Aeromonas salmonicida activates rainbow trout IgM+ B cells signalling through Toll like receptors

As B cells are singularly equipped with a B cell receptor (BCR) and a range of innate receptors, they are able to integrate both antigen-specific and innate signals, with the latter being essential to reach an adequate level of activation. Whether teleost B cells sense pathogens through innate mechanisms has not yet been explored, despite the fact that fish B cells display a wider array of innate receptors than many mammalian B cell subsets. Hence, in the current study, we have investigated the effects of inactivated Aeromonas salmonicida, a Gram negative rainbow trout pathogen, on trout splenic IgM+ B cells in vitro in the presence or absence of different inhibitors of Toll-like receptor (TLR) signalling, to establish to what degree innate signals are contributing to the activation of B cells in teleosts. Our results demonstrate that most of the effects that A. salmonicida exerts on trout IgM+ B cells are significantly blocked in the presence of inhibitors of MyD88 and TRIF, important nodes in TLR signal pathways. Thus, the data presented demonstrates that, also in teleost, TLR signalling is essential for the activation of IgM+ B cells. These results will be useful for the future optimization of novel vaccines and adjuvants.

The discovery of host-encoded pattern recognition receptors (PRRs) that sense common molecular patterns from infectious agents and the demonstration of their role in triggering early inflammatory processes, has been a key milestone to fully understand how the immune response is organized 1 . These receptors sense pathogenassociated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs) and activate cells to initiate a cascade of responses that leads to the production of cytokines or antimicrobial compounds that orchestrate an inflammatory response 2 . A further consequence of PRR engagement is the activation of antigen presenting cells to initiate antigen presentation to T cells. Therefore, the ligation of PRRs benefits the host in two ways. First, it activates an innate response aimed at directly killing pathogens, while at the same time, it initiates an antigen-specific adaptive immune response that provides a second layer of protection in case it is needed 1,2 . Despite having established their evident effects on the adaptive immune response, PRR-mediated responses have been historically associated with innate immune cell populations such as dendritic cells and macrophages. Evidence gathered in the recent years, however, has pointed to a key role of B cell-intrinsic PRR activation in modelling specific antibody responses 3 .
Toll-like receptors (TLRs) constitute the most studied group of PRRs, and have been identified in all animal groups from invertebrates to human 4 . TLRs are synthetized in the endoplasmic reticulum, transported to the Golgi complex, and then delivered to the cellular membrane or to intracellular compartments like endosomes. Thus, they are type I transmembrane proteins. They are formed by an ectodomain containing several leucine-rich repeats responsible for the recognition of PAMPs; a transmembrane domain; and an intracellular Toll-interleukin 1 (IL-1) receptor (TIR) domain that mediates downstream signal transduction 5 . Once activated, the TIR domain of the TLRs recruits specific adaptor molecules which provoke the subsequent activation of the inflammatory response. Two different TLR signalling pathways have been described depending on the adaptor molecule that is recruited by TIR, a Myeloid Differentiation Primary Response protein 88 (MyD88)-dependent pathway and a TRIF-dependent pathway which is completely independent of MyD88. In mammals, MyD88 is used by all TLRs

