Clarissa Rodrigues Nascimento

Monocytes from HTLV-1–infected patients are unable to fully mature into dendritic cells

Human T-cell lymphotropic virus type 1 (HTLV-1) is a causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1-associated myelopathy/tropical spastic paraparesis is a chronic inflammatory disease characterized by loss of motor movement in response to spinal marrow cell destruction by T lymphocytes. To perform their cellular function, T cells need to be activated by antigen-presenting cells, such as dendritic cells (DCs). The aim of this work was to analyze DC differentiation and activation from monocytes of HTLV-1-infected individuals. We demonstrated that monocytes from HTLV-1-infected patients who had been stimulated to differentiate had an impaired loss of CD14 expression, expressed low levels of CD1a, and maintained secretion of tumor necrosis factor-α compared with monocytes from noninfected donors. We further evaluated DC activation by tumor necrosis factor-α. We observed that in response to activation, DCs that were derived from noninfected donors had an increase in the percentage of CD83(+), CD86(+), and human leukocyte antigen-DR(+) cells, whereas in DCs derived from HTLV-1-infected patients, the percentage of CD83(+), CD86(+), and human leukocyte antigen-DR(+) cells remained similar to that of nonactivated cells. Moreover, these cells had an impaired capacity to stimulate allogeneic T lymphocytes. We demonstrated that DC maturation was altered in HTLV-1-infected patients, which could contribute to the development of HTLV-1-associated diseases.

Novel Family of Insect Salivary Inhibitors Blocks Contact Pathway Activation by Binding to Polyphosphate, Heparin, and Dextran Sulfate

Objective—Polyphosphate and heparin are anionic polymers released by activated mast cells and platelets that are known to stimulate the contact pathway of coagulation. These polymers promote both the autoactivation of factor XII and the assembly of complexes containing factor XI, prekallikrein, and high-molecular-weight kininogen. We are searching for salivary proteins from blood-feeding insects that counteract the effect of procoagulant and proinflammatory factors in the host, including elements of the contact pathway. Approach and Results—Here, we evaluate the ability of the sand fly salivary proteins, PdSP15a and PdSP15b, to inhibit the contact pathway by disrupting binding of its components to anionic polymers. We attempt to demonstrate binding of the proteins to polyphosphate, heparin, and dextran sulfate. We also evaluate the effect of this binding on contact pathway reactions. We also set out to determine the x-ray crystal structure of PdSP15b and examine the determinants of relevant molecular interactions. Both proteins bind polyphosphate, heparin, and dextran sulfate with high affinity. Through this mechanism they inhibit the autoactivation of factor XII and factor XI, the reciprocal activation of factor XII and prekallikrein, the activation of factor XI by thrombin and factor XIIa, the cleavage of high-molecular-weight kininogen in plasma, and plasma extravasation induced by polyphosphate. The crystal structure of PdSP15b contains an amphipathic helix studded with basic side chains that forms the likely interaction surface. Conclusions—The results of these studies indicate that the binding of anionic polymers by salivary proteins is used by blood feeders as an antihemostatic/anti-inflammatory mechanism.

The kallikrein-kinin system in experimental Chagas disease: a paradigm to investigate the impact of inflammatory edema on GPCR-mediated pathways of host cell invasion by Trypanosoma cruzi

Chronic chagasic myocarditis (CCM) depends on Trypanosoma cruzi persistence in the myocardium. Studies of the proteolytic mechanisms governing host/parasite balance in peripheral sites of T. cruzi infection revealed that tissue culture trypomastigotes (TCTs) elicit inflammatory edema and stimulate protective type-1 effector T cells through the activation of the kallikrein-kinin system. Molecular studies linked the proinflammatory phenotype of Dm28c TCTs to the synergistic activities of tGPI, a lipid anchor that functions as a Toll-like receptor 2 (TLR2) ligand, and cruzipain, a kinin-releasing cysteine protease. Analysis of the dynamics of inflammation revealed that TCTs activate innate sentinel cells via TLR2, releasing CXC chemokines, which in turn evoke neutrophil/CXCR2-dependent extravasation of plasma proteins, including high molecular weight kininogen (HK), in parasite-laden tissues. Further downstream, TCTs process surface bound HK, liberating lysyl-BK (LBK), which then propagates inflammatory edema via signaling of endothelial G-protein-coupled bradykinin B2 receptors (BK2R). Dm28 TCTs take advantage of the transient availability of infection-promoting peptides (e.g., bradykinin and endothelins) in inflamed tissues to invade cardiovascular cells via interdependent signaling of BKRs and endothelin receptors (ETRs). Herein we present a space-filling model whereby ceramide-enriched endocytic vesicles generated by the sphingomyelinase pathway might incorporate BK2R and ETRs, which then trigger Ca2+-driven responses that optimize the housekeeping mechanism of plasma membrane repair from cell wounding. The hypothesis predicts that the NF-κB-inducible BKR (BK1R) may integrate the multimolecular signaling platforms forged by ceramide rafts, as the chronic myocarditis progresses. Exploited as gateways for parasite invasion, BK2R, BK1R, ETAR, ETBR, and other G protein-coupled receptor partners may enable persistent myocardial parasitism in the edematous tissues at expense of adverse cardiac remodeling.

