RESUMO

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis.

Assuntos

RESUMO

We have reported that some lectins act as agonists of toll-like receptors (TLRs) and have immunomodulatory properties. The plant lectin ArtinM, for example, interacts with N-glycans of TLR2, whereas other lectins of microbial origin interact with TLR2 and TLR4. Expression of the receptors on the surface of antigen-presenting cells exposes N-glycans that may be targeted by lectins of different structures, specificities, and origins. In vitro, these interactions trigger cell signaling that leads to NF-κB activation and production of the Th1 polarizing cytokine IL-12. In vivo, a same sequence of events follows the administration of an active lectin to mice infected with an intracellular pathogen, conferring resistance to the pathogen. The lectins of the human pathogens Toxoplasma gondii (TgMIC1 and TgMIC4) and Paracoccidioides brasiliensis (Paracoccin), by recognition and activation of TLR2 and TLR4, induce cell events and in vivo effects comparable to the promoted by the plant lectin ArtinM. In this article, we highlight these two distinct mechanisms for activating antigen-presenting cells. On the one hand, TLRs act as sensors for the presence of conventional pathogen-associated molecular patterns, such as microbial lipids. On the other hand, we showed that TLR-mediated cell activation might be triggered by an alternative way, in which lectins bind to TLRs N-glycans and stimulate cells to increase the expression of pro-inflammatory cytokines. This process may lead to the development of new pharmaceutical tools that promote protective immune responses directed against intracellular pathogens and tumors.

RESUMO

BACKGROUND: Paracoccin (PCN) is an N-acetylglucosamine-binding lectin from the human pathogenic fungus Paracoccidioides brasiliensis. Recombinant PCN (rPCN) induces a T helper (Th) 1 immune response when prophylactically administered to BALB/c mice, protecting them against subsequent challenge with P. brasiliensis. In this study, we investigated the therapeutic effect of rPCN in experimental paracoccidioidomycosis (PCM) and the mechanism accounting for its beneficial action. METHODOLOGY/PRINCIPAL FINDINGS: Four distinct regimens of rPCN administration were assayed to identify which was the most protective, relative to vehicle administration. In all rPCN-treated mice, pulmonary granulomas were less numerous and more compact. Moreover, fewer colony-forming units were recovered from the lungs of rPCN-treated mice. Although all therapeutic regimens of rPCN were protective, maximal efficacy was obtained with two subcutaneous injections of 0.5 µg rPCN at 3 and 10 days after infection. The rPCN treatment was also associated with higher pulmonary levels of IL-12, IFN-γ, TNF-α, nitric oxide (NO), and IL-10, without IL-4 augmentation. Encouraged by the pulmonary cytokine profile of treated mice and by the fact that in vitro rPCN-stimulated macrophages released high levels of IL-12, we investigated the interaction of rPCN with Toll-like receptors (TLRs). Using a reporter assay in transfected HEK293T cells, we verified that rPCN activated TLR2 and TLR4. The activation occurred independently of TLR2 heterodimerization with TLR1 or TLR6 and did not require the presence of the CD14 or CD36 co-receptors. The interaction between rPCN and TLR2 depended on carbohydrate recognition because it was affected by mutation of the receptor's N-glycosylation sites. The fourth TLR2 N-glycan was especially critical for the rPCN-TLR2 interaction. CONCLUSIONS/SIGNIFICANCE: Based on our results, we propose that PCN acts as a TLR agonist. PCN binds to N-glycans on TLRs, triggers regulated Th1 immunity, and exerts a therapeutic effect against P. brasiliensis infection.

Assuntos