Identification of CD8α+ dendritic cells in rainbow trout (Oncorhynchus mykiss) intestine.

Dendritic cells (DCs) are professional antigen presenting cells located at mucosal surfaces and lymphoid tissues. Their main role is to present antigens to T cells and thus regulate the initiation of the acquired immune response and modulate tolerance mechanisms towards self-antigens. Despite their relevance, not many studies have addressed the identification and characterization of specific DC subsets in teleost fish. Previous studies in our group identified a DC subpopulation co-expressing CD8α and major histocompatibility complex II (MHC II) on the cell surface in rainbow trout (Oncorhynchus mykiss) skin and gills. A complete functional and phenotypical characterization of these cell subsets was then undertaken, unequivocally recognizing them as DCs (CD8+ DCs). In the current study, we report the identification of a homologous population in rainbow trout intestinal lamina propria (LP). We have studied the main features of these intestinal CD8+ DCs, comparing them to those of CD8+ DCs from another mucosal tissue (gills). Interestingly, intestinal CD8+ DCs exhibited significant phenotypical and functional differences when compared to gill CD8+ DCs, suggesting that the location of DCs strongly conditions their activation state. These results will contribute to further expand our knowledge on how intestinal immune responses are regulated in fish, helping us to rationally design oral vaccines in the future.

CpG Oligodeoxynucleotides Modulate Innate and Adaptive Functions of IgM+ B Cells in Rainbow Trout.

Oligodeoxynucleotides (ODN) containing unmethylated CpG motifs have been widely postulated as vaccine adjuvants both in mammals and teleost fish. However, to date, the effects that CpGs provoke on cells of the adaptive immune system remain mostly unexplored in fish. Given that rainbow trout (Oncorhynchus mykiss) IgM+ B cells from spleen and blood transcribe high levels of toll like receptor 9 (TLR9), the receptor responsible for CpG detection in mammals, in the current work, we have investigated the effects of CpGs on both spleen and blood IgM+ B cells from this species. CpGs were shown to exert strong proliferative effects on both spleen and blood IgM+ B cells, also increasing their survival. The fact that CpGs increase the size of IgM+ B cells, reduce the expression of surface IgM and IgD and up-regulate the number of IgM-secreting cells strongly suggest that IgM+ B cells differentiate to plasmablasts/plasma cells in response to CpG stimulation. Additionally, CpGs were shown to modulate the antigen presenting capacities of trout IgM+ B cells through an increased surface MHC II expression and transcriptional up-regulation of co-stimulatory molecules, although in this case, significant differences were observed between the effects exerted on spleen and blood cells. Similarly, differences were observed between spleen and blood IgM+ B cells when CpG stimulation was combined with B cell receptor (BCR) cross-linking. Finally, CpGs were also shown to affect innate functions of teleost IgM+ B cells such as their phagocytic capacity. These results demonstrate that CpGs regulate many adaptive and innate functions of teleost B cells, supporting their inclusion as adjuvants in novel vaccine formulations.

Rainbow Trout IgM+ B Cells Preferentially Respond to Thymus-Independent Antigens but Are Activated by CD40L.

In the absence of class switch recombination and germinal centers, the mechanisms through which B cells from teleost fish mount extrafollicular immunoglobulin M (IgM) memory responses remains mostly unexplored. In this report, we demonstrate that teleost IgM+ B cells respond to CD40L, a thymus-dependent activation signal, similarly to mammalian B2 cells. However, when stimulated with different types of antigens, fish IgM+ B cells only reach a general activation state in response to antigens cataloged as thymus-independent 1 (TI-1) in mammals, as established through both functional assays and RNA sequencing. Interestingly, fish IgM+ B cells remained completely unresponsive to TI-2 antigens, suggesting that the engagement of innate receptors provided by TI-1 antigens is required for the activation of teleost B cells. Finally, a synergy between CD40L and TI-1 antigens was also demonstrated, further supporting that there is no clear dichotomy between thymus-dependent and TI responses in teleost fish.

Different IgM+ B cell subpopulations residing within the peritoneal cavity of vaccinated rainbow trout are differently regulated by BAFF.

