Evaluation of Tritrichomonas foetus infection clearance in heifers immunized with a single intravaginal dose of formaldehyde fixed strain B1 cells.

Vaccines against Tritrichomonas foetus have been shown to reduce the time of infection after natural or experimental exposure. The object of this study was to assess the protection against T. foetus infection conferred by a single vaginal instillation of formaldehyde fixed T. foetus cells. Aberdeen Angus virgin heifers were randomly allocated to 3 groups of 12 individuals to receive placebo or formaldehyde fixed T. foetus cells prepared following one of two procedures (formalin or freshly prepared solution) and six weeks later they were challenged with 106T. foetus trophozoites. The median time for clearance among control heifers was 93.75 days while in animals immunized with formaldehyde fixed T. foetus it was 45 days. A single vaginal dose of cells fixed with fresh formaldehyde solution gave a rate of decay of infection per unit of time of 2.54 (CI 95% = 1.07;6.01).

Plasmodium DNA-mediated TLR9 activation of T-bet+ B cells contributes to autoimmune anaemia during malaria.

Infectious pathogens contribute to the development of autoimmune disorders, but the mechanisms connecting these processes are incompletely understood. Here we show that Plasmodium DNA induces autoreactive responses against erythrocytes by activating a population of B cells expressing CD11c and the transcription factor T-bet, which become major producers of autoantibodies that promote malarial anaemia. Additionally, we identify parasite DNA-sensing through Toll-like receptor 9 (TLR9) along with inflammatory cytokine receptor IFN-γ receptor (IFN-γR) as essential signals that synergize to promote the development and appearance of these autoreactive T-bet+ B cells. The lack of any of these signals ameliorates malarial anaemia during infection in a mouse model. We also identify both expansion of T-bet+ B cells and production of anti-erythrocyte antibodies in ex vivo cultures of naive human peripheral blood mononuclear cells (PBMC) exposed to P. falciprum infected erythrocyte lysates. We propose that synergistic TLR9/IFN-γR activation of T-bet+ B cells is a mechanism underlying infection-induced autoimmune-like responses.

Subversion of the B-cell compartment during parasitic, bacterial, and viral infections.

Recent studies on HIV infection have identified new human B-cell subsets with a potentially important impact on anti-viral immunity. Current work highlights the occurrence of similar B-cell alterations in other viral, bacterial, and parasitic infections, suggesting that common strategies have been developed by pathogens to counteract protective immunity. For this review, we have selected key examples of human infections for which B-cell alterations have been described, to highlight the similarities and differences in the immune responses to a variety of pathogens. We believe that further comparisons between these models will lead to critical progress in the understanding of B-cell mechanisms and will open new target avenues for therapeutic interventions.

Leishmania infantum amastigotes trigger a subpopulation of human B cells with an immunoregulatory phenotype.

Visceral leishmaniasis is caused by the protozoan parasites Leishmania infantum and Leishmania donovani. This infection is characterized by an uncontrolled parasitization of internal organs which, when left untreated, leads to death. Disease progression is linked with the type of immune response generated and a strong correlation was found between disease progression and serum levels of the immunosuppressive cytokine IL-10. Other studies have suggested a role for B cells in the pathology of this parasitic infection and the recent identification of a B-cell population in humans with regulatory functions, which secretes large amounts of IL-10 following activation, have sparked our interest in the context of visceral leishmaniasis. We report here that incubation of human B cells with Leishmania infantum amastigotes resulted in upregulation of multiple cell surface activation markers and a dose-dependent secretion of IL-10. Conditioned media from B cells incubated with Leishmania infantum amastigotes were shown to strongly inhibit CD4(+) T-cell activation, proliferation and function (i.e. as monitored by TNF and IFNγ secretion). Blockade of IL-10 activity using a soluble IL-10 receptor restored only partially TNF and IFNγ production to control levels. The parasite-mediated IL-10 secretion was shown to rely on the activity of Syk, phosphatidylinositol-3 kinase and p38, as well as to require intracellular calcium mobilization. Cell sorting experiments allowed us to identify the IL-10-secreting B-cell subset (i.e. CD19(+)CD24(+)CD27(-)). In summary, exposure of human B cells to Leishmania infantum amastigotes triggers B cells with regulatory activities mediated in part by IL-10, which could favor parasite dissemination in the organism.

B cells have distinct roles in host protection against different nematode parasites.

