Carolina L. Montes

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.

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.

A Trypanosoma cruzi antigen signals CD11b+ cells to secrete cytokines that promote polyclonal B cell proliferation and differentiation into antibody-secreting cells.

Microbial‐induced polyclonal activation of B cells is a common event in several forms of infections, and is believed to play a crucial role both for enhancing the production of specific antibodies and for maintenance of B cell memory. Therefore, a major challenge in biomedical research is the identification of pathogen‐derived products capable of rapidly mounting B cell expansion and differentiation. Here we report that glutamate dehydrogenase (GDH) stimulates polyclonal proliferation and differentiation of naive B cells. This stimulation was found to be T cell independent, but to absolutely require CD11b+ cells. Moreover, we demonstrate that stimulation of CD11b+ cells by GDH leads to the production of IL‐6, IL‐10 and B cell‐activating factor (BAFF), all of which combine to powerfully induce B cell expansion. Importantly, IL‐6 and IL‐10 further drive B cell terminal differentiation into plasma cells by up‐regulating critical transcription factors and immunoglobulin secretion. Our data provide the first evidence that a protozoan antigen can induce BAFF production by accessory cells, which in concert with other cytokines trigger polyclonal B cell activation.

Trypanosoma cruzi infection induces a massive extrafollicular and follicular splenic B-cell response which is a high source of non-parasite-specific antibodies.

Acute infection with Trypanosoma cruzi, the aetiological agent of Chagas’ disease, results in parasitaemia and polyclonal lymphocyte activation. It has been reported that polyclonal B‐cell activation is associated with hypergammaglobulinaemia and delayed parasite‐specific antibody response. In the present study we analysed the development of a B‐cell response within the different microenvironments of the spleen during acute T. cruzi infection. We observed massive germinal centre (GC) and extrafollicular (EF) responses at the peak of infection. However, the EF foci were evident since day 3 post‐infection (p.i.), and, early in the infection, they mainly provided IgM. The EF foci response reached its peak at 11 days p.i. and extended from the red pulp into the periarteriolar lymphatic sheath. The GCs were detected from day 8 p.i. At the peak of parasitaemia, CD138+ B220+ plasma cells in EF foci, red pulp and T‐cell zone expressed IgM and all the IgG isotypes. Instead of the substantial B‐cell response, most of the antibodies produced by splenic cells did not target the parasite, and parasite‐specific IgG isotypes could be detected in sera only after 18 days p.i. We also observed that the bone marrow of infected mice presented a strong reduction in CD138+ B220+ cells compared with that of normal mice. Hence, in acute infection with T. cruzi, the spleen appears to be the most important lymphoid organ that lodges plasma cells and the main producer of antibodies. The development of a B‐cell response during T. cruzi infection shows features that are particular to T. cruzi and other protozoan infection but different to other infections or immunization with model antigens.

Trypanosoma cruzi mitochondrial malate dehydrogenase triggers polyclonal B‐cell activation

It has been proposed that Trypanosoma cruzi, the aetiologic agent of Chagas’ disease, produces mitogenic substances responsible for the polyclonal B‐cell activation observed during the acute phase of the infection. Isolation and characterization of the molecules involved in the induction of polyclonal activation observed during infectious diseases have posed a great challenge for the immunologist over the last decade. In this work we report that a 33 kD protein obtained from an alkaline fraction of T. cruzi epimastigotes (FI) stimulates proliferation and promotes differentiation into antibody‐secreting cells of normal murine B cells in a T‐cell independent manner. By flow cytometry we also found that the 33 kDa protein induces an increase in the expression of MHC class II and B7.2 but not B7.1 molecules on the B‐cell surface. Sequencing by mass spectrometry identified the T. cruzi 33 kD protein as hypothetical oxidoreductase, a member of the aldo/ketoreductase family. In this report we demonstrate that this protein is also present in the infective bloodstream trypomastigote form of the parasite and was identified as T. cruzi mitochondrial malate dehydrogenase (mMDH) by enzyme activity and by Western blotting using a specific mMDH polyclonal antiserum. The biologic relevance of mMDH‐induced polyclonal activation concerning T. cruzi infection is discussed.

Depletion of immature B cells during Trypanosoma cruzi infection: involvement of myeloid cells and the cyclooxygenase pathway

The ability of a microorganism to elicit or evade B cell responses represents a determinant factor for the final outcome of an infection. Although pathogens may subvert humoral responses at different stages of B cell development, most studies addressing the impact of an infection on the B cell compartment have focused on mature B cells within peripheral lymphoid organs. Herein, we report that a protozoan infection, i.e. a Trypanosoma cruzi infection, induces a marked loss of immature B cells in the BM, which also compromises recently emigrated B cells in the periphery. The depletion of BM immature B cells is associated with an increased rate of apoptosis mediated by a parasite‐indirect mechanism in a Fas/FasL‐independent fashion. Finally, we demonstrated that myeloid cells play an important role in B cell depletion, since CD11b+ BM cells from infected mice secrete a product of the cyclooxygenase pathway that eliminates immature B cells. These results highlight a previously unrecognized maneuver used by a protozoan parasite to disable B cell generation, limiting host defense and favoring its chronic establishment.