Alessandra Granato

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.

Kininogens coordinate adaptive immunity through the proteolytic release of bradykinin, an endogenous danger signal driving dendritic cell maturation.

Strategically positioned in peripheral tissues, immune sentinel cells sense microbes and/or their shed products through different types of pattern‐recognition receptors. Upon secretion, pre‐formed pro‐inflammatory mediators activate the microvasculature, inducing endothelium/neutrophil adherence and impairing endothelium barrier function. As plasma proteins enter into peripheral tissues, short‐lived proinflammatory peptides are rapidly generated by limited proteolysis of complement components and the kininogens (i.e. kinin‐precursor proteins). While much emphasis has been placed on the studies of the vascular functions of kinins, their innate effector roles remain virtually unknown. A few years ago, we reported that exogenous bradykinin (BK) potently induces dendritic cell (DC) maturation, driving IL‐12‐dependent Th1 responses through the activation of G‐protein‐coupled BK B2 receptors (B2R). The premise that immature DC might sense kinin‐releasing pathogens through B2R was demonstrated in the subcutaneous mouse model of Trypanosoma cruzi infection. Analysis of the dynamics of parasite‐evoked inflammation revealed that activation of TLR2/neutrophils drives the influx of plasma proteins, including kininogens, into peripheral tissues. Once associated to cell surfaces and/or extracellular matrices, the surface‐bound kininogens are cleaved by T. cruzi cysteine proteases. Acting as short‐lived ‘danger’ signals, kinins activate DC via B2R, converting them into Th1 inducers. Fine tuned control of the extravascular levels of these natural peptide adjuvants is exerted by kinin‐degrading metallopeptidases, e.g. Angiotensin converting enzyme (ACE/CD143). In summary, the studies in the subcutaneous model of T. cruzi infection revealed that the peripheral levels of BK, a DC maturation signal, are controlled by TLR2/neutrophils and ACE, respectively characterized as positive and negative modulators of innate/adaptive immunity.

TLR4 Promotes B Cell Maturation: Independence and Cooperation with B Lymphocyte-Activating Factor

We have previously shown that TLR4 triggering promotes the generation of CD23+CD93+ transitional T2-like cells in vitro from mouse B cell precursors, suggesting a possible role for this receptor in B cell maturation. In this study, we perform an extensive study of cell surface markers and functional properties of B cells matured in vitro with LPS, comparatively with the well-known B cell maturation factor B lymphocyte-activating factor (BAFF). LPS increased generation of CD23+ transitional B cells in a TLR4-dependent way, upregulating IgD and CD21 and downregulating CD93, without inducing cell proliferation, in a manner essentially equivalent to BAFF. For both BAFF and LPS, functional maturation of the IgM+CD23+CD93+ cells was confirmed by their higher proliferative response to anti-CD40 plus IL-4 compared with IgM+CD23negCD93+ cells. BAFF-R-Fc–mediated neutralization experiments showed that TLR4-induced B cell maturation was independent of BAFF. Distinct from BAFF, maturation by LPS relied on the activation of canonical NF-κB pathway, and the two factors together had complementary effects, leading to higher numbers of IgM+CD23+CD93+ cells with their simultaneous addition. Importantly, BCR cross-linking abrogated the generation of CD23+ B cells by LPS or BAFF, indicating that signals mimicking central tolerance act on both systems. Addition of cyclosporin A reverted BCR-mediated inhibition, both for BAFF and LPS, suggesting similar regulation of signaling pathways by calcineurin. Finally, LPS-injected mice showed a rapid increase of mature B cells in the bone marrow, suggesting that TLR4 signaling may effectively stimulate B cell maturation in vivo, acting as an accessory stimulus in B cell development, complementary to the BAFF physiological pathway.

IL-4 Regulates Bim Expression and Promotes B Cell Maturation in Synergy with BAFF Conferring Resistance to Cell Death at Negative Selection Checkpoints