The short chain fatty acid sodium butyrate regulates the induction of CD1a in developing dendritic cells.

Dendritic cells (DCs) are professional antigen-presenting cells with attributes for priming/activating T cells and mediating immune responses. Considering the importance of DCs in the initiation of immune responses, it will be of interest to study their mechanisms of regulation. Histone-modifying enzymes, such as histone deacetylases (HDACs), are critical in controlling chromatin organization. The aim of our study was to investigate DC differentiation under the influence of sodium butyrate (NaB), a short chain fatty acid that is a histone deacetylase inhibitor. Monocytes from healthy individuals were differentiated into immature DCs with IL-4 and GM-CSF in the presence or absence of NaB. DC differentiation was evaluated by CD14 and CD1a expression by flow cytometry. We observed that monocytes stimulated to differentiate in the presence of NaB displayed colony formation and dendritic cell morphology, lost CD14 and showed decreased secretion of IL-1β. The acquisition of CD1a, however, was impaired. Being a natural short chain fatty acid, NaB may regulate CD1a acquisition independently of its HDAC inhibitory activity. We observed that the addition of peroxisome proliferator-activated receptor γ (PPAR-γ) antagonist (GW9662) did not reverse NaB effect, suggesting this was not the pathway involved. On the other hand, CD1a can also be induced by toll like receptors 2 (TLR 2) agonists, such as Pam3Cys, and NaB inhibited this effect. Our data suggest that the histone deacetylase inhibitor NaB instead of impairing DC differentiation inhibits the acquisition of CD1a induced both by cytokines and by TLR 2 agonist stimulus. Furthermore, this occurs at the transcriptional level as NaB led to a decrease in mRNA levels of CD1a and upregulation of CD1d.

Leukemic cell products down-regulate human dendritic cell differentiation

The microenvironment produced by solid tumors is inhibitory to the immune system, inducing dendritic cell (DC) alterations, but there is a paucity of information regarding haematological malignances. The aim of this study was to investigate DC differentiation under the influence of leukemic cell products. Monocytes from healthy volunteers were cultured in the presence of IL-4 and GM-CSF for the generation of immature DCs. Supernatants from leukemic cultures were added to monocyte cultures during differentiation. The lineages used were K562, a chronic myeloid leukemia, HL-60, a promyelocytic leukemia and DAUDI, originated from Burkitt lymphoma. It was observed that the expression of CD14 remained high and the CD1a was low in the presence of tumor supernatants, while non-malignant supernatants did not affect these parameters. Furthermore, IL-1β and TNF-α production by monocytes during differentiation was increased by the presence of tumor supernatants. The modifications on CD14 and CD1a expressions could be mimicked by the addition of exogenous IL-1β and partially inhibited by the neutralization of IL-1β. These results suggest that soluble products from leukemic cells interfere with DC differentiation and, in the present work, this effect could be mediated by monocyte-derived IL-1β in response to tumor supernatants.

ABCB1 (P-glycoprotein) but not ABCC1 (MRP1) is downregulated in peripheral blood mononuclear cells of spontaneously hypertensive rats

Although the kidney is a major target in hypertension, several studies have correlated important immune alterations with the development of hypertension in spontaneously hypertensive rats (SHR), like increased secretion of pro-inflammatory cytokines, inflammatory infiltration in kidneys and thymic atrophy. Because adenosine-triphosphate-binding cassette sub-family B member 1 (ABCB1; P-glycoprotein) and adenosine-triphosphate-binding cassette sub-family C member 1 (ABCC1; multidrug resistance protein 1), two proteins first described in multidrug resistant tumors, physiologically transport several immune mediators and are required for the adequate functioning of the immune system, we aimed to measure the expression and activity of these proteins in peripheral blood mononuclear cells (PBMC), thymocytes, and also kidneys of normotensive Wistar Kyoto rats and SHR. Our results showed that ABCB1, but not ABCC1, activity was diminished (nearly 50%) in PBMC. Moreover, Abcb1b gene was downregulated in PBMC and kidney of SHR and this was not counterbalanced by an upregulation of its homolog Abcb1a, suggesting that the diminished activity is due to downregulation of the gene. No alteration was detected in ABCB1 activity in SHR thymocytes, indicating that this downregulation occurs after lymphocytes leave the primary lymphoid organs. Even though it is not known at present which parameter(s) is(are) responsible for this downregulation, it may contribute for the altered immune response observed in hypertension and to possible altered drug disposition in hypertensive individuals, resulting in greater drug interaction and increased drug toxicity.

