
B1 cells require Blimp1. While some studies revealed that mammalian B1 cells secrete IgM independently of
Blimp157, the IgM production by B1 cells in Blimp1-decient animals was inhibited, suggesting the requirement
of Blimp1 for normal IgM production58.
To further study the eects of IL-6 on IgM secretion, we performed additional experiments in v ivo. Here again,
the in vivo administration of IL-6 alone signicantly increased the number of IgM-secreting cells in the spleen
at day 2 post-injection. is eect was not observed in the kidney and was lost in the spleen at later sampling
points, revealing a tissue-specic transitional eect. In mammals, some of the positive eects that IL-6 has on
IgG expression are also transitional, since IL-6 is required for the normal induction, but not for the maintenance
of plasma cell responses in vivo because the eects of dierent survival factors are redundant59. Additionally,
we studied how IL-6 aected the antibody response elicited by the injection of an IPNV vaccine, observing that
although IPNV by itself was unable to induce a signicant IgM response, when combined with IL-6, the number
of IgM-secreting cells in the spleen or head kidney signicantly increased in comparison to sh immunized
with IPNV alone. Although the stimulatory eect on the number of IgM-secreting cells was no longer visible at
day 15 post-injection, at this point we could still see a synergistic eect of IL-6 on the amount of IPNV-specic
antibodies in serum. While total IgM levels were not signicantly higher in sh injected with IPNV and IL-6,
the increase in the amount of IPNV-specic IgM went along with a signicant decrease in the quantity of IgM
with alternative specicities, such as BSA- or galactosidase-specic IgM. Increases in antigen-specic IgG titers
with constant total IgG titers have also been reported in humans and the authors postulated that specic IgG
levels were increased without having an eect on total IgG titers because the ratio of antigen-specic cells was
very low in relation to the total pool of B cells60. Similarly, it seems that trout IL-6, when combined with IPNV,
increases IgM secretion of a discrete number of B cells with no eect on overall IgM production. Furthermore,
it seems that the negative eect of IL-6 on the production of natural antibodies is a conserved eect, as antibod-
ies against phosphorylcholine and LPS are increased in IL-6 gene knockout mice20. Taken together, our results
clearly demonstrate that IL-6 is able to enhance antigen-specic IgM responses in sh as observed in mammals
for switched Ig isotypes17,53,55,56.
When mammalian B cells start their dierentiation towards plasma cells, a secretory switch in the mRNA pro-
vokes the down-regulation of surface Ig and an increase of secreted Ig, resulting in the lack of BCR once the cells
are fully dierentiated40. However, recent studies have demonstrated that whilst this phenomenon occurs in IgG
plasma cells it is not seen in IgM or IgA-secreting plasma cells, as the latter preserve a functional BCR in the cell
surface even if fully dierentiated41. Similarly, we observed that the IL-6- or LPS-induced dierentiation of trout
B cells to IgM ASCs was not associated with a decrease in surface IgM levels. As hypothesized in mammals41, the
presence of a functional BCR on IgM ASCs would allow them to respond directly to their specic antigen upon
secondary encounters, unlike IgG ASCs. e fact that IL-6 and LPS-activated B cells retain a functional BCR,
despite their dierentiation to ASCs was conrmed by the mobilization of intracellular calcium in response to
anti-IgM cross-linking. Interestingly, these stimulated cells mobilized intracellular calcium at levels signicantly
higher than control B cells, demonstrating that IL-6 and LPS predispose B cells to a posterior signaling through
the BCR despite their dierentiation to ASCs. is is the rst time such an eect is reported for IL-6 in B cells and
consequently it would be interesting to determine the eect of mammalian IL-6 on BCR signaling in IgM- and
IgA-secreting plasma cells.