Aeromonas salmonicida is phagocytized by igM + B cells.
Prior to characterizing the effects of A. salmonicida rainbow trout B cells, we studied whether this fish pathogen could be phagocytized by IgM + B cells. For this, we labelled inactivated A. salmonicida with Syto BC Green and incubated splenocytes with the labelled bacteria for 3 h. Thereafter, cells were labelled with a specific anti-IgM monoclonal antibody and analysed by confocal microscopy or flow cytometry. Our results show that A. salmonicida can be phagocytized by IgM + B cells (Fig. 1A,B), as well as by other non-IgM leukocytes (Fig. 1A,B). Our next step then was to establish if the TLR inhibitors used throughout this work had a negative impact on the capacity of rainbow trout IgM + B cells to phagocytize A. salmonicida. For this, we pre-incubated splenocytes for 1 h with the MyD88 inhibitor peptide or resveratrol. As negative controls, we used a control peptide or DMSO which is the solvent in which resveratrol is dissolved. After this time, we incubated the cells with labelled inactivated A. salmonicida for 3 h and analysed the phagocytic capacity by flow cytometry (Fig. 1B). The MyD88 inhibitor peptide did not have a negative effect on the capacity of IgM + B cells to internalize A. salmonicida (Fig. 1C), while resveratrol significantly inhibited the internalization (Fig. 1D). Similarly, resveratrol has been shown to reduce the phagocytic activity of human macrophages by down-regulating the expression of phagocytic receptors and NF-κB activity 20 . A. salmonicida increases igM + B cell survival and has lymphoproliferative effects through a TLR-dependent mechanism. Next, we investigated the effects of A. salmonicida on the survival of rainbow trout IgM + B cells. To this end, splenocytes were exposed to the different TLR inhibitors or their respective controls for 1 h and then incubated with the bacteria for 3 days. Controls without bacteria were also included. After this time, cells were labelled with anti-IgM and DAPI (to determine cell viability) and analysed following the gating strategy described in Supplementary Fig S1, after establishing that none of the treatments had a significant impact on cell viability (Fig. S1). Resveratrol provoked a moderate but non-significant decrease in the number of cells within the lymphoid gate, however the percentage of live cells within the gated population was never affected (Fig. S1). In these conditions, we established that A. salmonicida significantly increased the percentage of IgM + B cells in the cultures, effect that was reverted by the MyD88 inhibitor peptide but not by its respective control or by resveratrol ( Fig. 2A-C). Similar results were obtained when the absolute number of total IgM + B cells was determined (Fig. S2). To establish whether this increased percentage of B cells was due to a lymphoproliferative effect of A. salmonicida, a proliferation assay was carried out in parallel. As a first step, we established that none of the inhibitors or the controls used in the experiments were able to induce proliferation in the absence of A. salmonicida (Fig. S3). A. salmonicida, however, induced a low but significant proliferation of IgM + B cells (Fig. 2D-F). Although these mild proliferative effects were down-regulated in the presence of the MyD88 inhibitor peptide and resveratrol, the differences compared to the values reached by A. salmonicida www.nature.com/scientificreports/ alone were not significant. These results demonstrate that A. salmonicida is acting mainly as a survival factor for rainbow trout IgM + B cells, through a mechanism that involves MyD88.

A. salmonicida increases MHc ii surface expression in igM + B cells through a TLR-dependent mechanism. IgM + B cells as professional antigen presenting cells express MHC II on their cell surface 21 .
Thus, we decided to study if upon an encounter with A. salmonicida IgM + B cells modified the expression of MHC II on the surface and whether this effect was regulated by TLR signalling. For this, again we incubated splenocytes for 1 h with the inhibitors or their respective controls, and then stimulated the cells with inactivated A. salmonicida. After 72 h of incubation at 20 ºC, the cells were labelled with anti-IgM and anti-MHC II monoclonal antibodies, analysed by multicolor flow cytometry and the fluorescence intensity of MHC II measured. A clear effect of A. salmonicida on the levels of surface MHC II on IgM + B cells was visible ( Fig. 3A-C), effect that was reverted by resveratrol ( Fig. 3A-C). In this case, the MyD88 inhibitor peptide was not able of reverting the increase in the levels of surface MHC II provoked by the bacteria (Fig. 3A-C). Interestingly, in these cultures, the levels of surface MHC II were not significantly increased by the bacteria in IgMcells (Fig. 3D), suggesting a preferential role of IgM + B cells among splenocytes in the presentation of A. salmonicida.

