Adriana Gruppi

BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway

The B cell activating factor belonging to the tumor necrosis factor family (BAFF) is required for B cell survival and maturation. The mechanisms by which BAFF mediates B cell survival are less understood. We found that BAFF and a proliferation-inducing ligand (APRIL), which are related, block B cell antigen receptor (BCR)–induced apoptosis upstream of mitochondrial damage, which is consistent with a role for Bcl-2 family proteins. BCR ligation strongly increased expression of the proapoptotic Bcl-2 homology 3–only Bcl-2 protein Bim in both WEHI-231 and splenic B cells, and increases in Bim were reversed by BAFF or APRIL. Small interfering RNA vector–mediated suppression of Bim blocked BCR-induced apoptosis. BAFF also induced Bim phosphorylation and inhibited BCR-induced association of Bim with Bcl-2. BAFF induced delayed but sustained stimulation of extracellular signal–regulated kinase (ERK) and its activators, mitogen-activated protein kinase/ERK activating kinase (MEK) and c-Raf, and MEK inhibitors promoted accumulation and dephosphorylation of Bim. These results suggest that BAFF inhibits BCR-induced death by down-regulating Bim via sustained ERK activation, demonstrating that BAFF directly regulates Bim function. Although transitional immature type 1 (T1) B cell numbers are normal in Bim−/− mice, T2 and follicular mature B cells are elevated and marginal zone B cells are reduced. Our results suggest that mature B cell homeostasis is maintained by BAFF-mediated regulation of Bim.

Trypanosoma cruzi trans-sialidase initiates a program independent of the transcription factors RORγt and Ahr that leads to IL-17 production by activated B cells

We identified B cells as a major source for rapid, innate-like interleukin 17 (IL-17) production in vivo in response to Trypanosoma cruzi infection. IL-17+ B cells exhibited a plasmablast phenotype, outnumbered TH17 cells and were required for optimal response to this pathogen. Using both murine and human primary B cells, we demonstrate that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell surface mucin, CD45, leading to Btk-dependent signaling and IL-17A or IL-17F production via an ROR-γt and AHR-independent transcriptional program. Our combined data suggest that generation of IL-17+ B cells may be an unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.Here we identified B cells as a major source of rapid, innate-like production of interleukin 17 (IL-17) in vivo in response to infection with Trypanosoma cruzi. IL-17+ B cells had a plasmablast phenotype, outnumbered cells of the TH17 subset of helper T cells and were required for an optimal response to this pathogen. With both mouse and human primary B cells, we found that exposure to parasite-derived trans-sialidase in vitro was sufficient to trigger modification of the cell-surface mucin CD45, which led to signaling dependent on the kinase Btk and production of IL-17A or IL-17F via a transcriptional program independent of the transcription factors RORγt and Ahr. Our combined data suggest that the generation of IL-17+ B cells may be a previously unappreciated feature of innate immune responses required for pathogen control or IL-17-mediated autoimmunity.

Galectin-3 Mediates IL-4-Induced Survival and Differentiation of B Cells: Functional Cross-Talk and Implications during Trypanosoma cruzi Infection

The role of transcription factors in B cell survival and differentiation has been delineated during the last years. However, little is known about the intermediate signals and the intracellular pathways that control these events. In this study, we provide evidence both in vitro and in vivo, showing that galectin-3 (Gal-3), a β-galactoside-binding protein, is a critical mediator of B cell differentiation and survival. Although Gal-3 is not expressed in resting B cells from normal mice, its expression is markedly induced after activation with stimuli such as IL-4 and CD40 cross-linking. These signals promote survival and block the final differentiation of these cells, thus allowing the rising of a memory B cell phenotype. In addition, Gal-3 is expressed in B cells from Trypanosoma cruzi-infected mice, which received signals for activation and differentiation in vivo. By using an antisense strategy, we determined that Gal-3 is a critical signal mediating the effects of IL-4 on B cell fate. Blockade of intracellular Gal-3 in vitro abrogated IL-4-induced survival of activated B cells, favoring the differentiation toward a plasma cell pathway. Moreover, B cells with restrained endogenous Gal-3 expression failed to down-regulate the Blimp-1 transcription factor after IL-4 stimulation. Finally, inhibition of Gal-3 in vivo skewed the balance toward plasma cell differentiation, which resulted in increased Ig production and parasite clearance during T. cruzi infection. Thus, the present study provides evidence of a novel role for Gal-3 as an intracellular mediator of B cell survival and a checkpoint in IL-4-induced B cell commitment toward a memory phenotype.

