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Activation of invariant natural killer T (iNKT) cells by α-galactosylceramides (α-GalCers) stimulates strong immune responses and potent anti-tumor immunity. Numerous modifications of the glycolipid structure have been assessed to derive activating ligands for these T cells with altered and potentially advantageous properties in the induction of immune responses. Here, we synthesized variants of the prototypical α-GalCer, KRN7000, with amide-linked phenyl alkane substitutions on the C4″-position of the galactose ring. We show that these variants have weak iNKT cell stimulating activity in mouse models but substantially greater activity for human iNKT cells. The most active of the C4″-amides in our study showed strong anti-tumor effects in a partially humanized mouse model for iNKT cell responses. In silico analysis suggested that the tether length and degree of flexibility of the amide substituent affected the recognition by iNKT cell antigen receptors of the C4″-amide substituted glycolipids in complex with their antigen presenting molecule CD1d. Our findings establish the use of stable C4″-amide linked additions to the sugar moiety for further exploration of the immunological effects of structural modifications of iNKT cell activating glycolipids and highlight the critical need for more accurate animal models to assess these compounds for immunotherapeutic potential in humans.

Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) cause acute respiratory tract infections in children worldwide. Natural killer T (NKT) cells are unconventional T lymphocytes, and their TCRs recognize glycolipids bound to the MHC-I-like molecule, CD1d. These cells modulate the inflammatory response in viral infections. Here, we evaluated the contribution of NKT cells in both hRSV and hMPV infections. A significant decrease in the number of neutrophils, eosinophils, and CD103+DCs infiltrating to the lungs, as well as an increased production of IFN-γ, were observed upon hRSV-infection in CD1d-deficient BALB/c mice, as compared to wild-type control mice. However, this effect was not observed in the CD1d-deficient BALB/c group, upon infection with hMPV. Importantly, reduced expression of CD1d in CD11b+ DCs and epithelial cells was found in hRSV -but not hMPV-infected mice. Besides, a reduction in the expression of CD1d in alveolar macrophages of lungs from hRSV- and hMPV-infected mice was found. Such reduction of CD1d expression interfered with NKT cells activation, and consequently IL-2 secretion, as characterized by in vitro experiments for both hRSV and hMPV infections. Furthermore, increased numbers of NKT cells recruited to the lungs in response to hRSV- but not hMPV-infection was detected, resulting in a reduction in the expression of IFN-γ and IL-2 by these cells. In conclusion, both hRSV and hMPV might be differently impairing NKT cells function and contributing to the immune response triggered by these viruses.

MicroRNA-423 (miR-423) is highly expressed in breast cancer (BC). Previously, our group showed that the SNP rs6505162:C>A located in the pre-miR-423 was significantly associated with increased familial BC risk in patients with a strong family history of BC. Therefore, in this study, we evaluated the functional role of rs6505162 in mammary tumorigenesis in vitro to corroborate the association of this SNP with BC risk. We found that rs6505162:C>A upregulated expression of both mature miR-423 sequences (3p and 5p). Moreover, pre-miR-423-A enhanced proliferation, and promoted cisplatin resistance in BC cell lines. We also showed that pre-miR-423-A expression decreased cisplatin-induced apoptosis, and increased BC cell migration and invasion. We propose that the rs6505162-A allele promotes miR-423 overexpression, and that the rs6505162-A allele induces BC cell proliferation, viability, chemoresistance, migration, and invasion, and decreases cell apoptosis as a consequence. We suggest that rs6505162:C>A is a functional SNP site with potential utility as a marker for early diagnosis, prognosis, and treatment efficacy monitoring in BRCA1/2-negative BC patients, as well as a possible therapeutic target.