A. salmonicida increases the number of antibody secreting cells in splenocyte cultures through
a TLR-dependent mechanism. Next, to establish if A. salmonicida can induce on its own the differentiation of IgM + B cells to IgM-secreting plasmablasts/plasma cells, we determined the effect that A. salmonicida had in the number of IgM-secreting cells through an ELISPOT assay. A. salmonicida significantly increased the number or IgM secreting cells in splenocytes cultures after 3 days of treatment ( Fig. 4A), effect that was significantly reversed in the presence of the MyD88 inhibitor peptide or resveratrol, but not by their respective controls ( Fig. 4A,B). Because a differentiation of naïve B cells to plasma-like cells usually implies an increase in size, we also determined by flow cytometry the impact of A. salmonicida on the size (forward side scatter, FSC) of splenic IgM + B cells. Our results confirmed that there was a significant size increase in response to the bacteria, effect that significantly reverted in the presence of the TLR inhibitors but not in response to their controls ( Fig. 4C,D). In mammals, the B cell differentiation process implies a down-regulation of the Pax5 transcription factor, as a consequence of the increased production of Blimp1 22 . In fish, whether a similar differentiation program takes place is still partially unknown and increases in IgM secretion dependent 16 or independent 16,23,24 of Blimp1 transcription have been reported in rainbow trout. To establish whether A. salmonicida induced changes in the transcription of these factors, IgM + B cells exposed or not to the bacteria were isolated by flow cytometry (Fig.  S4), RNA extracted and the levels of transcription of Blimp-1 and Pax5 determined by real time PCR. In our experiments, A. salmonicida significantly down-regulated Pax5 transcription in IgM + B cells but had no effect on Blimp1 mRNA levels (Fig. 4E).
A. salmonicida LPS has lower lymphoproliferative capacities than the inactivated bacteria but stronger effects on MHC II expression. Having established that A. salmonicida exerts positive effects on the survival, IgM secretion and MHC II expression of rainbow trout IgM + B cells through a TLR-dependent mechanism, we wanted to determine whether the A. salmonicida LPS on its own was capable of exerting similar effects or whether the complete bacterial cell was required. For this, we isolated LPS from A. salmonicida and incubated splenocytes for 3 days with either the inactivated bacteria or an amount of LPS that corresponded to that same amount of bacterial cells. Both the inactivated bacteria and the LPS were capable of significantly increasing the percentage of IgM + B cells in splenocyte cultures after 3 days of stimulation (Fig. 5A,B). Regarding the lymphoproliferative effects, only the inactivated bacteria induced a significant proliferation of IgM + B cells while LPS did not (Fig. 5C,D). On the contrary, when we analysed the effects on the MHC II surface expression of IgM + B cells, although both stimuli significantly increased the expression levels, the effects provoked by LPS were significantly higher than those exerted by the bacteria (Fig. 5E,F).  www.nature.com/scientificreports/ significantly inhibited this activity as previously reported in human macrophages 20 , the MyD88 inhibitor had no effect on the capacity of IgM + B cells to internalize A. salmonicida. This implies that any changes in the response to the bacteria observed in the presence of the MyD88 inhibitor are strictly due to an interference with MyD88 signalling and cannot be attributed to a reduced internalization. In the case of resveratrol, however, it is not possible to rule out that the reduced activation of B cells in the presence of this inhibitor is at least in part due to the fact that the bacteria is not internalized as efficiently. Furthermore, it must be noted that resveratrol possesses www.nature.com/scientificreports/ a wide range of biological properties and can interfere with intracellular signalling during the inflammatory responses at different steps 20 . This might explain why most of the reversions of A. salmonicida-induced activation of B cells were much stronger in the case of resveratrol than in response to the MyD88 inhibitor. In fact, it should be noted that the precise effects of the two inhibitors used in this study in downstream TLR signalling has never been established in teleost fish due to a lack of specific reagents. In this context, although the work presented in this paper represents sufficient evidence of how both inhibitors are capable of blocking inflammatory responses also in fish, it might be possible that these inhibitors are not 100% efficacious in fish or that they have effects slightly different to those reported for mammalian cells.