Regulated expression of galectin-1 during B-cell activation and implications for T-cell apoptosis

Galectin‐1 (GAL‐1), a highly conserved β‐galactoside‐binding protein, has shown immunomodulatory properties. In this study, we investigated the regulation of GAL‐1 expression within the B‐cell compartment usingTrypanosoma cruzi infection as a natural model of in vivo B‐cell activation. GAL‐1 was found to be expressed on activated B cells from T. cruzi‐infected mice, mainly localized at the cytosolic compartment. Expression of this protein was found to be modulated according to the activation state of the cells, revealing a significant increase in stimulated B cells that received signals via cross‐linking of the B‐cell receptor and CD40. It was found that GAL‐1 was secreted by B cells to the extracellular milieu upon activation. Finally, purified GAL‐1 produced by activated B cells induced apoptosis of T cells but not B cells and also influenced interferon‐γ cytokine production. Hence, the present study describes a potential mechanism by which B cells can regulate T‐cell function and survival.

Galectins as immunoregulators during infectious processes: from microbial invasion to the resolution of the disease.

Recent evidence has implicated galectins, a family of evolutionarily conserved carbohydrate‐binding proteins, as regulators of immune cell homeostasis and host–pathogen interactions. Galectins operate at different levels of innate and adaptive immune responses, by modulating cell survival and cell activation or by influencing the Th1/Th2 cytokine balance. Furthermore, galectins may contribute to host–pathogen recognition and may serve as receptors for specific interactions of pathogens with their insect vectors. Here we will explore the influence of galectins in immunological processes relevant to microbial infection and will summarize exciting recent work related to the specific interactions between galectins and their glycoconjugate ligands as critical determinants of pathogen recognition. Understanding the role of galectin–sugar interactions during the course of microbial infections might contribute to defining novel targets for disease prevention and immune intervention.

Polyclonal B cell activation in infections: infectious agents’ devilry or defense mechanism of the host?

Polyclonal B cell activation is not a peculiar characteristic to a particular infection, as many viruses, bacteria, and parasites induce a strong polyclonal B cell response resulting in hyper‐γ‐globulinemia. Here, we discuss the different roles proposed for polyclonal B cell activation, which can be crucial for early host defense against rapidly dividing microorganisms by contributing antibodies specific for a spectrum of conserved structures present in the pathogens. In addition, polyclonal B cell activation can be responsible for maintenance of memory B cell responses because of the continuous, unrestricted stimulation of memory B cells whose antibody production may be sustained in the absence of the antigens binding‐specific BCR. Conversely, polyclonal activation can be triggered by microorganisms to avoid the host‐specific, immune response by activating B cell clones, which produce nonmicroorganism‐specific antibodies. Finally, some reports suggest a deleterious role for polyclonal activation, arguing that it could potentially turn on anti‐self‐responses and lead to autoimmune manifestations during chronic infections.

IL-17RA Signaling Reduces Inflammation and Mortality during Trypanosoma cruzi Infection by Recruiting Suppressive IL-10-Producing Neutrophils

Members of the IL-17 cytokine family play an important role in protection against pathogens through the induction of different effector mechanisms. We determined that IL-17A, IL-17E and IL-17F are produced during the acute phase of T. cruzi infection. Using IL-17RA knockout (KO) mice, we demonstrate that IL-17RA, the common receptor subunit for many IL-17 family members, is required for host resistance during T. cruzi infection. Furthermore, infected IL-17RA KO mice that lack of response to several IL-17 cytokines showed amplified inflammatory responses with exuberant IFN-γ and TNF production that promoted hepatic damage and mortality. Absence of IL-17RA during T. cruzi infection resulted in reduced CXCL1 and CXCL2 expression in spleen and liver and limited neutrophil recruitment. T. cruzi-stimulated neutrophils secreted IL-10 and showed an IL-10-dependent suppressive phenotype in vitro inhibiting T-cell proliferation and IFN-γ production. Specific depletion of Ly-6G+ neutrophils in vivo during T. cruzi infection raised parasitemia and serum IFN-γ concentration and resulted in increased liver pathology in WT mice and overwhelming wasting disease in IL-17RA KO mice. Adoptively transferred neutrophils were unable to migrate to tissues and to restore resistant phenotype in infected IL-17RA KO mice but migrated to spleen and liver of infected WT mice and downregulated IFN-γ production and increased survival in an IL-10 dependent manner. Our results underscore the role of IL-17RA in the modulation of IFN-γ-mediated inflammatory responses during infections and uncover a previously unrecognized regulatory mechanism that involves the IL-17RA-mediated recruitment of suppressive IL-10-producing neutrophils.