SUMMARY The immune system must work in an orchestrated way to achieve an optimal response upon detection of antigens. The cells comprising the immune response are traditionally divided into two major subsets, innate and adaptive, with particular characteristics for each type. Type I natural killer T (iNKT) cells are defined as innate-like T cells sharing features with both traditional adaptive and innate cells, such as the expression of an invariant T cell receptor (TCR) and several NK receptors. The invariant TCR in iNKT cells interacts with CD1d, a major histocompatibility complex class I (MHC-I)-like molecule. CD1d can bind and present antigens of lipid nature and induce the activation of iNKT cells, leading to the secretion of various cytokines, such as gamma interferon (IFN-γ) and interleukin 4 (IL-4). These cytokines will aid in the activation of other immune cells following stimulation of iNKT cells. Several molecules with the capacity to bind to CD1d have been discovered, including α-galactosylceramide. Likewise, several molecules have been synthesized that are capable of polarizing iNKT cells into different profiles, either pro- or anti-inflammatory. This versatility allows NKT cells to either aid or impair the clearance of pathogens or to even control or increase the symptoms associated with pathogenic infections. Such diverse contributions of NKT cells to infectious diseases are supported by several publications showing either a beneficial or detrimental role of these cells during diseases. In this article, we discuss current data relative to iNKT cells and their features, with an emphasis on their driving role in diseases produced by pathogenic agents in an organ-oriented fashion.

Human herpesviruses are a ubiquitous family of viruses that infect individuals of all ages and are present at a high prevalence worldwide. Herpesviruses are responsible for a broad spectrum of diseases, ranging from skin and mucosal lesions to blindness and life-threatening encephalitis, and some of them, such as Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein–Barr virus (EBV), are known to be oncogenic. Furthermore, recent studies suggest that some herpesviruses may be associated with developing neurodegenerative diseases. These viruses can establish lifelong infections in the host and remain in a latent state with periodic reactivations. To achieve infection and yield new infectious viral particles, these viruses require and interact with molecular host determinants for supporting their replication and spread. Important sets of cellular factors involved in the lifecycle of herpesviruses are those participating in intracellular membrane trafficking pathways, as well as autophagic-based organelle recycling processes. These cellular processes are required by these viruses for cell entry and exit steps. Here, we review and discuss recent findings related to how herpesviruses exploit vesicular trafficking and autophagy components by using both host and viral gene products to promote the import and export of infectious viral particles from and to the extracellular environment. Understanding how herpesviruses modulate autophagy, endolysosomal and secretory pathways, as well as other prominent trafficking vesicles within the cell, could enable the engineering of novel antiviral therapies to treat these viruses and counteract their negative health effects.

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong antitumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T-cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogues of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell–specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T-cell responses. The antitumor effects of iNKT-cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell–specific BiTEs activated iNKT cells without causing iNKT cell anergy or exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens. Significance: Covalently stabilized conjugates that engage the antigen receptors of iNKT cells and target a tumor antigen activate potent antitumor immunity without induction of anergy or depletion of the responding iNKT cells.

Vibrio cholerae is a human pathogen, which is transmitted by the consumption of contaminated food or water. V. cholerae strains belonging to the serogroups O1 and O139 can cause cholera outbreaks and epidemics, a severe life-threatening diarrheal disease. In contrast, serogroups other than O1 and O139, denominated as non-O1/non-O139, have been mainly associated with sporadic cases of moderate or mild diarrhea, bacteremia and wound infections. Here we investigated the virulence determinants and phylogenetic origin of a non-O1/non-O139 V. cholerae strain that caused a gastroenteritis outbreak in Santiago, Chile, 2018. We found that this outbreak strain lacks the classical virulence genes harboured by O1 and O139 strains, including the cholera toxin (CT) and the toxin-coregulated pilus (TCP). However, this strain carries genomic islands (GIs) encoding Type III and Type VI secretion systems (T3SS/T6SS) and antibiotic resistance genes. Moreover, we found these GIs are wide distributed among several lineages of non-O1/non-O139 strains. Our results suggest that the acquisition of these GIs may enhance the virulence of non-O1/non-O139 strains that lack the CT and TCP-encoding genes. Our results highlight the pathogenic potential of these V. cholerae strains.DATA SUMMARYSequence data were submitted to GenBank under the accession number SRLP00000000. The authors confirm that all supporting data and protocols have been provided within the article or through supplementary data files.Data statementAll supporting data, code and protocols have been provided within the article or through supplementary data files. Four supplementary tables are available with the online version of this article.