B cells can mediate protective responses against nematode parasites by supporting Th2 cell development and/or by producing Abs. To examine this, B cell-deficient mice were inoculated with Nippostrongylus brasiliensis or Heligmosomoides polygyrus. B cell-deficient and wild type mice showed similar elevations in Th2 cytokines and worm expulsion after N. brasiliensis inoculation. Worm expulsion was inhibited in H. polygyrus-inoculated B cell-deficient mice, although Th2 cytokine elevations in mucosal tissues were unaffected. Impaired larval migration and development was compromised as early as day 4 after H. polygyrus challenge, and administration of immune serum restored protective immunity in B cell-deficient mice, indicating a primary role for Ab. Immune serum even mediated protective effects when administered to naive mice prior to inoculation. This study suggests variability in the importance of B cells in mediating protection against intestinal nematode parasites, and it indicates an important role for Ab in resistance to tissue-dwelling parasites.

Identification and characterization of autoantibody-producing B220(low) B (B-1) cells appearing in malarial infection.

Mice with malaria showed unique immunological responses, including the expansion of NK1.1(-)TCR(int) cells (extrathymic T cells). Since TCR(int) cells with autoreactivity and autoantibody-producing B cells (B-1 cells) are often simultaneously activated under autoimmune conditions, it was examined whether B-1 cells were activated in the course of malarial infection. From days 14 after infection, B220(low) B-1 cells appeared in the liver and spleen. The number of B220(low) B cells was highest at day 14, but the ratio was highest at days 28-35. In parallel with the appearance of B220(low) cells, autoantibodies against HEp-2 cells and double-stranded DNA were detected in sera. These B220(low) cells had phenotypes of CD44(high), CD23(-) and CD62L(-). In sharp contrast, conventional B220(high) B cells (B-2 cells) were CD44(low), CD23(+) and CD62L(+). These results suggested that malaria immune responses were not mediated by conventional T and B cells but resembled the responses during autoimmune diseases.

Helminth antigens modulate immune responses in cells from multiple sclerosis patients through TLR2-dependent mechanisms.

To better understand the link between parasite infections and the course of multiple sclerosis (MS), we studied the role of TLRs in helminth product recognition by dendritic cells (DCs) and B cells. Baseline expression of TLR2 was significantly higher in infected-MS patients compared with uninfected MS subjects or healthy controls. Moreover, cells exposed to TLR2 agonists or to soluble egg Ag (SEA) from Schistosoma mansoni resulted in significant TLR2 up-regulation. SEA suppressed the LPS-induced DCs production of IL-1beta, IL-6, IL-12, and TNF-alpha and enhanced TGF-beta as well as IL-10 production. Similarly, after exposure to SEA, anti-CD40-activated B cells increased IL-10 production. Both processes were MyD88 dependent. In addition, SEA down-regulated the expression of LPS-induced costimulatory molecules on DCs in a MyD88-independent manner. DCs stimulation by SEA and TLR2 agonists induced increasing phosphorylation of the MAPK ERK1/2. Neither stimulus showed an effect on p38 and JNK1/2 phosphorylation, however. Addition of the ERK1/2 inhibitor U0126 was associated with dose-dependent inhibition of IL-10 and reciprocal enhancement of IL-12. Finally, cytokine effects and changes observed in DCs costimulatory molecule expression after SEA exposure were lost when TLR2 expression was silenced. Overall, these findings indicate that helminth molecules exert potent regulatory effects on both DCs and B cells through TLR2 regulation conducted via different signaling pathways. This knowledge could prove critical in developing novel therapeutic approaches for the treatment of autoimmune diseases such as MS.

Gradual decline in malaria-specific memory T cell responses leads to failure to maintain long-term protective immunity to Plasmodium chabaudi AS despite persistence of B cell memory and circulating antibody.

The mechanisms responsible for the generation and maintenance of immunological memory to Plasmodium are poorly understood and the reasons why protective immunity in humans is so difficult to achieve and rapidly lost remain a matter for debate. A possible explanation for the difficulty in building up an efficient immune response against this parasite is the massive T cell apoptosis resulting from exposure to high-dose parasite Ag. To determine the immunological mechanisms required for long-term protection against P. chabaudi malaria and the consequences of high and low acute phase parasite loads for acquisition of protective immunity, we performed a detailed analysis of T and B cell compartments over a period of 200 days following untreated and drug-treated infections in female C57BL/6 mice. By comparing several immunological parameters with the capacity to control a secondary parasite challenge, we concluded that loss of full protective immunity is not determined by acute phase parasite load nor by serum levels of specific IgG2a and IgG1 Abs, but appears to be a consequence of the progressive decline in memory T cell response to parasites, which occurs similarly in untreated and drug-treated mice with time after infection. Furthermore, by analyzing adoptive transfer experiments, we confirmed the major role of CD4(+) T cells for guaranteeing long-term full protection against P. chabaudi malaria.