IL-4 plays an essential role in the activation of mature B cells, but less is known about the role of IL-4 in B cell maturation and tolerance checkpoints. In this study, we analyzed the effect of IL-4 on in vitro B cell maturation, from immature to transitional stages, and its influence on BCR-mediated negative selection. Starting either from purified CD19+IgM− B cell precursors, or sorted bone marrow immature (B220lowIgMlowCD23−) and transitional (B220intIgMhighCD23−) B cells from C57BL/6 mice, we compared the maturation effects of IL-4 and BAFF. We found that IL-4 stimulated the generation of CD23+ transitional B cells from CD23− B cells, and this effect was comparable to BAFF. IL-4 showed a unique protective effect against anti-IgM apoptotic signals on transitional B cell checkpoint, not observed with BAFF. IL-4 and BAFF strongly synergized to promote B cell maturation, and IL-4 also rendered it refractory to BCR-mediated cell death. IL-4 blocked upregulation of proapoptotic Bim protein levels induced by BCR crosslinking, suggesting that diminished levels of intracellular Bim promote protection to BCR-induced cell death. Evidence was obtained indicating that downmodulation of Bim by IL-4 occurred in a posttranscriptional manner. Consistent with data obtained in vitro, IL-4 in vivo was able to inhibit Bim upregulation and prevent cell death. These results contribute to the understanding of the role of IL-4 in B lymphocyte physiology, unveiling a previously undescribed activity of this cytokine on the maturation of B cells, which could have important implications on the breaking of B cell central tolerance in autoimmunity.

GENETIC CONTROL OF THE B CELL RESPONSE TO LPS: OPPOSING EFFECTS IN PERITONEAL VERSUS SPLENIC B CELL POPULATIONS

Lipopolysaccharide (LPS) from gram-negative bacteria activates B cells, enabling them to proliferate and differentiate into plasma cells. This response is critically dependent on the expression of TLR4; but other genes, such as RP105 and MHC class II, have also been shown to contribute to B cell LPS response. Here, we have evaluated the role of genetic control of the B cell response to LPS at the single cell level. We compared the response to LPS of peritoneal cavity (PEC) and splenic B cells on the BALB/c genetic background (LPS-low responder) to those on the C57BL/6J background (LPS-high responder) and their F1 progeny (CB6F1). Both PEC and splenic B cells from B6 exhibited 100% clonal growth in the presence of LPS; whereas, BALB/c PEC and splenic B cells achieved only 50% and 23% clonal growth, respectively. Adding CpG to the LPS stimulus pushed PEC B cell clonal growth in the low responder strain BALB/c up to 90%, showing that the nonresponse to LPS is a specific effect. Surprisingly, PEC B cells on the F1 background behaved as high responders, while splenic B cells behaved as low responders to LPS. The data presented here reveals a previous unsuspected behavior in the genetic control of the B cell response to LPS with an opposing impact in splenic versus peritoneal cavity B cells. These results suggest the existence of an, as yet, unidentified genetic factor exclusively expressed by coelomic B cells that contributes to the control of the LPS signaling pathway in the B lymphocyte.

A rapid and quantitative method for the evaluation of V gene usage, specificities and the clonal size of B cell repertoires.

The quantitative simultaneous description of both variable region gene usage and antigen specificity of immunoglobulin repertoires is a major goal in immunology. Current quantitative assays are labor intensive and depend on extensive gene expression cloning prior to screening for antigen specificity. Here we describe an alternative method based on high efficiency single B cell cultures coupled with RT-PCR that can be used for rapid characterization of immunoglobulin gene segment usage, clonal size and antigen specificity. This simplified approach should facilitate the study of antibody repertoires expressed by defined B cell subpopulations, the analysis of immune responses to self and nonself-antigens, the development and screening of synthetic antibodies and the accelerated study and screening of neutralizing antibodies to pathogenic threats.

Crucial role for T cell-intrinsic IL-18R-MyD88 signaling in cognate immune response to intracellular parasite infection

MyD88 is the main adaptor molecule for TLR and IL-1R family members. Here, we demonstrated that T-cell intrinsic MyD88 signaling is required for proliferation, protection from apoptosis and expression of activation/memory genes during infection with the intracellular parasite Trypanosoma cruzi, as evidenced by transcriptome and cytometry analyses in mixed bone-marrow (BM) chimeras. The lack of direct IL-18R signaling in T cells, but not of IL-1R, phenocopied the absence of the MyD88 pathway, indicating that IL-18R is a critical MyD88-upstream pathway involved in the establishment of the Th1 response against an in vivo infection, a presently controvert subject. Accordingly, Il18r1−/− mice display lower levels of Th1 cells and are highly susceptible to infection, but can be rescued from mortality by the adoptive transfer of WT CD4+ T cells. Our findings establish the T-cell intrinsic IL-18R/MyD88 pathway as a crucial element for induction of cognate Th1 responses against an important human pathogen.

