Beatriz Rossetti Ferreira

Ticks produce highly selective chemokine binding proteins with antiinflammatory activity

Bloodsucking parasites such as ticks have evolved a wide variety of immunomodulatory proteins that are secreted in their saliva, allowing them to feed for long periods of time without being detected by the host immune system. One possible strategy used by ticks to evade the host immune response is to produce proteins that selectively bind and neutralize the chemokines that normally recruit cells of the innate immune system that protect the host from parasites. We have identified distinct cDNAs encoding novel chemokine binding proteins (CHPBs), which we have termed Evasins, using an expression cloning approach. These CHBPs have unusually stringent chemokine selectivity, differentiating them from broader spectrum viral CHBPs. Evasin-1 binds to CCL3, CCL4, and CCL18; Evasin-3 binds to CXCL8 and CXCL1; and Evasin-4 binds to CCL5 and CCL11. We report the characterization of Evasin-1 and -3, which are unrelated in primary sequence and tertiary structure, and reveal novel folds. Administration of recombinant Evasin-1 and -3 in animal models of disease demonstrates that they have potent antiinflammatory properties. These novel CHBPs designed by nature are even smaller than the recently described single-domain antibodies (Hollinger, P., and P.J. Hudson. 2005. Nat. Biotechnol. 23:1126–1136), and may be therapeutically useful as novel antiinflammatory agents in the future.

Complete genome sequence of the sugarcane nitrogen-fixing endophyte Gluconacetobacter diazotrophicus Pal5

BackgroundGluconacetobacter diazotrophicus Pal5 is an endophytic diazotrophic bacterium that lives in association with sugarcane plants. It has important biotechnological features such as nitrogen fixation, plant growth promotion, sugar metabolism pathways, secretion of organic acids, synthesis of auxin and the occurrence of bacteriocins.ResultsGluconacetobacter diazotrophicus Pal5 is the third diazotrophic endophytic bacterium to be completely sequenced. Its genome is composed of a 3.9 Mb chromosome and 2 plasmids of 16.6 and 38.8 kb, respectively. We annotated 3,938 coding sequences which reveal several characteristics related to the endophytic lifestyle such as nitrogen fixation, plant growth promotion, sugar metabolism, transport systems, synthesis of auxin and the occurrence of bacteriocins. Genomic analysis identified a core component of 894 genes shared with phylogenetically related bacteria. Gene clusters for gum-like polysaccharide biosynthesis, tad pilus, quorum sensing, for modulation of plant growth by indole acetic acid and mechanisms involved in tolerance to acidic conditions were identified and may be related to the sugarcane endophytic and plant-growth promoting traits of G. diazotrophicus. An accessory component of at least 851 genes distributed in genome islands was identified, and was most likely acquired by horizontal gene transfer. This portion of the genome has likely contributed to adaptation to the plant habitat.ConclusionThe genome data offer an important resource of information that can be used to manipulate plant/bacterium interactions with the aim of improving sugarcane crop production and other biotechnological applications.

Successive tick infestations selectively promote a T-helper 2 cytokine profile in mice

Several studies have revealed that T lymphocytes and cytokines play a crucial role in determining the outcome of parasitic infections in terms of protective immunity. In this study we found that Rhipicephalus sanguineus tick saliva stimulates transforming growth factor‐β (TGF‐β), and reduces interleukin‐12 (IL‐12) secretion by cells from normal C3H/HeJ mice. Moreover, murine lymph node cells harvested 6 days after the fourth infestation with ticks presented an 82·4% decrease in their proliferative response to concanavalin A (Con A) compared with the response of control cells. In addition, lymph node cells cultured in the presence of Con A showed a T‐helper 2‐type (Th2‐type) cytokine profile, represented by augmented IL‐4 and IL‐10 and TGF‐β. On the other hand, the IL‐2, interferon‐γ (IFN‐γ) and IL‐12 synthesis was significantly inhibited. These results indicate that ticks may modulate the host’s immune response through saliva injection. Consideringthat C3H/HeJ mice develop no protective immunity to R. sanguineus infestation, our results suggest that tick‐induced Th2‐type cytokines and a decreased proliferative response probably lead the host to a susceptible state to both tick and tick‐transmitted pathogens.