Helminth infections associated with multiple sclerosis induce regulatory B cells.

OBJECTIVE: To assess the importance of B-cell control during parasite infections in multiple sclerosis (MS) patients. METHODS: Peripheral blood CD19+ B cells from 12 helminth-infected MS patients, 12 MS patients without infection, 10 patients infected with Trypanosoma cruzi, 8 subjects infected with Paracoccidioides brasiliensis, and 12 healthy control subjects were purified using magnetic cell sorting. Interleukin (IL)-4, IL-6, IL-10, tumor necrosis factor-alpha, lymphotoxin, transforming growth factor-beta, brain-derived neurotrophic factor, and nerve growth factor secretion were evaluated after stimulation with CDw32 L cells and CD40 antibody using enzyme-linked immunosorbent assays. The production of anti-myelin oligodendrocyte glycoprotein IgG and IgM antibodies was evaluated by enzyme-linked immunosorbent spot assays. Cell phenotype was assessed by flow cytometry. RESULTS: Helminth infections in MS patients created a B-cell population producing high levels of IL-10, dampening harmful immune responses through a mechanism mediated, at least in part, by the ICOS-B7RP-1 pathway. The IL-10-producing B-cell phenotype detected expressed high levels of CD1d and was similar to the one observed in mature naive B2 cells (namely, CD11b(-), CD5(-), CD27(-), and IgD+). Moreover, B cells isolated from helminth-infected MS patients also produced greater amounts of brain-derived neurotrophic factor and nerve growth factor compared with those of normal subjects, T. cruzi-infected subjects, P. brasiliensis-infected subjects, or uninfected MS patients, raising the possibility that these cells may exert a neuroprotective effect on the central nervous system. INTERPRETATION: Increased production of B-cell-derived IL-10 and of neurotrophic factors are part of the parasite's regulation of host immunity and can alter the course of MS, potentially explaining environmental-related MS suppression observed in areas with low disease prevalence.

Trypanosoma cruzi infection beats the B-cell compartment favouring parasite establishment: can we strike first?

Trypanosoma cruzi, the causative agent of Chagas' disease, may sabotage humoral response by affecting B cells at the different stages of its development. The present review highlights the contributions of our laboratory in understanding how T. cruzi hinders B-cell generation and B-cell expansion limiting host defence and favouring its chronic establishment. We discuss how homoeostatic mechanisms can be triggered to control exacerbated B-cell proliferation that favour T. cruzi infection by eliminating parasite-specific B cells. Specific targeting of evasion mechanisms displayed in T. cruzi infection, as in vivo Fas/FasL blockade or Gal-3 expression inhibition, allowed us to modulate B-cell responses enhancing the anti-parasite humoral immune response. A comprehensive understanding of the biology of the B cell in health and disease is strictly required to devise immunointervention strategies aimed at enhancing protective immune responses during infections.

Characterization of the spleen B-cell compartment at the early and late blood-stage Plasmodium chabaudi malaria.

Polyclonal B-cell activation is a feature of the early spleen cell response to blood-stage Plasmodium chabaudi malaria. Immunity to blood-stage malaria is guaranteed by the generation of B cells able to produce parasite-specific antibodies mainly from the immunoglobulin (Ig)G2a isotype. In the present study, we characterized the spleen B-cell compartment during blood-stage P. chabaudi infection. The numbers of B220(+) and B220(LOW) CD138(+) (plasma) cells increased sharply between days 4 and 7 post-infection (p.i.). At this time B220(+) cells expressed surface (s)IgM, but nearly all B220(LOW) CD138(+) cells showed concomitantly intracellular (i)IgM and IgG2a. Both follicular and marginal zone B cells were activated expressing high amounts of CD69. At day 40 p.i., B220(LOW) CD138(+) cell population was still increased but, differently from acute infection, 61.1% of these cells were positive for iIgG2a while only 14.2% expressed iIgM. Moreover, at days 20 and 40 p.i., 29.2% and 13.0% of B220(+) cells expressed sIgG2a, respectively. According to cell size and expression of CD80, CD86, CD11b, CD44 and CD38, B220(+) sIgG2a(+) cells had a phenotype characteristic of activated/memory B cells. Furthermore, 14.1% of B220(+) sIgG2a(+) cells at day 30 p.i. expressed a marginal zone B-cell phenotype. Importantly, B cells from 40-day-infected mice were very efficient in presenting parasite antigens leading to proliferation of both CD4(+) and CD8(+) cells. Our results contribute for understanding the dynamics of B cells during P. chabaudi infection, underlying the mechanisms of antigen presentation and antibody production, which are essential for the acquisition of protective immunity against malaria.