In teleost fish, IgM+ B cells are one of the main responders against inflammatory stimuli in the peritoneal cavity, as IgM+ B cells dominate the peritoneum after intraperitoneal stimulation, also increasing the levels of secreted IgM. BAFF, a cytokine known to play a major role in B cell biology, has been shown to be up-regulated along with its receptors in the peritoneum of rainbow trout upon antigenic exposure, however, the regulatory mechanisms underneath this response remain unclear. In this study, we have identified two different IgM+ B cell types residing in the peritoneal cavity of previously vaccinated rainbow trout (Oncorhynchus mykiss): IgD+IgMhiMHCIIhi cells, resembling naïve B cells, and IgD-IgMloMHCIIlo cells, resembling antibody-secreting cells. Based on their membrane IgM levels, these cell types were named IgMhi and IgMlo B cells, respectively. As each of these B cell populations showed a distinct expression pattern for the different BAFF receptors, we studied the effect of BAFF individually on each cell subset. Recombinant BAFF promoted the survival of IgMlo but not IgMhi B cells in vitro, resulting in increased levels of IgM-secreting cells. In contrast, BAFF increased the levels of membrane MHC II only on IgMhi B cells, suggesting different functions on these B cell subsets. Moreover, we also showed that peritoneal IgMhi B cells expressed BAFF at levels comparable to those seen on myeloid cells. These results point to BAFF as a main regulator of B cell homeostasis in the peritoneal cavity, suggesting that this cytokine can trigger different signals on different peritoneal B cell subsets in a specific manner.

Regulation of IgM+ B Cell Activities by Rainbow Trout APRIL Reveals Specific Effects of This Cytokine in Lower Vertebrates.

Tumor necrosis factor ligand superfamily members such as B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL) have been identified in mammals as key regulators of B cell homeostasis and activation. However, the immune functions of APRIL are not as well defined as those of BAFF. Furthermore, while BAFF is present in all vertebrates, APRIL is missing in some animal groups, suggesting that BAFF has compensated the functions of APRIL in these species. In this context, we thought of great interest to explore the effects of APRIL on teleost B cells, given that APRIL appears for the first time in evolution in bony fish. Thus, in this study, we have performed an extensive analysis of the effect of APRIL on B cells using rainbow trout (Oncorhynchus mykiss) as a model species. Our results demonstrate that APRIL induces a specific proliferation of IgM+ B cells by itself and increases IgM secretion without promoting a terminal differentiation to plasma cells. APRIL also increased the levels of surface MHC II and augmented the capacity of these cells to process antigen, effects that were exclusively exerted on IgM+ B cells. Although our results point to a highly conserved role of APRIL on B cell homeostasis and activation throughout evolution, some specific differential effects have been observed in fish in comparison to the effects of APRIL previously described in mammals. Finally, the effects that APRIL induces on rainbow trout IgM+ B cells described in this paper have been compared with those previously reported in response to BAFF.

Novel Insights on the Regulation of B Cell Functionality by Members of the Tumor Necrosis Factor Superfamily in Jawed Fish.

Most ligands and receptors from the tumor necrosis factor (TNF) superfamily play very important roles in the immune system. In particular, many of these molecules are essential in the regulation of B cell biology and B cell-mediated immune responses. Hence, in mammals, it is known that many TNF family members play a key role on B cell development, maturation, homeostasis, activation, and differentiation, also influencing the ability of B cells to present antigens or act as regulators of immune responses. Evolutionarily, jawed fish (including cartilaginous and bony fish) constitute the first animal group in which an adaptive immune response based on B cells and immunoglobulins is present. However, until recently, not much was known about the expression of TNF ligands and receptors in these species. The sequences of many members of the TNF superfamily have been recently identified in different species of jawed fish, thus allowing posterior analysis on the role that these ligands and receptors have on B cell functionality. In this review, we summarize the current knowledge on the impact that the TNF family members have in different aspects of B cell functionality in fish, also providing an in depth comparison with functional aspects of TNF members in mammals, that will permit a further understanding of how B cell functionality is regulated in these distant animal groups.

Dysregulation of B Cell Activity During Proliferative Kidney Disease in Rainbow Trout.