Modulation of the immune system has been widely targeted for the treatment of several immune-related diseases, such as autoimmune disorders and cancer, due to its crucial role in these pathologies. Current available therapies focus mainly on symptomatic treatment and are often associated with undesirable secondary effects. For several years, remission of disease and subsequently recovery of immune homeostasis has been a major goal for immunotherapy. Most current immunotherapeutic strategies are aimed to inhibit or potentiate directly the adaptive immune response by modulating antibody production and B cell memory, as well as the effector potential and memory of T cells. Although these immunomodulatory approaches have shown some success in the clinic with promising therapeutic potential, they have some limitations related to their effectiveness in disease models and clinical trials, as well as elevated costs. In the recent years, a renewed interest has emerged on targeting innate immune cells for immunotherapy, due to their high plasticity and ability to exert a potent and extremely rapid response, which can influence the outcome of the adaptive immune response. In this review, we discuss the immunomodulatory potential of several innate immune cells, as well as they use for immunotherapy, especially in autoimmunity and cancer.

Macrophage activation is intimately linked to metabolic reprogramming. Inflammatory (M1) macrophages are able to sustain inflammatory responses and to kill pathogens, mostly by relying on aerobic glycolysis and fatty acid biosynthesis. Glycolysis is a fast way of producing ATP, and fatty acids serve as precursors for the synthesis of inflammatory mediators. On the opposite side, anti-inflammatory (M2) macrophages mediate the resolution of inflammation and tissue repair, switching their metabolism to fatty acid oxidation and oxidative phosphorylation. Over the years, this classical view has been challenged by recent discoveries pointing to a more complex metabolic network during macrophage activation. Lipid metabolism plays a critical role in the activation of both M1 and M2 macrophages. Recent evidence shows that fatty acid oxidation is also essential for inflammasome activation in M1 macrophages, and glycolysis is now known to fuel fatty acid oxidation in M2 macrophages. Ultimately, targeting lipid metabolism in macrophages can improve the outcome of metabolic diseases. Here, we review the main aspects of macrophage immunometabolism from the perspective of the metabolism of lipids. Building a reliable metabolic network during macrophage activation will bring us closer to targeting macrophages for improving human health.

Listeria monocytogenes causes severe diseases in humans, including febrile gastroenteritis and systemic infections that has a high mortality despite antibiotic treatment. This pathogen may cause massive outbreaks associated to the consumption of contaminated food products, which highlight its importance in public health. In the last decade, L. monocytogenes has emerged as a foodborne pathogen of major importance in Chile. A previous work showed that in Chile during 2008 and 2009, L. monocytogenes serotypes 1/2a, 1/2b and 4b were the most frequently identified in food and clinical strains. Here we report the molecular characterization of L. monocytogenes strains isolated from 2008 to 2017 in the country. Our results indicate that serotypes 1/2a, 1/2b and 4b continue to be the most commonly found in food products. In addition, we identify persistent and widespread PFGE subtypes. This study reports ten years of epidemiological surveillance ofL. monocytogenes in Chile.

BackgroundInitiation of antiretroviral therapy (ART) in human immunodeficiency virus (HIV)-infected individuals with cryptococcal meningitis places them at risk for Cryptococcus-associated immune reconstitution inflammatory syndrome (C-IRIS). The relationship between antibody immunity and C-IRIS risk has not been investigated.MethodsWe compared plasma levels of immunoglobulins, C. neoformans glucuronoxylomannan (GXM) capsule-specific and laminarin (Lam)-binding IgM and IgG, and percentages of peripheral blood total and memory B cells between 27 HIV-infected patients with CM who developed C-IRIS and 63 who did not, and evaluated associations of these parameters with risk of C-IRIS.ResultsPrior to initiation of ART, plasma IgM, Lam-binding IgM (Lam-IgM), Lam-IgG, and GXM-IgM levels were significantly lower in patients who developed C-IRIS than those who did not. Multivariate analysis revealed significant inverse associations between C-IRIS and IgM (P = .0003), Lam-IgM (P = .0005), Lam-IgG (P = .002), and GXM-IgM (P = .002) independent of age, sex, HIV viral load, CD4+ T-cell count, and cerebrospinal fluid fungal burden. There were no associations between C-IRIS and total or memory B cells.DiscussionAntibody profiles that include plasma IgM, Lam-IgM, Lam-IgG, and/or GXM-IgM may have value in furthering our understanding of C-IRIS pathogenesis and hold promise as candidate biomarkers of C-IRIS risk.

