Luciana Barros de Arruda

Expression of functional TLR4 confers proinflammatory responsiveness to Trypanosoma cruzi glycoinositolphospholipids and higher resistance to infection with T. cruzi.

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-κB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.

HIV-1 p55Gag Encoded in the Lysosome-associated Membrane Protein-1 as a DNA Plasmid Vaccine Chimera Is Highly Expressed, Traffics to the Major Histocompatibility Class II Compartment, and Elicits Enhanced Immune Responses

Several genetic vaccines encoding antigen chimeras containing the lysosome-associated membrane protein (LAMP) translocon, transmembrane, and cytoplasmic domain sequences have elicited strong mouse antigen-specific immune responses. The increased immune response is attributed to trafficking of the antigen chimera to the major histocompatibility class II (MHC II) compartment where LAMP is colocalized with MHC II. In this report, we describe a new form of an HIV-1 p55gag DNA vaccine, with the gag sequence incorporated into the complete LAMP cDNA sequence. Gag encoded with the translocon, transmembrane and cytoplasmic lysosomal membrane targeting sequences of LAMP, without the luminal domain, was poorly expressed, did not traffic to lysosomes or MHC II compartments of transfected cells, and elicited a limited immune response from DNA immunized mice. In contrast, addition of the LAMP luminal domain sequence to the construct resulted in a high level of expression of the LAMP/Gag protein chimera in transfected cells that was further increased by including the inverted terminal repeat sequences of the adeno-associated virus to the plasmid vector. This LAMP/Gag chimera with the complete LAMP protein colocalized with endogenous MHC II of transfected cells and elicited strong cellular and humoral immune responses of immunized mice as compared with the response to DNA-encoding native Gag, with a 10-fold increase in CD4+ responses, a 4- to 5-fold increase in CD8+ T-cell responses, and antibody titers of >100,000. These results reveal novel roles of the LAMP luminal domain as a determinant of Gag protein expression, lysosomal trafficking, and possibly of the immune response to Gag.

Cooperative Activation of TLR2 and Bradykinin B2 Receptor Is Required for Induction of Type 1 Immunity in a Mouse Model of Subcutaneous Infection by Trypanosoma cruzi

We have previously reported that exogenous bradykinin activates immature dendritic cells (DCs) via the bradykinin B2 receptor (B2R), thereby stimulating adaptive immunity. In this study, we show that these premises are met in a model of s.c. infection by Trypanosoma cruzi, a protozoan that liberates kinins from kininogens through its major protease, cruzipain. Intensity of B2R-dependent paw edema evoked by trypomastigotes correlated with levels of IL-12 produced by CD11c+ dendritic cells isolated from draining lymph nodes. The IL-12 response induced by endogenously released kinins was vigorously increased in infected mice pretreated with inhibitors of angiotensin converting enzyme (ACE), a kinin-degrading metallopeptidase. Furthermore, these innate stimulatory effects were linked to B2R-dependent up-regulation of IFN-γ production by Ag-specific T cells. Strikingly, the trypomastigotes failed to up-regulate type 1 immunity in TLR2−/− mice, irrespective of ACE inhibitor treatment. Analysis of the dynamics of inflammation revealed that TLR2 triggering by glycosylphosphatidylinositol-anchored mucins induces plasma extravasation, thereby favoring peripheral accumulation of kininogens in sites of infection. Further downstream, the parasites generate high levels of innate kinin signals in peripheral tissues through the activity of cruzipain. The demonstration that the deficient type 1 immune responses of TLR2−/− mice are rescued upon s.c. injection of exogenous kininogens, along with trypomastigotes, supports the notion that generation of kinin “danger” signals is intensified through cooperative activation of TLR2 and B2R. In summary, we have described a s.c. infection model where type 1 immunity is vigorously up-regulated by bradykinin, an innate signal whose levels in peripheral tissues are controlled by an intricate interplay of TLR2, B2R, and ACE.

Bradykinin B2 Receptors of Dendritic Cells, Acting as Sensors of Kinins Proteolytically Released by Trypanosoma cruzi, Are Critical for the Development of Protective Type-1 Responses

Although the concept that dendritic cells (DCs) recognize pathogens through the engagement of Toll-like receptors is widely accepted, we recently suggested that immature DCs might sense kinin-releasing strains of Trypanosoma cruzi through the triggering of G-protein-coupled bradykinin B2 receptors (B2R). Here we report that C57BL/6.B2R−/− mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates as compared to B2R+/+ mice. qRT-PCR revealed a 5-fold increase in T. cruzi DNA (14 d post-infection [p.i.]) in B2R−/− heart, while spleen parasitism was negligible in both mice strains. Analysis of recall responses (14 d p.i.) showed high and comparable frequencies of IFN-γ-producing CD4+ and CD8+ T cells in the spleen of B2R−/− and wild-type mice. However, production of IFN-γ by effector T cells isolated from B2R−/− heart was significantly reduced as compared with wild-type mice. As the infection continued, wild-type mice presented IFN-γ-producing (CD4+CD44+ and CD8+CD44+) T cells both in the spleen and heart while B2R−/− mice showed negligible frequencies of such activated T cells. Furthermore, the collapse of type-1 immune responses in B2R−/− mice was linked to upregulated secretion of IL-17 and TNF-α by antigen-responsive CD4+ T cells. In vitro analysis of tissue culture trypomastigote interaction with splenic CD11c+ DCs indicated that DC maturation (IL-12, CD40, and CD86) is controlled by the kinin/B2R pathway. Further, systemic injection of trypomastigotes induced IL-12 production by CD11c+ DCs isolated from B2R+/+ spleen, but not by DCs from B2R−/− mice. Notably, adoptive transfer of B2R+/+ CD11c+ DCs (intravenously) into B2R−/− mice rendered them resistant to acute challenge, rescued development of type-1 immunity, and repressed TH17 responses. Collectively, our results demonstrate that activation of B2R, a DC sensor of endogenous maturation signals, is critically required for development of acquired resistance to T. cruzi infection.