Proliferative kidney disease (PKD) is a widespread disease caused by the endoparasite Tetracapsuloides bryosalmonae (Myxozoa: Malacosporea). Clinical disease, provoked by the proliferation of extrasporogonic parasite stages, is characterized by a chronic kidney pathology with underlying transcriptional changes indicative of altered B cell responses and dysregulated T-helper cell-like activities. Despite the relevance of PKD to European and North American salmonid aquaculture, no studies, to date, have focused on further characterizing the B cell response during the course of this disease. Thus, in this work, we have studied the behavior of diverse B cell populations in rainbow trout (Oncorhynchus mykiss) naturally infected with T. bryosalmonae at different stages of preclinical and clinical disease. Our results show a clear upregulation of all trout immunoglobulins (Igs) (IgM, IgD, and IgT) demonstrated by immunohistochemistry and Western blot analysis, suggesting the alteration of diverse B cell populations that coexist in the infected kidney. Substantial changes in IgM, IgD, and IgT repertoires were also identified throughout the course of the disease further pointing to the involvement of the three Igs in PKD through what appear to be independently regulated mechanisms. Thus, our results provide strong evidence of the involvement of IgD in the humoral response to a specific pathogen for the first time in teleosts. Nevertheless, it was IgT, a fish-specific Ig isotype thought to be specialized in mucosal immunity, which seemed to play a prevailing role in the kidney response to T. bryosalmonae. We found that IgT was the main Ig coating extrasporogonic parasite stages, IgT+ B cells were the main B cell subset that proliferated in the kidney with increasing kidney pathology, and IgT was the Ig for which more significant changes in repertoire were detected. Hence, although our results demonstrate a profound dysregulation of different B cell subsets during PKD, they point to a major involvement of IgT in the immune response to the parasite. These results provide further insights into the pathology of PKD that may facilitate the future development of control strategies.

Identification of the First Teleost CD5 Molecule: Additional Evidence on Phenotypical and Functional Similarities between Fish IgM+ B Cells and Mammalian B1 Cells.

Despite teleost fish being the first animal group in which all elements of adaptive immunity are present, the lack of follicular structures, as well as the fact that systemic Ab responses rely exclusively on unswitched low-affinity IgM responses, strongly suggests that fish B cell responses resemble mammalian B1 cell responses rather than those of B2 cells. In line with this hypothesis, in the current study, we have identified a homolog of CD5 in teleost fish. This pan-T marker belonging to the scavenger receptor cysteine-rich family of receptors is commonly used in mammals to distinguish a subset of B1 cells. Subsequently, we have demonstrated that a very high percentage of teleost IgM+ B cells express this marker, in contrast to the limited population of CD5-expressing B1 cells found in most mammals. Furthermore, we demonstrate that fish IgM+ B cells share classical phenotypic features of mammalian B1 cells such as large size, high complexity, high surface IgM, and low surface IgD expression, regardless of CD5 expression. Additionally, fish IgM+ B cells, unlike murine B2 cells, also displayed extended survival in cell culture and did not proliferate after BCR engagement. Altogether, our results demonstrate that although fish are evolutionarily the first group in which all the elements of acquired immunity are present, in the absence of follicular structures, most teleost IgM+ B cells have retained phenotypical and functional characteristics of mammalian B1 cells.

Identification of a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells in Teleost Respiratory Surfaces.

Dendritic cells (DCs) are highly specialized antigen-presenting cells that bridge innate and adaptive immune responses in vertebrates, being key modulators in the initiation of specific responses. Although teleost fish present the main elements of a fully developed adaptive immune system, not many studies have focused on identifying specific DC subsets in teleost species. Previous work from our group identified in rainbow trout (Oncorhynchus mykiss) skin a DC subpopulation co-expressing CD8α and major histocompatibility complex II ß on the cell surface. Interestingly, these CD8+ DCs expressed common unique markers of mammalian cross-presenting DCs, a DC subset with an important role in antigen presentation and activation of CD8+ T cytotoxic lymphocytes. In this study, we have identified a similar DC subset in rainbow trout gills that also transcribes molecules uniquely expressed on diverse mammalian cross-presenting DC populations such as CD8, CD103, CD141, Batf3, IFN regulatory protein 8, and toll-like receptor 3. Hence, we have undertaken a broad phenotypic and functional characterization of this new DC subset that includes the confirmation of novel capacities for DCs in teleost, such an IgM-binding capacity and responsiveness to CD40 ligand. Furthermore, our results show that in gills, this DC subset shows some different phenotypic and functional characteristics when compared with their homologs in the skin, suggesting an adaptation of the cells to different mucosal tissues or different maturation status depending on their location. Our findings contribute to increase our knowledge on fish cross-presenting DCs, an important cell population to take into account for the future design of mucosal vaccination strategies.

