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Summary We have characterized a Leishmania protein belonging to the silent information regulator 2 (SIR2) family [SIR2 related protein 1 (SIR2RP1)] that might play an immunoregulatory role during infection through its capacity to trigger B-cell effector functions. We report here that SIR2RP1 leads to the proliferation of activated B cells, causing increased expression of major histocompatibility complex (MHC) II and the costimulatory molecules CD40 and CD86, which are critical ligands for T-cell cross-talk during the development of adaptive immune responses. In contrast, B cells isolated from Toll-like receptor 2 (TLR2) knockout mice were unable to respond to the SIR2RP1 stimulus. Similarly, SIR2RP1 induced the maturation of dendritic cells (DCs) in a TLR2-dependent manner with the secretion of pro-inflammatory cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)-α] and enhanced the costimulatory properties of DCs. Nevertheless, immunization assays demonstrated that TLR2-deficient mice were able to mount a specific humoral response to SIR2RP1. Interestingly, further investigations showed that macrophages were activated by SIR2RP1 even in the absence of TLR2. Therefore, a different type of interplay between SIR2RP1 and the major antigen-presenting cells in vivo could explain the immune response observed in TLR2-deficient mice. Together, these results demonstrate that TLR2 signalling contributes to SIR2RP1 recognition by innate immune host cells.

The intracellular protozoan parasite Trypanosoma cruzi is the aetiological agent of Chagas' disease. We have previously identified a T. cruzi-released protein called Tc52, which is crucial for parasite survival and virulence. In the present study, we attempted to define the Tc52 epitope(s) responsible for its immunoregulatory function. A naturally occurring major peptide fragment of molecular mass 28 kDa (Tc28k) was identified, which was localized in the C-terminal portion of Tc52 and was inhibitory for T-cell activation. Synthetic peptides corresponding to amino acid sequences in Tc52 were evaluated for their ability to modulate T-cell proliferation and cytokine production. Results obtained using five peptides spanning the N-terminal or C-terminal domain of the Tc52 protein indicated that the activity mapped to Tc52 residues 432-445. Moreover, it was found that the peptide, when coupled to a carrier protein (ovalbumin), exhibited increased inhibitory activity on T-lymphocyte activation. Incubation with 8 nm ovalbumin-coupled peptide 432-445 resulted in approximately the same levels (75%) of inhibition of T-cell proliferation as 5 micro g/ml Tc28k. Furthermore, we showed that the coupled peptide significantly down-regulated the secretion of interferon-gamma (IFN-gamma) and interleukin-2 (IL-2). Likewise, in immunized mice, the coupled peptide 432-445 was a very poor B- and T-cell antigen compared with the other Tc52-derived peptides. These results suggest that the immunomodulatory portion of the T. cruzi Tc52 virulent factor may reside, at least in part, in a conserved sequence within its C-terminal domain, which could minimize its antigenicity.

Trypanosoma cruzi, the causative agent of Chagas' disease, is a protozoan parasite that infects humans and other mammals in Central and Latin America. Several alterations of the immune response after infection have been described, such as severe immunosuppression of both cellular and humoral responses and massive polyclonal B- and T-cell activation, including the expansion of self-reactive clones. We have investigated the effects of the intraperitoneal injection of a recombinant 24,000 MW T. cruzi-specific antigen (rTc24) on the immune response of normal and deficient strains of mice. We analysed the in vivo and ex vivo levels of lymphocyte activation and the proliferative responses to rTc24 by determining the expression of CD69 activation marker and the levels of thymidine incorporation by spleen cells. The numbers of antibody-producing cells were determined by ELISPOT and the levels of immunoglobulin in the sera by isotype-specific enzyme-linked immunosorbent assay. We observed an increased [3H]thymidine ([3H]TdR) incorporation by spleen cells after rTc24 stimulation in vivo and in vitro. This proliferative activity induced by rTc24 was independent of the mouse strain used in the experiments (including C3H/HeJ mice) and ruled out the possibility that rTc24 preparations were contaminated by lipopolysaccharide. The injection of rTc24 protein induced preferentially the activation of B cells, as determined by the increased expression of CD69 molecules on IgM+ spleen cells. Considerable increases of IgM-secreting B cells were determined in both athymic and euthymic BALB/c mice. Mice that are deficient in B cells (BALB.Xid) responded to rTc24 but to a lesser extent. These increases in IgM B-cell numbers were accompanied by elevated levels of IgM immunoglobulins in the sera of injected animals. Our results suggest a role for rTc24 in B-cell activation.

In previous studies, we identified a gene product belonging to the silent information regulatory 2 protein (SIR2) family. This protein is expressed by all Leishmania species so far examined (L. major, L. infantum, L. amazonensis, L. mexicana) and found to be crucial for parasite survival and virulence. In the present study, we investigated whether a Leishmania SIR2 recombinant protein (LmSIR2) would affect T- and B-cell functions in a murine model. In vitro treatment of spleen cells from normal BALB/c mice with LmSIR2 showed increased expression of CD69 on B cells. This effect was not abolished by the addition of polymyxin B. Intravenous injection of LmSIR2 into BALB/c mice induced increased spleen B cell number by a factor of about approximately 1.6, whereas no modification occurred at the level of CD4(+) and CD8(+) cells. Furthermore, intraperitoneal injection of LmSIR2 alone without adjuvant into BALB/c mice or nude mice triggered the production of elevated levels of LmSIR2-specific antibodies. The analysis of specific isotype profiles showed a predominance of immunoglobulin G1 (IgG1) and IgG2a antibody responses in BALB/c mice, and IgM in nude mice. Moreover, the anti-LmSIR2 mouse antibodies in the presence of complement induced the in vitro lysis of L. infantum amastigotes. In the absence of complement, the antibodies induced significant inhibition of amastigotes developpement inside macrophages. Together, the current study provides the first evidence that a Leishmania protein belonging to the SIR2 family may play a role in the regulation of immune response through its capacity to trigger B-cell effector function.

A glycoprotein of apparent molecular weight 58,000 (unreduced)/68,000 (in its reduced form) (gp 58/68), which is one of the fibronectin/collagen receptors of Trypanosoma cruzi, was purified to homogeneity from the trypomastigote forms of the Tehuantepec and Y strains of the parasite. Purified gp 58/68 inhibited formation of cell-bound and fluid-phase alternative pathway C3 convertase in a dose-dependent fashion, as assessed using purified human complement components. Gp 58/68 differed from the human regulatory proteins H, DAF, MCP and CR1 and from previously reported regulatory proteins on the parasite membrane in that it was unable to enhance decay-dissociation of preformed alternative pathway C3 convertase sites, did not serve as a co-factor for I-mediated cleavage of C3b and had no inhibitory activity on the classical pathway convertases. The inhibitory effect of gp 58/68 was most likely dependent on an interaction of the protein with factor B rather than with C3b. Gp 58/68 provides trypomastigotes with an additional potential mechanism for escaping complement lysis by the human alternative pathway.