DNA vaccine encoding human immunodeficiency virus-1 Gag, targeted to the major histocompatibility complex II compartment by lysosomal-associated membrane protein, elicits enhanced long-term memory response

Antigen presentation by major histocompatibility complex type II (MHC II) molecules and activation of CD4+ helper T cells are critical for the generation of immunological memory. We previously described a DNA vaccine encoding human immunodeficiency virus‐1 p55Gag as a chimera with the lysosome‐associated membrane protein (LAMP/gag). The LAMP/gag chimera protein traffics to the MHC II compartment of transfected cells and elicits enhanced immune responses as compared to a DNA vaccine encoding native gag not targeted to the MHC II compartment. We have now investigated the long‐term responses of immunized mice and show that the LAMP/gag DNA vaccine promotes long‐lasting B cell‐ and CD4+ and CD8+ T‐cell memory responses and elicits a potent Gag‐specific CD8+ recall response to challenge with vaccinia virus encoding gag, even 11 months after immunization. In contrast, the immune responses induced by DNA encoding non‐targeted Gag decay rapidly and elicit very low or undetectable levels of Gag‐specific CD4+ and CD8+ memory cells. A single priming immunization with LAMP/gag DNA is sufficient to generate T‐cell memory. Following this initial priming immunization with LAMP/gag DNA, booster immunizations with native gag DNA or the LAMP/gag chimera are equally efficient in eliciting B‐ and T‐cell secondary responses, results in accordance with observations that secondary expansion of CD8+ cells in the boost phase does not require additional CD4+ help. These findings underscore the significance of targeting DNA‐encoded vaccine antigens to the MHC II processing compartments for induction of long‐term immunological memory.

Dendritic Cell-Lysosomal-Associated Membrane Protein (LAMP) and LAMP-1-HIV-1 Gag Chimeras Have Distinct Cellular Trafficking Pathways and Prime T and B Cell Responses to a Diverse Repertoire of Epitopes

Ag processing is a critical step in defining the repertoire of epitope-specific immune responses. In the present study, HIV-1 p55Gag Ag was synthesized as a DNA plasmid with either lysosomal-associated membrane protein-1 (LAMP/gag) or human dendritic cell-LAMP (DC-LAMP/gag) and used to immunize mice. Analysis of the cellular trafficking of these two chimeras demonstrated that both molecules colocalized with MHC class II molecules but differed in their overall trafficking to endosomal/lysosomal compartments. Following DNA immunization, both chimeras elicited potent Gag-specific T and B cell immune responses in mice but differ markedly in their IL-4 and IgG1/IgG2a responses. The DC-LAMP chimera induced a stronger Th type 1 response. ELISPOT analysis of T cell responses to 122 individual peptides encompassing the entire p55gag sequence (15-aa peptides overlapping by 11 residues) showed that DNA immunization with native gag, LAMP/gag, or DC-LAMP/gag induced responses to identical immunodominant CD4+ and CD8+ peptides. However, LAMP/gag and DC-LAMP/gag plasmids also elicited significant responses to 23 additional cryptic epitopes that were not recognized after immunization with native gag DNA. The three plasmids induced T cell responses to a total of 39 distinct peptide sequences, 13 of which were induced by all three DNA constructs. Individually, DC-LAMP/gag elicited the most diverse response, with a specific T cell response against 35 peptides. In addition, immunization with LAMP/gag and DC-LAMP/gag chimeras also promoted Ab secretion to an increased number of epitopes. These data indicate that LAMP-1 and DC-LAMP Ag chimeras follow different trafficking pathways, induce distinct modulatory immune responses, and are able to present cryptic epitopes.

Essential role of RIG-I in the activation of endothelial cells by dengue virus

Dengue virus (DENV) infection is associated to exacerbated inflammatory response and structural and functional alterations in the vascular endothelium. However, the mechanisms underlying DENV-induced endothelial cell activation and their role in the inflammatory response were not investigated so far. We demonstrated that human brain microvascular endothelial cells (HBMECs) are susceptible to DENV infection, which induces the expression of the cytoplasmic pattern recognition receptor (PRR) RIG-I. Infection of HBMECs promoted an increase in the production of type I IFN and proinflammatory cytokines, which were abolished after RIG-I silencing. DENV-infected HBMECs also presented a higher ICAM-1 expression dependent on RIG-I activation as well. On the other hand, ablation of RIG-I did not interfere with virus replication. Our data suggest that RIG-I activation by DENV may participate in the disease pathogenesis through the modulation of cytokine release and expression of adhesion molecules, probably contributing to leukocyte recruitment and amplification of the inflammatory response.