Background Bacillus Calmette-Guerin (BCG) vaccine is widely used for the prevention of tuberculosis, despite limited efficacy. Most immunological studies of BCG or Mycobacterium tuberculosis strains grow bacteria in the presence of detergent, which also strips the mycobacterial capsule. The impact of the capsule on vaccine efficacy has not been explored. Methods We tested the influence of detergent in cultures of BCG and M. tuberculosis strains on the outcome of vaccination experiments on mice and transcriptional responses on M. tuberculosis Results Vaccination of mice with encapsulated BCG promoted a more potent immune response relative to vaccination with unencapsulated BCG, including higher polysaccharide-specific capsule antibody titers, higher interferon γ and interleukin 17 splenic responses, and more multifunctional CD4(+) T cells. These differences correlated with variability in the bacterial burden in lung and spleen of mice infected with encapsulated or unencapsulated M. tuberculosis The combination of vaccination and challenge with encapsulated strains resulted in the greatest protection efficacy. The transcriptome of encapsulated M. tuberculosis was similar to that of starvation, hypoxia, stationary phase, or nonreplicating persistence. Conclusions The presence of detergent in growth media and a capsule on BCG were associated with differences in the outcome of vaccination, implying that these are important variables in immunological studies.

Glycosylceramides that activate CD1d-restricted invariant natural killer T (iNKT) cells have potential therapeutic applications for augmenting immune responses against cancer and infections. Previous studies using mouse models identified sphinganine variants of α‐galactosylceramide as promising iNKT cell activators that stimulate cytokine responses with a strongly pro-inflammatory bias. However, the activities of sphinganine variants in mice have generally not translated well to studies of human iNKT cell responses. Here we show that strongly proinflammatory and anti-tumor iNKT cell responses were achieved in mice by a variant of α‐galactosylceramide that combines a sphinganine base with a hydrocinnamoyl ester on C6″ of the sugar. Importantly, the activities observed with this variant were largely preserved for human iNKT cell responses. Structural and in silico modeling studies provided a mechanistic basis for these findings, and suggested basic principles for capturing useful properties of sphinganine analogues of synthetic iNKT cell activators in the design of immunotherapeutic agents.

The importance of antibody immunity in protection against Cryptococcus neoformans remains unresolved. We measured serum C neoformans-specific and total antibody levels and peripheral blood B cell subsets of 12 previously healthy patients with cryptococcosis (cases) and 21 controls. Before and after adjustment for age, sex, and race, cryptococcal capsular polysaccharide immunoglobulin G was higher in cases than controls, whereas total B and memory B cell levels were lower. These associations parallel previous findings in patients with human immunodeficiency virus-associated cryptococcosis and suggest that B cell subset perturbations may also associate with disease in previously normal individuals with cryptococcosis.

Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cell-mediated immunity (CMI). In contrast, most approved vaccines against other bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with either Abg85b-AM or PA-AM conjugates elicited an AM-specific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice reacted against a broad spectrum of AM structural variants and specifically recognized arabinan fragments. Conjugate vaccine immunized mice infected with Mtb had lower bacterial numbers in lungs and spleen, and lived longer than control mice. These findings provide additional evidence that humoral immunity can contribute to protection against Mtb., Author summary Vaccine design in the TB field has been driven by the imperative of attempting to elicit strong cell-mediated responses. However, in recent decades evidence has accumulated that humoral immunity can protect against many intracellular pathogens through numerous mechanisms. In this work, we demonstrate that immunization with mycobacterial capsular arabinomannan (AM) conjugates elicited responses that contributed to protection against Mtb infection. We developed two different conjugates including capsular AM linked to the Mtb related protein Ag85b or the Mtb unrelated PA from B. anthracis and found that immunization with AM conjugates elicited antibody populations with different specificities. These surface-specific antibodies could directly modify the transcriptional profile and metabolism of mycobacteria. In addition, we observed a prolonged survival and a reduction in bacterial numbers in lungs and spleen in mice immunized with Ag85b-AM conjugates after infection with Mtb and that the presence of AM-binding antibodies was associated with modest prolongation in survival and a marked reduction in mycobacterial dissemination. Finally, we show that AM is antigenically variable and could potentially form the basis for a serological characterization of mycobacteria based on serotypes.