Repression of Salmonella host cell invasion by aromatic small molecules from the human fecal metabolome

ABSTRACT The human microbiome is a collection of microorganisms that inhabit every surface of the body that is exposed to the environment, generally coexisting peacefully with their host. These microbes have important functions, such as producing vitamins, aiding in maturation of the immune system, and protecting against pathogens. We have previously shown that a small-molecule extract from the human fecal microbiome has a strong repressive effect on Salmonella enterica serovar Typhimurium host cell invasion by modulating the expression of genes involved in this process. Here, we describe the characterization of this biological activity. Using a series of purification methods, we obtained fractions with biological activity and characterized them by mass spectrometry. These experiments revealed an abundance of aromatic compounds in the bioactive fraction. Selected compounds were obtained from commercial sources and tested with respect to their ability to repress the expression of hilA, the gene encoding the master regulator of invasion genes in Salmonella. We found that the aromatic compound 3,4-dimethylbenzoic acid acts as a strong inhibitor of hilA expression and of invasion of cultured host cells by Salmonella. Future studies should reveal the molecular details of this phenomenon, such as the signaling cascades involved in sensing this bioactive molecule. IMPORTANCE Microbes constantly sense and adapt to their environment. Often, this is achieved through the production and sensing of small extracellular molecules. The human body is colonized by complex communities of microbes, and, given their biological and chemical diversity, these ecosystems represent a platform where the production and sensing of molecules occur. In previous work, we showed that small molecules produced by microbes from the human gut can significantly impair the virulence of the enteric pathogen Salmonella enterica. Here, we describe a specific compound from the human gut that produces this same effect. The results from this work not only shed light on an important biological phenomenon occurring in our bodies but also may represent an opportunity to develop drugs that can target these small-molecule interactions to protect us from enteric infections and other diseases.

Enlarged colitogenic T cell population paradoxically supports colitis prevention through the B-lymphocyte-dependent peripheral generation of CD4 + Foxp3 + Treg cells

Intestinal inflammation can be induced by the reconstitution of T/B cell-deficient mice with low numbers of CD4+ T lymphocytes depleted of CD25+Foxp3+ regulatory T cells (Treg). Using RAG-knockout mice as recipients of either splenocytes exclusively depleted of CD25+ cells or FACS-purified CD4+CD25−Foxp3− T cells, we found that the augmentation of potentially colitogenic naïve T cell numbers in the inoculum was unexpectedly beneficial for the suppression of colon disease and maintenance of immune homeostasis. Protection against T cell-mediated colitis correlated with a significant increment in the frequency of peripherally-induced CD4+CD25+Foxp3+ T (pTreg) cells, especially in the mesenteric lymph nodes, an effect that required the presence of B cells and CD4+CD25−Foxp3+ cells in physiological proportions. Our findings support a model whereby the interplay between B lymphocytes and a diversified naïve T cell repertoire is critical for the generation of CD4+CD25+Foxp3+ pTreg cells and colitis suppression.

Circulating Plasma MicroRNA-208a as Potential Biomarker of Chronic Indeterminate Phase of Chagas Disease

Chagas cardiomyopathy is the most severe clinical manifestation of chronic Chagas disease. The disease affects most of the Latin American countries, being considered one of the leading causes of morbidity and death in the continent. The pathogenesis of Chagas cardiomyopathy is very complex, with mechanisms involving parasite-dependent cytopathy, immune-mediated myocardial damage and neurogenic disturbances. These pathological changes eventually result in cardiac myocyte hypertrophy, arrhythmias, congestive heart failure and stroke during chronic infection phase. Herein, we show that miR-208a, a microRNA that is a key factor in promoting cardiovascular dysfunction during cardiac hypertrophy processes of heart failure, has its circulating levels increased during chronic indeterminate phase when compared to cardiac (CARD) clinical forms in patients with Chagas disease. In contrast, we have not found altered serum levels of miR-34a, a microRNA known to promote pro-apoptotic role in myocardial infarction during degenerative process of cardiac injuries thus indicating intrinsic differences in the nature of the mechanisms underlying the heart failure triggered by Trypanosoma cruzi infection. Our findings support that the chronic indeterminate phase is a progressive phase involved in the genesis of chagasic cardiopathy and point out the use of plasma levels of miR-208a as candidate biomarker in risk-prediction score for the clinical prognosis of Chagas disease.