e transcription factor NF-κ B is critically involved in many cellular processes such as inammation, immune
response, proliferation or apoptosis61. In our experiments, we have established that IL-6 and LPS canonically
activate NF-κ B in IgM+ B cells from unstimulated sh. In mammals, BCR cross-linking, CD40 ligation62 or stim-
ulation with LPS or phorbol ester63 have been shown to activate NF-κ B in B cells. However, to our knowledge,
this is the rst report of IL-6-mediated NF-κ B activation in B cells, although IL-6 has been shown to activate this
transcription factor in epithelial cells43. As a consequence of NF-κ B activation, dierent immune genes should
be transcriptionally up-regulated. However, only LPS induced the transcription of typically NF-κ B-regulated
genes such as IL-1β , IL-8 and TNF-α , suggesting that although IL-6 activated cells translocate p65 to the nucleus,
additional cellular mechanisms modify the downstream eects. Of course, the up-regulation of IgM synthesis
observed in IL-6-stimulated B cells could be regulated by NF-κ B as the promoter of the IgM light chain is one of
the main targets for NF-κ B in mammals64. Since it is known that dierent NF-κ B components can be activated
through a variety of mechanisms with dierent downstream eects61, it seems possible that IL-6 and LPS are
modulating distinct NF-κ B activation pathways, and this should be studied further.
As trout IL-6 induced the transcription of several antimicrobial peptides in trout macrophages29 and taking
into account that B cells in sh have antimicrobial properties associated to their phagocytic activity9, we also
studied the eect of IL-6 and LPS on CATH-1, CATH-2 and hepcidin transcription. While CATH-1 and CATH-2
were up-regulated by LPS, IL-6 only up-regulated CATH-2, as occurred in macrophages29. Despite the fact that
IL-6 modulates hepcidin transcription in macrophages29, IL-6 had no eects on hepcidin expression in IgM+ B
cells. Nevertheless, these results highlight the potent antimicrobial properties of sh B cells. Similar to other anti-
gen presenting cells, B cells express MHC II on the cell surface. us, we determined the eects of IL-6 and LPS
on MHC II surface expression levels and the phagocytic capacity of trout B cells. Again the eects exerted by IL-6
and LPS were quite dierent, all of them summarized in Table1. While IL-6 signicantly down-regulated MHC-II
surface expression on B cells, as expected in an ASC65, LPS provoked a signicant up-regulation of MHC-II

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expression and increased mRNA levels of the co-stimulatory molecules CD80/86 and CD83. e activation of
IL-6/STAT3 also induces the suppression of antigen presentation in human dendritic cells66. In contrast, and as
seen in our studies, mammalian B cells stimulated with LPS up-regulate MHC-II surface expression together
with co-stimulatory molecules (CD86, CD40)67 and plasma cells dierentiated in response to TI antigens retain
MHC-II expression and a functional antigen presenting machinery68. us, it seems that plasma cells generated
in response to LPS or other TI antigens, are not exclusively specialized in antibody secretion and still play a role
in other immune functions such as antigen presentation and pathogen clearance, and in line with this hypothesis,
LPS provoked a slight but signicant increase in the number of phagocytic B cells in the spleen cultures. is
increase was observed from 24 to 72 h post-treatment, whereas IL-6 had no eect. Since mammalian B1 cells are
also phagocytic, it would be interesting to determine if LPS is capable of increasing their phagocytic capacity.
Overall our results demonstrate that in contrast to mammals, sh IL-6 can trigger unstimulated B cells to
initiate their dierentiation towards ASCs. Consequently, trout IL-6 has mitogenic eects on IgM+ cells and these
proliferating cells increase in size, augment their secretion of IgM, up-regulate Blimp1 and decrease their surface
MHC-II expression. On the other hand, LPS shows a potent mitogenic activity and increases the secretion of IgM,
while driving the cells towards a quite dierent prole that predicts additional functions such as increased anti-
gen presentation (given the increased MHC-II surface expression, up-regulated transcription of co-stimulatory
molecules and higher phagocytic activity) and a pro-inammatory role (through the up-regulation of IL-1ß, IL-8
and TNF-α ). ese results highlight that IgM secretion can be induced through quite distinct dierentiation
pathways in sh.