B Cell-Activating Factor Regulates Different Aspects of B Cell Functionality and Is Produced by a Subset of Splenic B Cells in Teleost Fish.

In mammals, B cell functionality is greatly influenced by cytokines released by innate cells, such as macrophages or dendritic cells, upon the early recognition of common pathogen patterns through invariant receptors. B cell-activating factor (BAFF) is one of these innate B cell-helper signals and plays a key role in the survival and differentiation of B cells. Although, evolutionarily, teleost fish constitute the first animal group in which adaptive immunity based on Ig receptors is present, fish still rely greatly on innate responses. In this context, we hypothesized that BAFF would play a key role in the control of B cell responses in fish. Supporting this, our results show that teleost BAFF recapitulates mammalian BAFF stimulating actions on B cells, upregulating the expression of membrane MHC II, improving the survival of fish naïve B cells and antibody-secreting cells, and increasing the secretion of IgM. Surprisingly, we also demonstrate that BAFF is not only produced in fish by myeloid cells but is also produced by a subset of splenic B cells. Thus, if this B cell-produced BAFF proves to be actively regulating this same B cell subset, our findings point to an ancient mechanism to control B cell differentiation and survival in lower vertebrates, which has been silenced in mammals in physiological conditions, but reemerges under pathological conditions, such as B cell lymphomas and autoimmune diseases.

Characterization of BAFF and APRIL subfamily receptors in rainbow trout (Oncorhynchus mykiss). Potential role of the BAFF / APRIL axis in the pathogenesis of proliferative kidney disease.

Proliferative kidney disease (PKD) is a parasitic infection of salmonid fish characterized by hyper-secretion of immunoglobulins in response to the presence of the myxozoan parasite, Tetracapsuloides bryosalmonae. In this context, we hypothesized that the BAFF/APRIL axis, known to play a major role in B cell differentiation and survival in mammals, could be affected by the parasite and consequently be involved in the apparent shift in normal B cell activity. To regulate B cell activity, BAFF and APRIL bind to transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B cell maturation antigen (BCMA), whereas BAFF also binds to BAFF receptor (BAFF-R). In teleost fish, although some BAFF and APRIL sequences have been reported, their receptors have not been identified. Thus, as a first step in the current work, we have identified homologues to mammalian TACI, BCMA and BAFF-R in rainbow trout (Oncorhynchus mykiss), that constitute the first report of BAFF and APRIL receptor sequences in fish. Subsequently we studied the transcriptional modulation of BAFF, APRIL, and the fish-specific related cytokine, BALM and their putative receptors in fish naturally exposed to T. bryosalmonae. Finally, to gain further insights on the functional role that these cytokines play during the course of PKD, we have studied their effect on the survival of kidney IgM+ B cells and on immunoglobulin transcription. Our results support the premise that the BAFF / APRIL axis could play an important role during PKD, which may open the possibility of new therapeutic treatments against the disease.

The BAFF / APRIL axis plays an important role in virus-induced peritoneal responses in rainbow trout.

IgM+ B cells have been recently demonstrated to be key regulators of peritoneal inflammation in teleost, as a large number of them occupy the peritoneal cavity after 48 h of antigenic stimulation. Despite this, the number of studies addressing the mechanism through which this cell population expands and differentiates in response to stimuli has been scarcely addressed. Because the BAFF/APRIL axis is known to play a major role in B cell survival and differentiation in mammals, we hypothesized that it could be affected in the peritoneal cavity in response to an inflammatory stimulus. To verify this hypothesis, we studied how BAFF, APRIL and the fish-specific related cytokine BALM as well as their putative receptors are regulated in rainbow trout after intraperitoneal (i.p.) injection of viral hemorrhagic septicemia virus (VHSV). When the transcriptional analysis was performed in total cells from the peritoneum, we observed that VHSV provoked an up-regulation of both BAFF and BAFF receptor (BAFF-R) mRNA levels. However, when we examined how isolated peritoneal IgM+ B cells were transcriptionally affected by VHSV i.p. injection, we found that APRIL, BALM and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) were also up-regulated in response to the virus. IgM- cells, on the other hand, only up-regulated BALM transcription in response to VHSV. Finally, to gain further insight on the role that these cytokines play in the peritoneum, we have studied their effect on the survival of peritoneal IgM+ B cells. This work demonstrates a key role for the BAFF/APRIL axis in the peritoneal inflammatory response and contributes to further understanding how IgM+ B cells are regulated at this specific peripheral site.