CCR5 Plays a Critical Role in the Development of Myocarditis and Host Protection in Mice Infected with Trypanosoma cruzi

Abstract The pathogenesis of myocarditis during Trypanosoma cruzi infection is poorly understood. We investigated the role played by chemokine receptor 5 (CCR5) in the influx of T cells to the cardiac tissue of T. cruzi—infected mice. mRNA and protein for the CCR5 ligands CCL3, CCL4, and CCL5 were detected in the hearts of infected mice in association with CD4+ and CD8+ T cells. There was a high level of CCR5 expression on CD8+ T cells in the hearts of infected mice. Moreover, CCR5 expression on CD8+ T cells was positively modulated by T. cruzi infection. CCR5-deficient mice infected with T. cruzi experienced a dramatically inhibited migration of T cells to the heart and were also more susceptible to infection. These results suggest that CCR5 and its ligands play a central role in the control of T cell influx in T. cruzi-infected mice. Knowledge of the mechanisms that trigger and control the migration of cells to the heart in patients with Chagas disease may help in the design of drugs that prevent myocarditis and protect against the development of severe disease.

Molecular cloning and characterization of a highly selective chemokine-binding protein from the tick Rhipicephalus sanguineus

Ticks are blood-feeding parasites that secrete a number of immuno-modulatory factors to evade the host immune response. Saliva isolated from different species of ticks has recently been shown to contain chemokine neutralizing activity. To characterize this activity, we constructed a cDNA library from the salivary glands of the common brown dog tick, Rhipicephalus sanguineus. Pools of cDNA clones from the library were transfected into HEK293 cells, and the conditioned media from the transfected cells were tested for chemokine binding activity by chemical cross-linking to radiolabeled CCL3 followed by SDS-PAGE. By de-convolution of a single positive pool of 270 clones, we identified a full-length cDNA encoding a protein of 114 amino acids, which after signal peptide cleavage was predicted to yield a mature protein of 94 amino acids that we called Evasin-1. Recombinant Evasin-1 was produced in HEK293 cells and in insect cells. Using surface plasmon resonance we were able to show that Evasin-1 was exquisitely selective for 3 CC chemokines, CCL3 and CCL4 and the closely related chemokine CCL18, with KD values of 0.16, 0.81, and 3.21 nm, respectively. The affinities for CCL3 and CCL4 were confirmed in competition receptor binding assays. Analysis by size exclusion chromatography demonstrated that Evasin-1 was monomeric and formed a 1:1 complex with CCL3. Thus, unlike the other chemokine-binding proteins identified to date from viruses and from the parasitic worm Schistosoma mansoni, Evasin-1 is highly specific for a subgroup of CC chemokines, which may reflect a specific role for these chemokines in host defense against parasites.

Saliva of Rhipicephalus sanguineus tick impairs T cell proliferation and IFN-γ-induced macrophage microbicidal activity

