Cristina Cunha

TLR3 essentially promotes protective class I–restricted memory CD8+ T-cell responses to Aspergillus fumigatus in hematopoietic transplanted patients

Aspergillus fumigatus is a model fungal pathogen and a common cause of severe infections and diseases. CD8⁺ T cells are present in the human and murine T-cell repertoire to the fungus. However, CD8⁺ T-cell function in infection and the molecular mechanisms that control their priming and differentiation into effector and memory cells in vivo remain elusive. In the present study, we report that both CD4⁺ and CD8⁺ T cells mediate protective memory responses to the fungus contingent on the nature of the fungal vaccine. Mechanistically, class I MHC-restricted, CD8⁺ memory T cells were activated through TLR3 sensing of fungal RNA by cross-presenting dendritic cells. Genetic deficiency of TLR3 was associated with susceptibility to aspergillosis and concomitant failure to activate memory-protective CD8⁺ T cells both in mice and in patients receiving stem-cell transplantations. Therefore, TLR3 essentially promotes antifungal memory CD8⁺ T-cell responses and its deficiency is a novel susceptibility factor for aspergillosis in high-risk patients.

Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity

Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.

CD4+ T cell vaccination overcomes defective cross-presentation of fungal antigens in a mouse model of chronic granulomatous disease

Aspergillus fumigatus is a model fungal pathogen and a common cause of infection in individuals with the primary immunodeficiency chronic granulomatous disease (CGD). Although primarily considered a deficiency of innate immunity, CGD is also linked to dysfunctional T cell reactivity. Both CD4(+) and CD8(+) T cells mediate vaccine-induced protection from experimental aspergillosis, but the molecular mechanisms leading to the generation of protective immunity and whether these mechanisms are dysregulated in individuals with CGD have not been determined. Here, we show that activation of either T cell subset in a mouse model of CGD is contingent upon the nature of the fungal vaccine, the involvement of distinct innate receptor signaling pathways, and the mode of antigen routing and presentation in DCs. Aspergillus conidia activated CD8(+) T cells upon sorting to the Rab14(+) endosomal compartment required for alternative MHC class I presentation. Cross-priming of CD8(+) T cells failed to occur in mice with CGD due to defective DC endosomal alkalinization and autophagy. However, long-lasting antifungal protection and disease control were successfully achieved upon vaccination with purified fungal antigens that activated CD4(+) T cells through the endosome/lysosome pathway. Our study thus indicates that distinct intracellular pathways are exploited for the priming of CD4(+) and CD8(+) T cells to A. fumigatus and suggests that CD4(+) T cell vaccination may be able to overcome defective antifungal CD8(+) T cell memory in individuals with CGD.

Immunometabolic Pathways in BCG-Induced Trained Immunity

Summary The protective effects of the tuberculosis vaccine Bacillus Calmette-Guerin (BCG) on unrelated infections are thought to be mediated by long-term metabolic changes and chromatin remodeling through histone modifications in innate immune cells such as monocytes, a process termed trained immunity. Here, we show that BCG induction of trained immunity in monocytes is accompanied by a strong increase in glycolysis and, to a lesser extent, glutamine metabolism, both in an in-vitro model and after vaccination of mice and humans. Pharmacological and genetic modulation of rate-limiting glycolysis enzymes inhibits trained immunity, changes that are reflected by the effects on the histone marks (H3K4me3 and H3K9me3) underlying BCG-induced trained immunity. These data demonstrate that a shift of the glucose metabolism toward glycolysis is crucial for the induction of the histone modifications and functional changes underlying BCG-induced trained immunity. The identification of these pathways may be a first step toward vaccines that combine immunological and metabolic stimulation.

Dectin-1 isoforms contribute to distinct Th1/Th17 cell activation in mucosal candidiasis

The recognition of β-glucans by dectin-1 has been shown to mediate cell activation, cytokine production and a variety of antifungal responses. Here, we report that the functional activity of dectin-1 in mucosal immunity to Candida albicans is influenced by the genetic background of the host. Dectin-1 was required for the proper control of gastrointestinal and vaginal candidiasis in C57BL/6, but not BALB/c mice; in fact, the latter showed increased resistance in the absence of dectin-1. The susceptibility of dectin-1-deficient C57BL/6 mice to infection was associated with defects in IL-17A and aryl hydrocarbon receptor-dependent IL-22 production and in adaptive Th1 responses. In contrast, the resistance of dectin-1-deficient BALB/c mice was associated with increased IL-17A and IL-22 production and the skewing towards Th1/Treg immune responses that provide immunological memory. Disparate canonical/noncanonical NF-κB signaling pathways downstream of dectin-1 were activated in the two different mouse strains. Thus, the net activity of dectin-1 in antifungal mucosal immunity is dependent on the hosts genetic background, which affects both the innate cytokine production and the adaptive Th1/Th17 cell activation upon dectin-1 signaling.

Non-hematopoietic cells contribute to protective tolerance to Aspergillus fumigatus via a TRIF pathway converging on IDO.