Expansion and differentiation of IgM+ B cells in the rainbow trout peritoneal cavity in response to different antigens.

To date, intraperitoneal (i.p.) injection seems to be the most effective vaccination route in aquaculture, as many i.p. administered fish vaccines are capable of conferring strong and long-lasting immune responses. Despite this, how peritoneal leukocytes are regulated upon antigen encounter has only been scarcely studied in fish. Although, in the past, myeloid cells were thought to be the main responders to peritoneal inflammation, a recent study revealed that IgM+ B cells are one of the main cell types in the teleost peritoneal cavity in response to pathogenic bacteria. Thus, in the current work, we have focused on establishing how IgM+ B cells are recruited into the peritoneum in rainbow trout (Oncorhynchus mykiss) comparing different antigens: Escherichia coli as a bacterial model, E. coli-derived lipopolysaccharide (LPS) or viral hemorrhagic septicemia virus (VHSV). In addition to studying their capacity to dominate the peritoneal cavity, we have established how these IgM+ B cells are regulated in response to the different antigens, determining their levels of IgM secretion, surface MHC II expression, cell size and phagocytic abilities. Our results reveal that IgM+ B cells are one of the main cell types amplified in the peritoneum in response to either bacterial or viral antigens and that these immunogenic stimulations provoke a differentiation of some of these cells towards plasmablasts/plasma cells whereas others seem to be implicated in antigen presentation. These findings contribute to a better understanding of the immune processes that regulate peritoneal inflammation in teleost fish.

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

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.

Rainbow trout CK9, a CCL25-like ancient chemokine that attracts and regulates B cells and macrophages, the main antigen presenting cells in fish.

CK9 is a rainbow trout (Oncorhynchus mykiss) CC chemokine phylogenetically related to mammalian CCL25. Although CK9 is known to be transcriptionally regulated in response to inflammation particularly in mucosal tissues, its functionality has never been revealed. In the current work, we have demonstrated that CK9 is chemoattractant for antigen presenting cells (APCs) expressing major histocompatibility complex class II (MHC II) on the cell surface. Among these APCs, CK9 has a strong chemotactic capacity for both B cells (IgM+ and IgT+) and macrophages. Along with its chemotactic capacities, CK9 modulated the MHC II turnover of B lymphocytes and up-regulated the phagocytic capacity of both IgM+ cells and macrophages. Although CK9 had no lymphoproliferative effects, it increased the survival of IgT+ lymphocytes. Furthermore, we have established that the chemoattractant capacity of CK9 is strongly increased after pre-incubation of leukocytes with a T-independent antigen, whereas B cell receptor (BCR) cross-linking strongly abrogated their capacity to migrate to CK9, indicating that CK9 preferentially attracts B cells at the steady state or under BCR-independent stimulation. These results point to CK9 being a key regulator of B lymphocyte trafficking in rainbow trout, able to modulate innate functions of teleost B lymphocytes and macrophages.

Distribution of T Cells in Rainbow Trout (Oncorhynchus mykiss) Skin and Responsiveness to Viral Infection.

Although the skin constitutes the first line of defense against waterborne pathogens, there is a great lack of information regarding the skin associated lymphoid tissue (SALT) and whether immune components of the skin are homogeneously distributed through the surface of the fish is still unknown. In the current work, we have analyzed the transcription of several immune genes throughout different rainbow trout (Oncorhynchus mykiss) skin areas. We found that immunoglobulin and chemokine gene transcription levels were higher in a skin area close to the gills. Furthermore, this skin area as well as other anterior sections also transcribed significantly higher levels of many different immune genes related to T cell immunity such as T cell receptor α (TCRα), TCRγ, CD3, CD4, CD8, perforin, GATA3, Tbet, FoxP3, interferon γ (IFNγ), CD40L and Eomes in comparison to posterior skin sections. In agreement with these results, immunohistochemical analysis revealed that anterior skin areas had a higher concentration of CD3(+) T cells and flow cytometry analysis confirmed that the percentage of CD8(+) T lymphocytes was also higher in anterior skin sections. These results demonstrate for the first time that T cells are not homogeneously distributed throughout the teleost skin. Additionally, we studied the transcriptional regulation of these and additional T cell markers in response to a bath infection with viral hemorrhagic septicemia virus (VHSV). We found that VHSV regulated the transcription of several of these T cell markers in both the skin and the spleen; with some differences between anterior and posterior skin sections. Altogether, our results point to skin T cells as major players of teleost skin immunity in response to waterborne viral infections.