Discussion
Our results demonstrated that A. salmonicida was capable of increasing the survival of IgM + B cells in splenocyte cultures. This increase seemed to be at least partially due to a lymphoproliferative effect of the bacteria on IgM + B cells, however, the proliferation rates were not very high, suggesting that A. salmonicida is acting mainly as a survival factor for rainbow trout IgM + B cells. In consequence, only the increased IgM + B cell survival observed in response to the bacteria, was significantly reverted by the MyD88 inhibitor, demonstrating that the BCR alone does not account for this effect. The increase in B cell survival in response to the bacteria could be mediated by the recognition of A. salmonicida LPS, as similar B cell survival rates were obtained in response to the complete bacteria or its LPS. However, the complete bacteria seems to be a more suitable antigen for the induction of proliferation, as significantly lower responses were observed in response to LPS. Interestingly, previous investigations point to salmonids lacking TLR4, the main receptor responsible for LPS sensing in mammals 14 , thus, the receptor that is mediating these effects in B cells is still unknown and is an issue that should be addressed in future studies.
A. salmonicida also increased MHC II expression on the surface of IgM + B cells, similarly to what had been previously reported in response to E. coli LPS 16 or CpGs 18 . In this case, however, only resveratrol and not the MyD88 inhibitor, was capable of significantly reverting this effect. Whether this is a consequence of surface MHC II expression up-regulation requiring the internalization of the bacteria or because this effect is mediated through a TRIF-dependent mechanism is still undetermined and deserves further investigation. Remarkably, we found that the bacterial LPS on its own was capable of inducing higher levels of surface MHC II expression in IgM + B cells than those observed in response to the intact bacteria. Hence, these results suggests that an internalization of the bacteria is not required to induce surface MHC II expression.
Our results also demonstrate a differentiation of rainbow trout naïve IgM + B cells towards IgM-secreting cells in response to inactivated A. salmonicida. The increased number of IgM-secreting cells in the splenocyte cultures treated with the bacteria could be a result of an increased survival of already-existing IgM-secreting cells in the cultures as previously established in peritoneal B cells treated with BAFF 28 . However, the fact that A. salmonicida also increased the size of B cells and down-regulated their Pax5 mRNA levels suggested in fact a differentiation of naïve cells towards an IgM-secreting cell. All these effects were significantly reverted in the presence of both TLR inhibitors. In mammals, the contribution of B cell-intrinsic TLRs to antibody secretion has been widely documented. For example, Pasare and Medzhitov transferred purified B cells from wild type, MyD88 knockout, TLR4 knockout and CD40 knockout mice into mice that lacked mature B cells 29 . In these experiments, mice that received wild-type B cells were able to generate antigen-specific IgM and IgG responses to a combination of human serum albumin and LPS, whereas antibody production was significantly impaired in mice that received TLR4 knockout or MyD88 knockout B cells. Interestingly, mice that received CD40 knockout B cells had normal IgM titres but impaired IgG production 29 . Completely different results were obtained in another study in which TLR-deficient mice were immunized with a T cell dependent antigen (TNP-KLH) in combination with different adjuvants 30 . In this case, similar antibody responses were obtained in TLR-deficient mice when compared to wild type mice. These contradictory results led other authors to speculate that the degree of TLR help needed is highly dependent on the antigen, being TLR engagement required to a higher degree when BCR stimulation or T-cell help is limiting 31 . Given the scatter distribution of B and T cells in teleost immune tissues, lacking organized lymphoid structures such as cognate germinal centers 32 , and knowing that BCR signalling provokes less effects in fish B cells than in mammalian conventional B cells 26 , it seems plausible to hypothesize that TLR engagement plays a quite prominent role in teleost B cells. This hypothesis seems confirmed by the fact that the degree of reversion exerted by the TLR inhibitors on the effects provoked by A. salmonicida on trout IgM + B cells was quite high, completely reverting many of them.
In conclusion, we have demonstrated that upon recognition of inactivated A. salmonicida, rainbow trout IgM + B cells increase in number, proliferate, increase surface MHC II expression and differentiate towards IgM-secreting cells. Among these effects, the MyD88 inhibitor significantly reverted the increased IgM + B cell survival and the up-regulated IgM secretion, whereas resveratrol significantly reverted the higher surface MHC II levels and the increased IgM secretion. These results highlight a large contribution of TLR signalling in the activation of B cells by the bacteria. Finally, we have also established that A. salmonicida LPS by itself has stronger effects on MHC II surface expression than the complete bacteria, while having lower lymphoproliferative effects. Understanding how fish B cells sense antigens is essential for the development of effective vaccines and adjuvants, therefore our work will hopefully contribute to the design of a more effective A. salmonicida vaccine in the near future.