DNA Encoding an HIV-1 Gag/Human Lysosome-Associated Membrane Protein-1 Chimera Elicits a Broad Cellular and Humoral Immune Response in Rhesus Macaques

Previous studies of HIV-1 p55Gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type II (MHC II) complex molecules by use of a DNA antigen formulation encoding Gag as a chimera with the mouse lysosome-associated membrane protein (mLAMP/gag). In the present study, we have analyzed the magnitude and breadth of Gag-specific T-lymphocyte and antibody responses elicited in Rhesus macaques after immunization with DNA encoding a human LAMP/gag (hLAMP/gag) chimera. ELISPOT analyses indicated that the average Gag-specific IFN-γ response elicited by the hLAMP/gag chimera was detectable after only two or three naked DNA immunizations in all five immunized macaques and reached an average of 1000 spot-forming cells (SFC)/106 PBMCs. High IFN-γ ELISPOT responses were detected in CD8+-depleted cells, indicating that CD4+ T-cells play a major role in these responses. The T-cell responses of four of the macaques were also tested by use of ELISPOT to 12 overlapping 15-amino acids (aa) peptide pools containing ten peptides each, encompassing the complete Gag protein sequence. The two Mamu 08 immunized macaques responded to eight and twelve of the pools, the Mamu B01 to six, and the other macaque to five pools indicating that the hLAMP/gag DNA antigen formulation elicits a broad T-cell response against Gag. Additionally, there was a strong HIV-1-specific IgG response. The IgG antibody titers increased after each DNA injection, indicating a strong amnestic B-cell response, and were highly elevated in all the macaques after three immunizations. Moreover, the serum of each macaque recognized 13 of the 49 peptides of a 20-aa peptide library covering the complete Gag amino acid sequence. In addition, HIV-1-specific IgA antibodies were present in the plasma and external secretions, including nasal washes. These data support the findings of increased immunogenicity of genetic vaccines encoded as LAMP chimeras, including the response to DNA vaccines by non-human primates.

IL‐22 modulates IL‐17A production and controls inflammation and tissue damage in experimental dengue infection

Dengue virus (DENV), a mosquito‐borne flavivirus, is a public health problem in many tropical countries. IL‐22 and IL‐17A are key cytokines in several infectious and inflammatory diseases. We have assessed the contribution of IL‐22 and IL‐17A in the pathogenesis of experimental dengue infection using a mouse‐adapted DENV serotype 2 strain (P23085) that causes a disease that resembles severe dengue in humans. We show that IL‐22 and IL‐17A are produced upon DENV‐2 infection in immune‐competent mice. Infected IL‐22−/− mice had increased lethality, neutrophil accumulation and pro‐inflammatory cytokines in tissues, notably IL‐17A. Viral load was increased in spleen and liver of infected IL‐22−/− mice. There was also more severe liver injury, as seen by increased transaminases levels and tissue histopathology. γδ T cells and NK cells are sources of IL‐17A and IL‐22, respectively, in liver and spleen. We also show that DENV‐infected HepG2 cells treated with rhIL‐22 had reduced cell death and decreased IL‐6 production. IL‐17RA−/− mice were protected upon infection and IL‐17A‐neutralizing‐Ab‐treatment partially reversed the phenotype observed in IL‐22−/−‐infected mice. We suggest that disrupting the balance between IL‐22 and IL‐17A levels may represent an important strategy to reduce inflammation and tissue injury associated with severe dengue infection.

Interplay between Inflammation and Cellular Stress Triggered by Flaviviridae Viruses

The Flaviviridae family comprises several human pathogens, including Dengue, Zika, Yellow Fever, West Nile, Japanese Encephalitis viruses, and Hepatitis C Virus. Those are enveloped, single-stranded positive sense RNA viruses, which replicate mostly in intracellular compartments associated to endoplasmic reticulum (ER) and Golgi complex. Virus replication results in abundant viral RNAs and proteins, which are recognized by cellular mechanisms evolved to prevent virus infection, resulting in inflammation and stress responses. Virus RNA molecules are sensed by Toll-like receptors (TLRs), RIG-I-like receptors (RIG-I and MDA5) and RNA-dependent protein kinases (PKR), inducing the production of inflammatory mediators and interferons. Simultaneously, the synthesis of virus RNA and proteins are distinguished in different compartments such as mitochondria, ER and cytoplasmic granules, triggering intracellular stress pathways, including oxidative stress, unfolded protein response pathway, and stress granules assembly. Here, we review the new findings that connect the inflammatory pathways to cellular stress sensors and the strategies of Flaviviridae members to counteract these cellular mechanisms and escape immune response.