Trypanosoma cruzi Infection Selectively Renders Parasite-Specific IgG+ B Lymphocytes Susceptible to Fas/Fas Ligand-Mediated Fratricide

The control of B cell expansion has been thought to be solely regulated by T lymphocytes. We show in this study that Trypanosoma cruzi infection induces up-regulation of both Fas and Fas ligand (FasL) molecules on B cells and renders them susceptible to B cell-B cell killing (referred to as fratricide throughout this paper) mediated via Fas/FasL. Moreover, by in vivo administration of anti-FasL blocking mAb we demonstrate that Fas-mediated B cell apoptosis is an ongoing process during this parasitic infection. We also provide evidence that B cells that have switched to IgG isotype are the preferential targets of B cell fratricide. More strikingly, this death pathway selectively affects IgG+ B cells reactive to parasite but not self Ags. Parasite-specific but not self-reactive B cells triggered during this response are rescued after either in vitro or in vivo FasL blockade. Fratricide among parasite-specific IgG+ B lymphocytes could impair the immune control of T. cruzi and possibly other chronic protozoan parasites. Our results raise the possibility that the blockade of Fas/FasL interaction in the B cell compartment of T. cruzi-infected mice may provide a means for enhancing antiparasitic humoral immune response without affecting host tolerance.

Trypanosoma cruzi-induced immunosuppression : B cells undergo spontaneous apoptosis and lipopolysaccharide (LPS) arrests their proliferation during acute infection

Acute infection with Trypanosoma cruzi is characterized by multiple manifestations of immunosuppression of both cellular and humoral responses. B cells isolated at the acute stage of infection have shown marked impairment in their response to polyclonal activators in vitro. The present work aims at studying the B cell compartment in the context of acute T. cruzi infection to provide evidence for B cell activation, spontaneous apoptosis and arrest of the cell cycle upon mitogenic stimulation as a mechanism underlying B cell hyporesponse. We found that B cells from acutely infected mice, which fail to respond to the mitogen LPS, showed spontaneous proliferation and production of IgM, indicating a high level of B cell activation. Furthermore, these activated B cells also exhibited an increase in Fas expression and apoptosis in cultures without an exogenous stimulus. On the other hand, B cells from early acute and chronic infected mice did not present activation or apoptosis, and were able to respond properly to the mitogen. Upon in vitro stimulation with LPS, B cells from hyporesponder mice failed to progress through the cell cycle (G0/G1 arrest), nor did they increase the levels of apoptosis. These results indicate that B cell apoptosis and cell cycle arrest could be the mechanisms that control intense B cell expansion, but at the same time could be delaying the emergence of a specific immune response against the parasite.

BAFF and LPS cooperate to induce B cells to become susceptible to CD95/Fas‐mediated cell death

Microorganisms with pathogen‐associated molecular patterns (PAMP) activate B cells directly by binding to TLR and also indirectly by inducing APC to release cytokines such as BAFF that promote B cell survival. We found that murine B cells activated concomitantly with LPS (TLR‐4 ligand) and BAFF are protected from spontaneous apoptosis, but are more susceptible to Fas/CD95‐mediated cell death. This increased susceptibility to Fas‐induced apoptosis is associated with a dramatic coordinated up‐regulation of Fas/CD95 and IRF‐4 expression through a mechanism mediated, at least in part, by inhibition of the MEK/ERK pathway. Up‐regulation of Fas/CD95 by BAFF is restricted to B cells activated through TLR‐4, but not through TLR‐9, BCR or CD40. TLR ligands differ in the BAFF family receptors (R) they induce on B cells: BAFF‐R is increased by the TLR4 ligand, LPS, but not by the TLR9 ligand, CpG‐containing oligodeoxynucleotides, which, in contrast, strongly up‐regulates transmembrane activator and CAML interactor (TACI). This suggests the up‐regulation of Fas by BAFF is mediated by BAFF‐R and not by TACI. Consistently, APRIL, which binds to TACI and B cell maturation antigen but not BAFF‐R, did not enhance Fas expression on LPS‐activated B cells. Increased susceptibility to Fas‐mediated killing of B cells activated with LPS and BAFF may be a fail‐safe mechanism to avoid overexpansion of nonspecific or autoreactive B cells.