Materials and methods
Experimental fish. Healthy specimens of rainbow trout (Oncorhynchus mykiss) of approximately 50-70 g were obtained from Centro de Acuicultura El Molino (Madrid, Spain). As previously described 18,24 , fish were maintained at the Animal Health Research Centre (CISA-INIA) laboratory at 16 ºC with a re-circulating water system and 12:12 h light:dark photoperiod. Fish were fed twice a day with a commercial diet (Skretting, Spain). Prior to any experimental procedure, fish were acclimatized to laboratory conditions for 2 weeks and during Spleen leukocyte isolation. Rainbow trout were killed by benzocaine (Sigma-Aldrich) overdose and the spleen collected. Single cell suspensions were obtained as previously reported 18,24 using 100 µm nylon cell strainers (BD Biosciences) and Leibovitz medium (L-15, Invitrogen) supplemented with 100 I.U./ml penicillin and 100 µg/ml streptomycin (P/S, Life Technologies), 10 units/ml heparin (Sigma) and 5% foetal calf serum (FCS, Life Technologies). Cell suspensions were placed onto 30/51% discontinuous Percoll (GE Healthcare) density gradients and centrifuged at 500 × g for 30 min at 4 ºC. The interface cells were washed twice in L-15 with 2% FCS and cells were resuspended in L-15 with 5% FCS. The viable cell concentration was determined by Trypan blue (Sigma-Aldrich) exclusion, adjusting the concentration to 2 × 10 6 cells/ml.

Bacteria and LPS.
The Gram-negative fish pathogen A. salmonicida subsp. salmonicida CECT4237 was aerobically grown in Tryptone Soya Broth (Oxoid) at 25 ˚C. To stimulate the rainbow trout splenocytes, A. salmonicida grown in broth overnight to exponential phase was heat-inactivated at 65 °C for 1 h. In some experiments, LPS obtained from A. salmonicida was used to stimulate the cells. For this, LPS was isolated using a commercial extraction kit (iNtRON Biotechnology) following the manufacturer's protocol. The absence of DNA and protein contamination was confirmed by SDS-PAGE and agarose electrophoresis.

Reagents.
A MyD88 inhibitor peptide was purchased from Novusbio and used at a concentration of 100 µM.
A control peptide was used as a control at the same concentration. Resveratrol, obtained from Sigma, was diluted in DMSO and was used in cells at a final concentration of 50 µM. In this case, the same volume of DMSO was added to cell cultures as a negative control.
flow cytometry. IgM + B cells populations were identified following a method previously described 18,24 . For this, leukocytes were incubated for 30 min with anti-trout IgM [mAb mouse IgG 1 coupled to fluorescein (FITC) or allophycocyanin (APC) at 0.5 μg/ml] and anti-trout MHC II [mAb mouse IgG 1 coupled to APC at 2 μg/ml] antibodies in staining buffer (phenol red-free L-15 media supplemented with P/S and 2% FCS). Both antibodies have been previously characterised 33,34 . After the incubation, cells were washed twice with staining buffer and analysed on a FACS Celesta flow cytometer (BD Biosciences) equipped with FACS DIVA software. In all cases, isotype controls for mouse mAbs (BD Biosciences) were tested in parallel to discard unspecific binding. Cells were stained with 4′,6-diamidino-2-phenylindole (DAPI; 0.2 μg/ml) to check cell viability and doublets were excluded following the gating strategy described in Supplementary Fig. S1. Flow cytometry analysis was performed with FlowJo 10 (TreeStar).
phagocytic activity. Spleen leukocytes seeded in 96-well plates (Nunc) at a cell density of 4 × 10 5 cells/well were incubated with the MyD88 inhibitor peptide (10 µM), with resveratrol (50 µM) or with their respective controls at 20 °C. After 1 h, splenocytes were exposed to inactivated A. salmonicida previously labelled with 1 µM Syto BC Green (Thermo Fischer Scientific) at a cell: bacteria ratio of 1:2 or without bacteria in the case of negative controls and the phagocytosis assay was conducted following a method previously described 18 . After 3 h of incubation at 20 ºC, cells were harvested by gently pipetting and non-ingested beads removed by centrifugation (100 × g for 10 min at 4 ºC) over a cushion of 3% (weight/volume) BSA (Fraction V; Fisher Scientific) in PBS supplemented with 4.5% (weight/volume) D-glucose (Sigma). Cells were then resuspended in staining buffer, labelled with anti-trout IgM-APC (0.5 µg/ml) and analysed on a FACS Celesta flow cytometer following the gating strategy previously described (Fig. S1). For confocal analysis, cells labelled with anti-trout IgM-APC (5 µg/ml) were washed with serum-free L-15 medium, seeded on poly-L-lysine coated slides, and incubated at 20 °C for 30 min. Laser scanning confocal microscopy images (0.3 μm thickness) were acquired with an inverted Zeiss Axiovert LSM 880 microscope. Images were analysed with Zen 2.0 (Carl Zeiss) and Fiji (NIH) software packages.