Comparative Study on the Effects of Cyclosporin A in Renal Cells in Culture

Background: Although cyclosporin A (CSA) inhibits P-glycoprotein (ABCB1), the relationship between this inhibition and CSA-induced nephrotoxicity is not established. Methods: Three renal cell lines were used to investigate the effects of CSA in cellular viability and accumulation of rhodamine 123 (Rho123): LLC-PK1, which does not express ABCB1 substantially; MDCK, expressing moderate amounts of this protein, and Ma104 cells, which express high amounts of ABCB1. Results: The viability was significantly reduced in the three cell lines after treatment with CSA concentrations >10 µM. Ma104 was the more resistant and LLC-PK1 the more sensitive. CSA increased Rho123 accumulation in the three cell lines when incubated simultaneously, MDCK presenting the higher increase. However, different results were achieved when the periods of incubation with Rho123 and CSA were disconnected: a post-incubation with CSA was more effective in Ma104 cells, while MDCK and LLC-PK1 showed no difference between pre-, co- and post-incubation with CSA. Conclusions: Our results suggest that the effects of CSA may be divided into two groups: ABCB1-independent (direct injury), and ABCB1-dependent toxicity, due to modulation of its activity. This could result in increased accumulation of noxious ABCB1 substrates, contributing to CSA-induced nephrotoxicity. Furthermore, the mechanisms of ABCB1 modulation by CSA may be different for different cell lines.

Mast cell coupling to the kallikrein-kinin system fuels intracardiac parasitism and worsens heart pathology in experimental chagas disease

During the course of Chagas disease, infectious forms of Trypanosoma cruzi are occasionally liberated from parasitized heart cells. Studies performed with tissue culture trypomastigotes (TCTs, Dm28c strain) demonstrated that these parasites evoke neutrophil/CXCR2-dependent microvascular leakage by activating innate sentinel cells via toll-like receptor 2 (TLR2). Upon plasma extravasation, proteolytically derived kinins and C5a stimulate immunoprotective Th1 responses via cross-talk between bradykinin B2 receptors (B2Rs) and C5aR. Awareness that TCTs invade cardiovascular cells in vitro via interdependent activation of B2R and endothelin receptors [endothelin A receptor (ETAR)/endothelin B receptor (ETBR)] led us to hypothesize that T. cruzi might reciprocally benefit from the formation of infection-associated edema via activation of kallikrein–kinin system (KKS). Using intravital microscopy, here we first examined the functional interplay between mast cells (MCs) and the KKS by topically exposing the hamster cheek pouch (HCP) tissues to dextran sulfate (DXS), a potent “contact” activator of the KKS. Surprisingly, although DXS was inert for at least 30 min, a subtle MC-driven leakage resulted in factor XII (FXII)-dependent activation of the KKS, which then amplified inflammation via generation of bradykinin (BK). Guided by this mechanistic insight, we next exposed TCTs to “leaky” HCP—forged by low dose histamine application—and found that the proinflammatory phenotype of TCTs was boosted by BK generated via the MC/KKS pathway. Measurements of footpad edema in MC-deficient mice linked TCT-evoked inflammation to MC degranulation (upstream) and FXII-mediated generation of BK (downstream). We then inoculated TCTs intracardiacally in mice and found a striking decrease of parasite DNA (quantitative polymerase chain reaction; 3 d.p.i.) in the heart of MC-deficient mutant mice. Moreover, the intracardiac parasite load was significantly reduced in WT mice pretreated with (i) cromoglycate (MC stabilizer) (ii) infestin-4, a specific inhibitor of FXIIa (iii) HOE-140 (specific antagonist of B2R), and (iv) bosentan, a non-selective antagonist of ETAR/ETBR. Notably, histopathology of heart tissues from mice pretreated with these G protein-coupled receptors blockers revealed that myocarditis and heart fibrosis (30 d.p.i.) was markedly and redundantly attenuated. Collectively, our study suggests that inflammatory edema propagated via activation of the MC/KKS pathway fuels intracardiac parasitism by generating infection-stimulatory peptides (BK and endothelins) in the edematous heart tissues.