Methods
Animals. Healthy specimens of female rainbow trout (Oncorhynchus mykiss) of approximately 50–70 g were
obtained from Centro de Acuicultura El Molino (Madrid, Spain). Fish were maintained at the Animal Health
Research Center (CISA-INIA) laboratory at 14 °C with a re-circulating water system and 12:12 h light:dark photo-
period. Fish were fed twice a day with a commercial diet (Skretting, Spain). Prior to any experimental procedure,
sh were acclimatized to laboratory conditions for 2 weeks and during this period no clinical signs were ever
observed. e experiments described comply with the Guidelines of the European Union Council (2010/63/
EU) for the use of laboratory animals and were previously approved by the Ethics committee from the Instituto
Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA; Code CEEA 2011/044).
Leukocyte isolation. Rainbow trout were killed via benzocaine (Sigma) overdose and blood was extracted
with a heparinized needle from the caudal vein and diluted 10 times with Leibovitz medium (L-15, Life
Technologies) supplemented with 100 I.U./ml penicillin, 100 ug/ml streptomycin (P/S), 10 units/ml heparin and
5% fetal calf serum (FCS) (all supplements also obtained from Life Technologies). Spleen and kidney were col-
lected and single cell suspensions generated using 100 μ m nylon cell strainers (BD Biosciences). Blood cell sus-
pensions were placed onto 51% Percoll (GE Healthcare) cushions whereas kidney and spleen suspensions were
placed onto 30/51% discontinuous density gradients. All suspensions were then centrifuged at 500 × g for 30 min
at 4 °C. e interface cells were collected and washed twice with L-15 containing 5% FCS.
Immune function IL-6 LPS
STAT3 transcription Up-regulated Up-regulated
SOCS3 transcription Up-regulated No eect
STAT3 phosphorylation Activated Activated
Mitogenic eects Signicant Very signicant
IgM secretion Increased Increased
mIgM expression Increased Increased
Total IgM expression Increased Increased
Blimp1 transcription Up-regulated No eect
ACKR2 transcription Up-regulated No eect
p65 translocation to nucleus Ye s Ye s
IL-1β transcription No eect Up-regulated
IL-8 transcription No eect Up-regulated
TNF-α transcription No eect Up-regulated
CATH1 transcription No eect Up-regulated
CATH2 transcription Up-regulated Up-regulated
Hepcidin transcription No eect No eect
BCR signaling to anti-IgM Increased Increased
MHC-II expression Decreased Increased
Phagocytic activity No eect Increased
CD80/86 transcription No eect Up-regulated
CD83 transcription No eect Up-regulated
Table 1. Summary table comparing the eects of IL-6 and LPS on trout spleen IgM+ B cells.

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Production of rainbow trout recombinant IL-6. e sequence encoding the mature peptide of trout IL-669
was amplied from spleen cDNA prepared from Aeromonas salmonicida infected sh70 and cloned to the pET/
Duet-1 vector (Novagen, UK). e N-terminal ATG (M) codon and a C-terminal His tag (GSGHHHHHHHHHH)
were incorporated from the vector for translation initiation and for purication of the recombinant protein. us,
the rtIL-6 had 211 amino acids, with a predicted molecular weight of 23.8 kDa and pI of 7.27. Sequence analysis
of three clones (pET/IL-6a, b, c) revealed that they were identical but diered by four nucleotides, of which two
resulted in two amino acid changes from the published IL-669, presumably due to polymorphism. e sequence has
been submitted to EMBL/GenBank/DDBJ databases under the accession number FR715329. A plasmid was used to
transform BL21 Star (DE3) competent cells (Invitrogen) and protein expression was induced by IPTG and puried
under denaturing conditions as described previously71. e puried, denatured rtIL-6 was refolded in refolding
buer containing 50 mM Tris–HCl, pH8.0, 0.5 M arginine, 0.5% Triton-100 and 5 mM β -mercaptoethanol at 4 °C for
2 days. e refolded rtIL-6 was repuried under native conditions and eluted at concentrations of up to 0.5 mg/ml.