Plasmodium yoelii can ablate vaccine-induced long-term protection in mice.

Malaria is a serious cause of morbidity and mortality for people living in endemic areas, but unlike many other infections, individuals exposed to the parasite do not rapidly become resistant to subsequent infections. High titers of Ab against the 19-kDa C-terminal fragment of the merozoite surface protein-1 can mediate complete protection in model systems; however, previous studies had not determined whether this vaccine generated long-term protection. In this study, we report that functional memory cells generated by merozoite surface protein-1, per se, do not offer any protection. This is because the parasite induces deletion of vaccine-specific memory B cells as well as long-lived plasma cells including those specific for bystander immune responses. Our study demonstrates a novel mechanism by which Plasmodium ablates immunological memory of vaccines, which would leave the host immuno-compromised.

Helminth infection protects mice from anaphylaxis via IL-10-producing B cells.

Modulation of the immune system by infection with helminth parasites, including schistosomes, is proposed to reduce the levels of allergic responses in infected individuals. In this study we investigated whether experimental infection with Schistosoma mansoni could alter the susceptibility of mice to an extreme allergic response, anaphylaxis. We formally demonstrate that S. mansoni infection protects mice from an experimental model of systemic fatal anaphylaxis. The worm stage of infection is shown to mediate this protective effect. In vivo depletion studies demonstrated an imperative role for B cells and IL-10 in worm-mediated protection. Furthermore, worm infection of mice increases the frequency of IL-10-producing B cells compared with that in uninfected mice. However, transfer of B cells from worm-infected mice or in vitro worm-modulated B cells to sensitized recipients exacerbated anaphylaxis, which was attributed to the presence of elevated levels of IL-4-producing B cells. Worm-modulated, IL-10-producing B cells from IL-4-deficient, but not IL-5-, IL-9- or IL-13-deficient, mice conferred complete resistance to anaphylaxis when transferred to naive mice. Therefore, we have dissected a novel immunomodulatory mechanism induced by S. mansoni worms that is dependent on an IL-10-producing B cell population that can protect against allergic hypersensitivity. These data support a role for helminth immune modulation in the hygiene hypothesis and further illustrate the delicate balance between parasite induction of protective regulatory (IL-10) responses and detrimental (IL-4) allergic responses.

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 beta-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.

Plasmodium chabaudi chabaudi infection in mice induces strong B cell responses and striking but temporary changes in splenic cell distribution.

B cells and Abs play a key role in controlling the erythrocytic stage of malaria. However, little is known about the way the humoral response develops during infection. We show that Plasmodium chabaudi chabaudi causes major, but temporary changes in the distribution of leukocytes in the spleen. Despite these changes, an ordered response to infection develops, which includes vigorous extrafollicular growth of plasmablasts and germinal center formation. Early in the response, the lymphocytes in the T zone and follicles become widely spaced, and the edges of these compartments blur. This effect is maximal around the peak of parasitemia. Germinal centers are apparent by day 8, peak at day 20, and persist through day 60. Extrafollicular foci of plasmablasts are visible from day 4 and initiate a very strong plasma cell response. Initially, the plasma cells have a conventional red pulp distribution, but by day 10 they are unconventionally sited in the periarteriolar region of the white pulp. In this region they form clusters occupying part of the area normally filled by T cells. B cells are absent from the marginal zone for at least 30 days after the peak of infection, although flow cytometry shows their continued presence in the spleen throughout infection. Relatively normal splenic architecture is regained by day 60 of infection. These results show that the changes in splenic cell distribution are linked to the presence of parasites and do not seem to interfere with the development of the humoral response.

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.

Theileria annulata in CD5(+) macrophages and B1 B cells.

Theileria parasites infect and transform bovine leukocytes. We have analyzed laboratory-established Theileria sp.-infected leukocyte lines and observed that transformed macrophages express CD5. Low-level expression of CD5 by macrophages was further confirmed on three independent Theileria annulata clinical isolates from Tunisia. Interestingly, the fourth CD5(+) clinical isolate (MB2) was morphologically different, expressed surface immunoglobulin M (IgM) and BoLA class II, and had rearranged Ig light-chain genes. To demonstrate that MB2 did indeed contain CD5(+) B cells, individual clonal lines were obtained by limiting dilution, and CD5 expression and Ig gene rearrangement were confirmed. This suggests that in natural infections T. annulata can invade and transform CD5(+) B cells.