Identification of Teleost Skin CD8α+ Dendritic-like Cells, Representing a Potential Common Ancestor for Mammalian Cross-Presenting Dendritic Cells.

Although fish constitute the most ancient animal group in which an acquired immune system is present, the presence of dendritic cells (DCs) in teleosts has been addressed only briefly, and the identification of a specific DC subset in teleosts remained elusive because of the lack of specific Abs. In mice, DCs expressing CD8α(+) in lymphoid tissues have the capacity to cross-present extracellular Ags to T cells through MHC I, similarly to tissue-derived CD103(+) DCs and the human CD141(+) DC population. In the current study, we identified a large and highly complex subpopulation of leukocytes coexpressing MHC class II and CD8α. This CD8α(+) MHC II(+) DC-like subpopulation constituted ∼1.2% of the total leukocyte population in the skin, showing phenotypical and functional characteristics of semimature DCs that seem to locally regulate mucosal immunity and tolerance in a species lacking lymph nodes. Furthermore, we identified trout homologs for CD141 and CD103 and demonstrated that, in trout, this skin CD8(+) DC-like subpopulation expresses both markers. To our knowledge, these results provide the first evidence of a specific DC-like subtype in nonimmune tissue in teleosts and support the hypothesis of a common origin for all mammalian cross-presenting DCs.

A cannabigerol quinone alleviates neuroinflammation in a chronic model of multiple sclerosis.

Phytocannabinoids like ∆(9)-tetrahydrocannabinol (THC) and cannabidiol (CBD) show a beneficial effect on neuroinflammatory and neurodegenerative processes through cell membrane cannabinoid receptor (CBr)-dependent and -independent mechanisms. Natural and synthetic cannabinoids also target the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARγ), an attractive molecular target for the treatment of neuroinflammation. As part of a study on the SAR of phytocannabinoids, we have investigated the effect of the oxidation modification in the resorcinol moiety of cannabigerol (CBG) on CB(1), CB(2) and PPARγ binding affinities, identifying cannabigerol quinone (VCE-003) as a potent anti-inflammatory agent. VCE-003 protected neuronal cells from excitotoxicity, activated PPARγ transcriptional activity and inhibited the release of pro-inflammatory mediators in LPS-stimulated microglial cells. Theiler's murine encephalomyelitis virus (TMEV) model of multiple sclerosis (MS) was used to investigate the anti-inflammatory activity of this compound in vivo. Motor function performance was evaluated and the neuroinflammatory response and gene expression pattern in brain and spinal cord were studied by immunostaining and qRT-PCR. We found that VCE-003 ameliorated the symptoms associated to TMEV infection, decreased microglia reactivity and modulated the expression of genes involved in MS pathophysiology. These data lead us to consider VCE-003 to have high potential for drug development against MS and perhaps other neuroinflammatory diseases.

Asymmetric segregation of polarized antigen on B cell division shapes presentation capacity.

During the activation of humoral immune responses, B cells acquire antigen for subsequent presentation to cognate T cells. Here we show that after mouse B cells accumulate antigen, it is maintained in a polarized distribution for extended periods in vivo. Using high-throughput imaging flow cytometry, we observed that this polarization is preserved during B cell division, promoting asymmetric antigen segregation among progeny. Antigen inheritance correlates with the ability of progeny to activate T cells: Daughter cells receiving larger antigen stores exhibit a prolonged capacity to present antigen, which renders them more effective in competing for T cell help. The generation of progeny with differential capacities for antigen presentation may have implications for somatic hypermutation and class switching during affinity maturation and as B cells commit to effector cell fates.