Innate responses combine with adaptive immunity to generate the most effective form of anti-Aspergillus immune resistance. Whereas the pivotal role of dendritic cells in determining the balance between immunopathology and protective immunity to the fungus is well established, we determined that epithelial cells (ECs) also contributes to this balance. Mechanistically, EC-mediated protection occurred through a Toll-like receptor 3/Toll/IL-1 receptor domain-containing adaptor-inducing interferon (TLR3/TRIF)-dependent pathway converging on indoleamine 2,3-dioxygenase (IDO) via non-canonical nuclear factor-κB activation. Consistent with the high susceptibility of TRIF-deficient mice to pulmonary aspergillosis, bone marrow chimeric mice with TRIF unresponsive ECs exhibited higher fungal burdens and inflammatory pathology than control mice, underexpressed the IDO-dependent T helper 1/regulatory T cell (Th1/Treg) pathway and overexpressed the Th17 pathway with massive neutrophilic inflammation in the lungs. Further studies with interferon (IFN)-γ, IDO or IL-17R unresponsive cells confirmed the dependency of immune tolerance to the fungus on the IFN-γ/IDO/Treg pathway and of immune resistance on the MyD88 pathway controlling the fungal growth. Thus, distinct immune pathways contribute to resistance and tolerance to the fungus, to which the hematopoietic/non-hematopoietic compartments contribute through distinct, yet complementary, roles.

Cdc42p controls yeast-cell shape and virulence of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis is characterized by a multiple budding phenotype and a polymorphic cell growth, leading to the formation of cells with extreme variations in shape and size. Since Cdc42 is a pivotal molecule in establishing and maintaining polarized growth for diverse cell types, as well as during pathogenesis of certain fungi, we evaluated its role during cell growth and virulence of the yeast-form of P. brasiliensis. We used antisense technology to knock-down PbCDC42s expression in P. brasiliensis yeast cells, promoting a decrease in cell size and more homogenous cell growth, altering the typical polymorphism of wild-type cells. Reduced expression levels also lead to increased phagocytosis and decreased virulence in a mouse model of infection. We provide genetic evidences underlying Pbcdc42p as an important protein during host-pathogen interaction and the relevance of the polymorphic nature and cell size in the pathogenesis of P. brasiliensis.

TLR9 Activation Dampens the Early Inflammatory Response to Paracoccidioides brasiliensis, Impacting Host Survival

Background Paracoccidioides brasiliensis causes paracoccidioidomycosis, one of the most prevalent systemic mycosis in Latin America. Thus, understanding the characteristics of the protective immune response to P. brasiliensis is of interest, as it may reveal targets for disease control. The initiation of the immune response relies on the activation of pattern recognition receptors, among which are TLRs. Both TLR2 and TLR4 have been implicated in the recognition of P. brasiliensis and regulation of the immune response. However, the role of TLR9 during the infection by this fungus remains unclear. Methodology/Principal findings We used in vitro and in vivo models of infection by P. brasiliensis, comparing wild type and TLR9 deficient (−/−) mice, to assess the contribution of TLR9 on cytokine induction, phagocytosis and outcome of infection. We show that TLR9 recognizes either the yeast form or DNA from P. brasiliensis by stimulating the expression/production of pro-inflammatory cytokines by bone marrow derived macrophages, also increasing their phagocytic ability. We further show that TLR9 plays a protective role early after intravenous infection with P. brasiliensis, as infected TLR9−/− mice died at higher rate during the first 48 hours post infection than wild type mice. Moreover, TLR9−/− mice presented tissue damage and increased expression of several cytokines, such as TNF-α and IL-6. The increased pattern of cytokine expression was also observed during intraperitoneal infection of TLR9−/− mice, with enhanced recruitment of neutrophils. The phenotype of TLR9−/− hosts observed during the early stages of P. brasiliensis infection was reverted upon a transient, 48 hours post-infection, neutrophil depletion. Conclusions/Significance Our results suggest that TLR9 activation plays an early protective role against P. brasiliensis, by avoiding a deregulated type of inflammatory response associated to neutrophils that may lead to tissue damage. Thus modulation of TLR9 may be of interest to potentiate the host response against this pathogen.

The C allele of rs5743836 polymorphism in the human TLR9 promoter links IL-6 and TLR9 up-regulation and confers increased B-cell proliferation

In humans, allelic variants in Toll-like receptors (TLRs) associate with several pathologies. However, the underlying cellular and molecular mechanisms of this association remain largely unknown. Analysis of the human TLR9 promoter revealed that the C allele of the rs5743836 polymorphism generates several regulatory sites, including an IL-6-responding element. Here, we show that, in mononuclear cells carrying the TC genotype of rs5743836, IL-6 up-regulates TLR9 expression, leading to exacerbated cellular responses to CpG, including IL-6 production and B-cell proliferation. Our study uncovers a role for the rs5743836 polymorphism in B-cell biology with implications on TLR9-mediated diseases and on the therapeutic usage of TLR9 agonists/antagonists.

The soluble pattern recognition receptor PTX3 links humoral innate and adaptive immune responses by helping marginal zone B cells

Cerutti and collaborators show that the humoral arms of the innate and adaptive immune systems are functionally interconnected by pentraxin 3, a soluble pattern recognition receptor that couples innate immune recognition with antibody-inducing function.