LPS contamination was checked by examining the expression of a number of LPS-responsive genes, including
IL-1β , TNF-α , IL-8, IL-10 and IL-11, and by undertaking the Limulus amoebocyte lysate assay (Sigma) as per the
manufacturer’s instructions.
Cell stimulation. Total leukocyte populations were dispensed in 24-well plates at a density of 2 × 106 cells per ml
and incubated with the appropriate stimulus: recombinant trout IL-6 (200 ng/ml), LPS (100 μ g/ml; > 95% purity)
(Sigma), TNP-KLH (5 μ g/ml) (Biotools), TNP-LPS (5 μ g/ml) (Biotools) or anti-IgM Ab (2 μ g/μ l) (clone 1.14)72 at
concentrations previously optimized. Non-stimulated controls were always included. Cells were always incubated
at 20 °C for dierent periods of time depending on specic experiments.
Cell sorting. IgM+ B cells were sorted from spleen, blood or kidney leukocyte suspensions using a BD FACSAria
III (BD Biosciences) cell sorter. For this, leukocytes were incubated for 30 min on ice with an anti-trout IgM mAb
(1.14) coupled to phycoerythrin (PE) in Staining buer (PBS containing 1% FCS and 0.5% sodium azide) that
prevents cell activation. Following two washing steps, cells were resuspended in FACS buer and IgM+ B cells iso-
lated based on their FSC/SSC proles (to exclude the granulocyte gate) and then on the basis of the uorescence
emitted by the anti-trout IgM antibody. IgM+ and IgM− cells were then collected in Trizol for subsequent RNA
isolation.
Real time PCR analysis of sorted cells. Total RNA was isolated from IgM+ sorted populations using
Tri-reagent (Life Technologies). e cDNA synthesis and real-time PCR analysis were performed as described
previously33,71. e primers (Supplementary Table S1) for real-time-PCR were designed so that at least one primer
crossed an intron, to ensure that genomic DNA could not be amplied under the PCR conditions used. e
expression level of each gene was rst normalized against the expression level of EF-1α and then expressed as a
fold change that was calculated as the average expression of the IL-6/LPS stimulated samples divided by that of
the controls.
Detection of phosphorylated STAT3. To conrm the phosphorylation of STAT3 in IL-6 and LPS-treated
B cells and demonstrate direct eects on IgM+ B cells, we rst sorted IgM+ cells from splenocyte cultures as
described above and then incubated them with the dierent stimuli in the absence of other cell types. For this,
sorted cells adjusted to 2 × 106 cells per ml were disposed in 96 well plates and incubated with IL-6 (200 ng/ml),
LPS (100 μ g/ml) or media alone for 1 h at 20 °C. Aer the incubation, cells were lysed using RIPA buer contain-
ing protease inhibitors (Roche). Proteins were fractionated onto a denaturing 12% SDS-PAGE gel and transferred
onto a polyvinylidene diuoride (PVDF) membrane (Immobilon-P; Millipore Merck). Aer blocking in PBS with
5% skim milk for 1 h, the membrane was incubated with a mAb against phospho-STAT3 (Ser727) (Santa Cruz
Biotechnology) in blocking solution at 4 °C overnight. Aer three washing steps, the membrane was incubated
for 1 h with the secondary antibody, a goat anti-mouse IgG-HRP conjugate (GE Healthcare Life Sciences). e
reactive bands were visualized with the ECL system (GE Healthcare Life Sciences).