Background: Immune suppression in the tumour microenvironment remains a major limitation to successful immunotherapy of cancer. In the current study, we analysed whether the natural killer T cell-activating glycolipid α-galactosylceramide could overcome immune suppression and improve vaccination against metastatic breast cancer. Methods: Mice with metastatic breast cancer (4T1 model) were therapeutically treated with a Listeria monocytogenes-based vaccine expressing tumour-associated antigen Mage-b followed by α-galactosylceramide as separate agents, or as a complex of α-galactosylceramide stably incorporated into Listeria-Mage-b. Effects on metastases, tumour weight, toxicity and immune responses were determined. Results: Sequential treatments of mice with established 4T1 breast carcinomas using Listeria-Mage-b followed by α-galactosylceramide as a separate agent was highly effective at reducing metastases, but was accompanied by severe liver toxicity. In contrast, combined therapy using Listeria-Mage-b modified by incorporation of α-galactosylceramide resulted in nearly complete elimination of metastases without toxicity. This was associated with a significant increase in the percentage of natural killer T cells in the spleen, and an increase in natural killer cell activity and in T cell responses to Mage-b. Conclusions: Our results suggest that direct incorporation of α-galactosylceramide into a live bacterial vaccine vector is a promising non-toxic new approach for the treatment of metastatic breast cancer.

NKT cells are a subpopulation of T lymphocytes with phenotypic properties of both T and NK cells and a wide range of immune effector properties. In particular, one subset of these cells, known as invariant NKT cells (iNKT cells), has attracted substantial attention because of their ability to be specifically activated by glycolipid antigens presented by a cell surface protein called CD1d. The development of synthetic α-galactosylceramides as a family of powerful glycolipid agonists for iNKT cells has led to approaches for augmenting a wide variety of immune responses, including those involved in vaccination against infections and cancers. Here, we review basic, preclinical and clinical observations supporting approaches to improving immune responses through the use of iNKT cell-activating glycolipids. Results from preclinical animal studies and preliminary clinical studies in humans identify many promising applications for this approach in the development of vaccines and novel immunotherapies.

Summary Many hematopoietic cell types express CD1d and are capable of presenting glycolipid antigens to invariant natural killer T cells (iNKT cells). However, the question of which cells are the principal presenters of glycolipid antigens in vivo remains controversial, and it has been suggested that this might vary depending on the structure of a particular glycolipid antigen. Here we have shown that a single type of cell, the CD8α+ DEC-205+ dendritic cell, was mainly responsible for capturing and presenting a variety of different glycolipid antigens, including multiple forms of α-galactosylceramide that stimulate widely divergent cytokine responses. After glycolipid presentation, these dendritic cells rapidly altered their expression of various costimulatory and coinhibitory molecules in a manner that was dependent on the structure of the antigen. These findings show flexibility in the outcome of two-way communication between CD8α+ dendritic cells and iNKT cells, providing a mechanism for biasing toward either proinflammatory or anti-inflammatory responses., Graphical Abstract, Highlights • Complexes of antigenic glycolipids bound to CD1d have been visualized in situ • A single DC subset predominates in presentation of a variety of glycolipids • Antigen presentation to iNKT cells rapidly alters accessory molecules on APCs • Reciprocal induction of CD70 and PD-L2 controls cytokine bias of iNKT cell responses

Invariant natural killer T (iNKT) cells are a specialized group of unconventional T cells that recognize glycolipids presented by the surface protein CD1d, expressed by most antigen-presenting cells. This results in the rapid secretion of a wide array of cytokines without the need for prior antigen immunization, thus making iNKT cells an attractive target for immunotherapy. Marine sponge sphingolipid α-galactosylceramide (α-GalCer) is a potent agonist for iNKT cell activation, characterized for inducing both Th1- and Th2-like cytokine secretion profiles. There is an increasing interest for the development of Th1- and Th2-polarizing ligands for more focused immunotherapeutic applications. Our aim is to evaluate the iNKT cell activation ability of C6” modified α-GalCer derivatives. We generated and isolated clones from iNKT cell hybridomas derived from a partially humanized mouse, consisting of a knock-in of the human CD1d gene. We selected by flow cytometry iNKT cell populations reactive to α-GalCer presented in the context of the human CD1d molecule. Additionally, we evaluated activation of iNKT cells by C6” modified α-GalCer derivatives and obtained a diverse interleukin-2 response depending on the structural modifications of the ligands and the individuality of the iNKT cell clones. In several cases, this response was more potent than that induced by stimulation with α-GalCer. Our results highlight differences between mouse and human lipid antigen presentation, as well as differences in the magnitude of the cytokine response amongst clonal populations. Our ongoing work aims to elucidate the mechanism for this observation by performing Next Generation Sequencing of the TCR chains of the responding clones.