B cell proliferation. The Click-IT EdU Alexa Fluor 488 Flow Cytometry Assay Kit (Life technologies) was
used to measure the proliferation of IgM + B cells as described before 18,24 . For this, splenocytes at a concentration of 2 × 10 6 cells per ml were incubated for 1 h with the different TLR inhibitors, their controls or media alone as described above. The cells were then stimulated with inactivated A. salmonicida for 3 days at 20 ºC as described above or left unstimulated. After this time, EdU (1 µM) was added to the cultures and the cells were incubated for an additional 24 h. At this point, cells were collected and stained with anti-trout IgM-APC (0.5 μg/ml). To analyse the incorporation of EdU, cells were then fixed and permeabilised with Cytofix/Cytoperm buffer for 15 min at room temperature (RT). Finally, the incorporation of EdU to the DNA was detected following the manufacturer´s instructions and then analysed by flow cytometry on a FACS Celesta flow cytometer.
ELISPOT analysis. ELISPOT  www.nature.com/scientificreports/ blocked with 2% BSA in PBS for 1 h at RT. After blocking, biotinylated anti-trout IgM was added to the plates and incubated at 1 µg/ml for 1 h at RT. Following additional washing steps (5 times in PBS) the plates were developed using streptavidin-HRP at 100 ng/ml (Thermo Fischer Scientific) at RT for 1 h, washed again with PBS and incubated with 3-amino 9-ethylcarbazole (Sigma-Aldrich) for 30 min at RT in the dark. Substrate reaction was stopped by washing the plates with tap water. Once the membranes were dried, the number of spots in each well was determined using an AID iSpot Reader System (Autoimmun Diagnostika GmbH).

transcriptional analysis of isolated igM + B cells. IgM + B cells populations were isolated by flow
cytometry in a BD FACSAria III cell sorter (BD Biosciences) after staining spleen leukocytes with anti-trout IgM-APC as described above, using their FSC/SSC and fluorescence characteristics. In this case 7-AAD (BD Biosciences) at 2.5 µg/ml was used to check the cell viability. Total cellular RNA was isolated from cell populations using the Power SYBR Green Cells-to-Ct Kit (Invitrogen) following manufacturer´s instructions and as described before 18,24 . RNA was treated with DNase during the process to remove genomic DNA that might interfere with the PCR reactions. Reverse transcription was also performed using the Power SYBR Green Cells-to-Ct Kit (Invitrogen) following manufacturer's instructions. To evaluate the levels of transcription of the different genes, real time PCR was performed with a LightCycler 96 System instrument (Roche) using SYBR Green PCR core Reagents (Applied Biosystems) and specific primers previously described 24 . Each sample was measured in duplicate under the following conditions: 10 min at 95 ºC, followed by 40 amplification cycles (15 s at 95 ºC and 1 min at 60 ºC). A melting curve for each primer set was obtained by reading fluorescence every degree between 60 ºC and 95 ºC to ensure only a single product had been amplified. The expression of individual genes was normalized to the relative expression of trout housekeeping gene elongation factor 1α (EF-1α), and the expression levels were calculated using the 2 -ΔCt method, where ΔCt is determined by subtracting the EF-1α value from the target Ct. No template negative controls and minus reverse transcriptase controls were included in all the experiments. Statistical analysis. Statistical analyses were performed using the Graphpad prism version 6 (Graphpad software). All values were verified to be normally distributed. In all experiments, one-way ANOVA followed by Tukey's test as a post-hoc was performed. The differences between the mean values were considered significant on different degrees, where * means P ≤ 0.05, ** means P ≤ 0.01 and *** means P ≤ 0.005.