B cell proliferation. e BrdU Flow Kit (Becton Dickinson) was used to measure the proliferation of IgM+
cells following manufacturer’s instructions. Splenocytes at a concentration of 2 × 106 cells per ml were incubated
for 3 days at 20 °C with the dierent stimuli as described above. Bromodeoxyuridine (BrdU, 10 μ M) was then
added to the cultures and the cells were incubated for an additional 24 h. Aer that time, trout cells were collected
and stained with anti-IgM-PE (1.14) antibody and then xed and permeabilized with Cytox/Cytoperm Buer
for 15 min on ice. Aerwards, cells were incubated with Cytoperm Permeabilization Buer Plus for 10 min on ice
and re-xed with Cytox/Cytoperm Buer for 5 min at RT. Cells were then incubated with DNase (30 μ g/106 cells)
for 1 h at 37 °C to expose the incorporated BrdU. Finally, cells were stained with FITC anti-BrdU antibody for
20 min at RT and analysed by ow cytometry (BD FACSCalibur, BD Biosciences).
ELISPOT analysis. ELISPOT was used to quantify the number of IgM-secreting B cells. For this, ELISPOT
plates containing Inmobilon-P membranes (Millipore) were activated with 70% ethanol for 30 s, coated with
anti-trout IgM mAb (clone 4C10) at 2 μ g/ml in PBS and incubated overnight at 4 °C. To block non-specic bind-
ing to the membrane, plates were then incubated with 2% bovine serum albumin (BSA) in PBS for 2 h at RT.
Stimulated (with IL-6 or LPS as a positive control) or unstimulated splenocytes from individual sh were added
to the wells in triplicate at a concentration of 1 × 105 cells per well. Aer 72 h of incubation at 20 °C, cells were
washed away 5 times with PBS and plates were blocked again with 2% BSA in PBS for 1 h at room temperature.
Aer blocking, biotinylated anti-trout IgM mAb (clone 4C10) was added to the plates and incubated at 1 μ g/ml fo r
1 h at RT. Following additional washing steps (5 times in PBS) the plates were developed using streptavidin-HRP
(ermo Scientic) at RT for 1 h, washed again with PBS and incubated with 3-amino 9-ethylcarbazole (Sigma

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Aldrich) for 30 min at RT in the dark. Substrate reaction was stopped by washing the plates with tap water. Once
the membranes had dried, they were digitally scanned and spot counts determined by the ImmunoSpot Series 45
Micro ELISPOT Analyzer.
Determination of total IgM levels. To determine total (both intracellular and extracellular) IgM levels,
cells were xed for 5 min with 4% paraformaldehyde in PBS, then permeabilized for 30 min in permeabilizitation
buer (staining buer containing 0.1% saponin) and thereaer incubated with an anti-IgM-PE antibody in per-
meabilization buer for another 30 min. Aer incubation, cells were washed three times with staining buer and
analyzed by ow cytometry.
NF-kB activation. Trout splenocytes were isolated and seeded in complete MGFL-15 medium (MGFL-15
supplemented with P/S, 100 μ g/ml gentamicin, 10% newborn calf serum (Gibco) and 5% carp serum). Cells were
incubated for 24 h with complete media containing IL-6, LPS or control media alone. Following stimulation,
cells were xed in 1% formaldehyde, and washed twice in PBS with 2% calf serum and 0.1% saponin (perme-
abilization buer). To determine nuclear translocation in IgM+ cells, splenocytes were stained with anti-p65
(Santa Cruz Biotechnology) and anti-trout IgM (1.14) for 30 min at 4 °C followed by 20 min at RT. Following the
primary staining, cells were washed and stained with goat anti-rabbit APC (Jackson ImmunoResearch) and rab-
bit anti-mouse FITC (Jackson ImmunoResearch). Prior to acquisition, Hoechst33342 nuclear stain (Molecular
Probes) was added as per the manufacturer’s recommendations. Data were collected on an ImageStream MKII
and analyzed using IDEAS soware (Amnis), as described previously11.