In this study, we investigated tick saliva effects on T cell proliferation, antigen presentation and IFN-gamma-induced macrophage activation, events which are associated with host immune defense mechanisms. Mice repeatedly infested with Rhipicephalus sanguineus ticks, similarly to dogs, did not develop resistance to further infestations. We determined that R. sanguineus tick saliva inhibited, in a dose-dependent manner, both Con-A and specific antigen-induced splenic T cell proliferation. Tick saliva diluted twenty times (64 microg/ml) inhibited Con-A-induced and antigen-specific T cell proliferation in 83% and 69%, respectively. In addition, the inhibition of cell proliferation correlated with a decrease in IL-2 production. Microconcentrator fractionated saliva was tested on a Con-A-induced cell proliferation assay, and showed that one fraction between 3 and 10 kDa and another smaller than 3 kDa can be responsible for the inhibition of T cell proliferation. Although saliva inhibited cell proliferation, it did not impair antigen presentation. Tick saliva further abrogated the killing of intracellular forms of Trypanosoma cruzi by IFN-gamma-activated macrophages. Moreover, saliva-induced macrophage inhibition of IFN-gamma-induced-trypanocidal activity was paralleled with 69% less nitric oxide (NO) production. Finally, tick saliva doubled the production of IL-10 and reduced 84.6% production of IFN-gamma by splenocytes cultured with T. cruzi, suggesting that decreased macrophage NO production may be due to a saliva-induced cytokine imbalance, leading to decreased NO synthase activity. Together, these data indicate that tick saliva can modulate host immune response, thus, contributing to its feeding success and favoring the transmission of tick-borne pathogens.

Tick saliva inhibits differentiation, maturation and function of murine bone‐marrow‐derived dendritic cells

Haematophagous arthropod vectors such as mosquitoes, tsetse flies, sandflies and ticks have evolved salivary immunomodulatory factors that prevent the vertebrate host from rejecting them meanwhile enhancing pathogen transmission. As dendritic cells (DC) play a major role in host immune responses, we studied the effects of Rhipicephalus sanguineus tick saliva on DC differentiation and maturation. Flow cytometry analysis revealed that the addition of saliva to bone marrow cells inhibits the differentiation of DC and decreased the population of differentiated immature DC, increasing the levels of major histocompatibility complex (MHC) class II while not altering the expression of costimulatory (CD40, CD80 and CD86) and adhesion (CD54) molecules. Furthermore, maturation of DC stimulated by lipopolysaccharide (LPS) in the presence of saliva resulted in a lower expression of costimulatory molecules, but did not alter the up‐regulation of MHC class II and CD54. The lipopolysaccharide (LPS)‐matured DC cultured with saliva also presented reduced production of interleukin‐12, whereas interleukin‐10 production was unaltered. Assessment of the function of DC cultured with tick saliva revealed them to be poor stimulators of cytokine production by antigen‐specific T cells. Our data indicate a novel modulatory role for the saliva of arthropod vectors at an initial step of the immune response through the inhibition of differentiation and maturation of DC into functional antigen‐presenting cells.

Deconstructing Tick Saliva: NON-PROTEIN MOLECULES WITH POTENT IMMUNOMODULATORY PROPERTIES*

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-α while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of ∼110 pmol/μl) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E2 (PGE2 ∼100 nm) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE2 inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE2 were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE2, but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE2 in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.

The involvement of CD4+CD25+ T cells in the acute phase of Trypanosoma cruzi infection

The infection with Trypanosoma cruzi leads to a vigorous and apparently uncontrolled inflammatory response in the heart. Although the parasites trigger specific immune response, the infection is not completely cleared out, a phenomenon that in other parasitic infections has been attributed to CD4+CD25+ T cells (Tregs). Then, we examined the role of natural Tregs and its signaling through CD25 and GITR in the resistance against infection with T. cruzi. Mice were treated with mAb against CD25 and GITR and the parasitemia, mortality and heart pathology analyzed. First, we demonstrated that CD4+CD25+GITR+Foxp3+ T cells migrate to the heart of infected mice. The treatment with anti-CD25 or anti-GITR resulted in increased mortality of these infected animals. Moreover, the treatment with anti-GITR enhanced the myocarditis, with increased migration of CD4+, CD8+, and CCR5+ leukocytes, TNF-alpha production, and tissue parasitism, although it did not change the systemic nitric oxide synthesis. These data showed a limited role for CD25 signaling in controlling the inflammatory response during this protozoan infection. Also, the data suggested that signaling through GITR is determinant to control of the heart inflammation, parasite replication, and host resistance against the infection.

Chemokine Production and Leukocyte Recruitment to the Lungs of Paracoccidioides brasiliensis-Infected Mice Is Modulated by Interferon-γ

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.