Analysis of MHC-II expression. e levels of MHC-II expression on the surface of IgM+ B cells were
measured via ow cytometry using a mAb against trout MHC-II73. Stimulated or unstimulated splenocytes were
washed in Staining Buer and co-incubated with PE-conjugated anti-trout IgM and the Alexa 647-conjugated
anti-MHC-II antibody for 30 min at 4 °C protected from light. Finally cells were washed twice with the same
buer and analysed by ow cytometry.
Phagocytic activity. Puried splenocytes (2 × 106) were cultured in 1 ml/well in 24-well plates in the pres-
ence or absence of IL-6 or LPS for 24, 48 or 72 h. Aer each time point, cells were incubated for 16 h at 20 °C
with uorescent beads (FluoSpheres Microspheres, 1.0 μ m, Crimson Red Fluorescent 625/645, 2% solids; Life
Technologies) at a cell/bead ratio of 1:10 or without beads as negative controls. Non-ingested beads were removed
by centrifugation (100 × g for 10 min at 4 °C) over a cushion of 3% (w/v) BSA (Fraction V; Fisher Scientic) in
PBS supplemented with 4.5 (w/v) D-glucose (Sigma). Aerwards, cells were resuspended in staining buer, labe-
led with PE-anti-IgM mAb (30 min at 4 °C), washed with the same buer and analyzed by ow cytometry.
Calcium ux. For calcium ux analysis, the calcium indicator Fluo-3 AM (Life Technologies) was used, fol-
lowing the manufacturer’s instructions. Briey, Fluo-3 was dissolved in DMSO and further diluted in an equal
volume of 20% (w/v) Pluronic F-127 (Life Technologies). Splenocytes were cultured in the presence or absence
of LPS or IL-6 during 24, 48 or 72 h. Aer each time point, cells were diluted in L-15 medium without FCS and
incubated with Fluo-3 AM at a nal concentration of 5 μ M for 1 h. Cells were then collected and washed, and a
baseline reading for 30 s acquired in a FACSCalibur ow cytometer. en 0.5 μ g/ml of anti-IgM were added to the
tube, and the emission of uorescence (525 nm) determined for 180 s in each sample.
Bioactivity of IL-6 in vivo. To assess the bioactivity of rtIL-6 in vivo, rainbow trout of approximately 5–7 g
were divided into three groups of 24 sh and injected intraperitoneally (i.p.) with 100 μ l of PBS, 100 μ l of PBS with
IL-6 (100 ng/sh), 100 μ l of PBS containing 2 × 1010 TCID50/ml inactivated IPNV (kindly donated by Professor
Øystein Evensen), or 100 μ l of PBS with a combination of IPNV and IL-6 at the same concentration as above. At
days 2, 6 and 15, six trout from each group were killed by benzocaine overdose. Blood was collected from the
caudal vein to determine antibody concentration and thereaer splenocytes and kidney leukocytes were isolated
to determine the number of IgM-secreting cells in ELISPOT assays as described above.
Antibody production. Serum samples were obtained aer blood clotting at RT for 1–2 h followed by incu-
bation overnight at 4 °C. Aerwards, the clot was centrifuged at 4000 rpm for 10 min and serum samples were
collected in a new tube that was centrifuged again at 10000 rpm for 10 min. Supernatants were nally collected in
dierent tubes and stored at − 20 °C until use. e production of total IgM, IPNV-specic IgM and natural IgM
antibodies (with reactivity to TNP-BSA, galactosidase, phosphorylcholine and poly I:C) was then established by
capture ELISA.
To assess total IgM levels, 96-well ELISA plates were coated overnight with 100 μ l of 2 μ g/ml mouse anti-trout
Ig mAb 4C10. Wells were then blocked with 100 ul of 1% BSA in 1% Tween-20 PBS for 1 h at RT. Plates were
washed 3 times with PBS-1% Tween-20 and serum samples were diluted 1:100 in PBS-1% BSA and added to the
wells. Samples were incubated 1 h at RT and washed 3 times in PBS-1% Tween-20. en, 50 μ l of biotinylated
4C10 mAb (1 μ g/ml) diluted in blocking buer were added to the wells and samples incubated for 1 h at RT. Aer
three washing steps, plates were incubated with 50 μ l of Streptavidin-HRP (1:2000 in PBS-1% BSA) for 1 h at RT.
Wells were washed again 3 times and then 50 μ l of TMB substrate (Sigma) were added. Absorbance at OD405 was
measured in a FLUO Star Omega Microplate Reader.
An ELISA method was also used to measure IPNV-specic antibodies. Plates were coated with 100 μ l of poly-
clonal anti-IPNV antibody diluted at 1:5000 in diluent buer (1% fat free dry milk-PBS) and incubated overnight
at 4 °C. Plates were washed 3 times in PBS-0.05% Tween-20 and blocked with 5% fat free dry milk-PBS for 2 h
at RT. Aer 3 washing steps, serum samples were diluted at 1:100 in diluent buer and incubated overnight at

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Scientific RepoRts | 6:30004 | DOI: 10.1038/srep30004
4 °C. Aer that, biotinylated 4C10 mAb and Streptavidin-HRP were used as described before. OPD substrate was
added to the wells and samples were measured at OD492 nm.
In order to measure the levels of natural IgM antibodies, plates were coated with 100 μ l of 15 μ g/ml of
TNP-BSA, 10 μ g/ml galactosidase, 20 μ g/ml phosphorylcholine or 20  g/ml poly I:C diluted in PBS and incu-
bated overnight at 4 °C. Plates were then blocked with 100 μ l of 1% BSA in 1% Tween-20 in PBS for 1 h at RT. Aer
washing steps in PBS-1% Tween-20, the serum samples were diluted 1:100 in PBS-1% BSA and added to the wells.
Biotinylated 4C10 mAb and Streptavidin-HRP were also used as described in total anti-IgM detection ELISA.
Finally, OPD substrate was added to the wells and samples were measured at OD492 nm.
Statistical analysis. Statistical analyses were performed using a two-tailed Student’s t test with Welch’s cor-
rection when the F test indicated that the variances of both groups diered signicantly. e dierences between
the mean values were considered signicant on dierent degrees, where * means P ≤ 0.05, * * means P ≤ 0.01 and
* * * means P ≤ 0.005 (GraphPad Prism 4 soware).
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Acknowledgements
We would like to thank Lucia Gonzalez and Maria Sanz for technical assistance. Professor Øystein Evensen
is also acknowledged for providing us with the inactivated IPNV. is work was supported by the European
Research Council (ERC Starting Grant 2011 280469) and by the European Commission under the 7th Framework
Programme for Research and Technological Development (FP7) of the European Union (Grant Agreement
311993 TARGETFISH). T.W. received funding from the MASTS pooling initiative (e Marine Alliance for
Science and Technology for Scotland). MASTS is funded by the Scottish Funding Council (grant reference
HR09011).
Author Contributions
B.A. performed most of the experimental work. T.W. and C.J.S. produced the recombinant IL-6 and performed
all transcriptional analysis. R.C., E.L., A.G.G. and A.L assisted B.A. in tissue sampling and processing, especially

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Scientific RepoRts | 6:30004 | DOI: 10.1038/srep30004
during in vivo experiments. J.H. and D.B. analyzed NF-κ B activation in stimulated cells. C.T. designed the
experiments and wrote the main body of the paper, with contributions from all other authors.
Additional Information
Supplementary information accompanies this paper at http://www.nature.com/srep
Competing nancial interests: e authors declare no competing nancial interests.
How to cite this article: Abós, B. et al. Distinct Dierentiation Programs Triggered by IL-6 and LPS in Teleost
IgM+ B Cells in e Absence of Germinal Centers. Sci. Rep. 6, 30004; doi: 10.1038/srep30004 (2016).
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