Made available in DSpace on 2018-12-11T17:13:42Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-10-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Systemic therapies are preferred for treating dermal dermatophytosis due to inadequate penetration of topical agents. However, systemic antifungals are associated with off-target effects and limited tissue penetration, and antimicrobial resistance is a growing concern. To address this, we investigated topical nitric oxide-releasing nanoparticles (NO-np), which have been used against superficial fungal infections and bacterial abscesses. In addition to enhanced penetration and permeation conferred by nanoparticles, nitric oxide, a broad-spectrum multi-mechanistic antimicrobial agent, offers decreased likelihood of resistance development. In the current study, NO-np inhibited Trichophyton rubrum in vitro, as well as in a murine model of dermal dermatophytosis. In mice, NO-np reduced fungal burden after three days, with complete clearance after seven. Furthermore, NO-np decreased tissue IL-2, 6, 10 and TNFα, indicating earlier attenuation of the host inflammatory response and decreased tissue morbidity. Thus, topical NO-np represent an attractive alternative to systemic therapy against dermal T. rubrum infection. Department of Medicine (Division of Dermatology) Albert Einstein College of Medicine Department of Medicine (Division of Infectious Diseases) Albert Einstein College of Medicine Departamento de Análises Clínicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista (UNESP) Department of Microbiology & Immunology Albert Einstein College of Medicine Millennium Institute on Immunology and Immunotherapy Programa de Inmunología Instituto de Ciencias Biomédicas Facultad de Medicina Universidad de Chile Department of Physiology & Biophysics Albert Einstein College of Medicine Department of Dermatology George Washington School of Medicine and Health Sciences Departamento de Análises Clínicas Faculdade de Ciências Farmacêuticas Universidade Estadual Paulista (UNESP) CNPq: 150261/2016-0 CAPES: 99999.007910/2014-02

Suppression of major histocompatibility complex (MHC) class II antigen presentation is believed to be among the major mechanisms used by Mycobacterium tuberculosis to escape protective host immune responses. Through a genome-wide screen for the genetic loci of M. tuberculosis that inhibit MHC class II-restricted antigen presentation by mycobacteria-infected dendritic cells, we identified the PE_PGRS47 protein as one of the responsible factors. Targeted disruption of the PE_PGRS47 (Rv2741) gene led to attenuated growth of M. tuberculosis in vitro and in vivo, and a PE_PGRS47 mutant showed enhanced MHC class II-restricted antigen presentation during in vivo infection of mice. Analysis of the effects of deletion or over-expression of PE_PGRS47 implicated this protein in the inhibition of autophagy in infected host phagocytes. Our findings identify PE_PGRS47 as a functionally relevant, non-redundant bacterial factor in the modulation of innate and adaptive immunity by M. tuberculosis, suggesting strategies for improving antigen presentation and the generation of protective immunity during vaccination or infection.

Certain types of glycolipids have been found to have remarkable immunomodulatory properties as a result of their ability to activate specific T lymphocyte populations with an extremely wide range of immune effector properties. The most extensively studied glycolipid reactive T cells are known as invariant natural killer T (iNKT) cells. The antigen receptors of these cells specifically recognize certain glycolipids, most notably glycosphingolipids with α-anomeric monosaccharides, presented by the major histocompatibility complex class I-like molecule CD1d. Once activated, iNKT cells can secrete a very diverse array of pro- and anti-inflammatory cytokines to modulate innate and adaptive immune responses. Thus, glycolipid-mediated activation of iNKT cells has been explored for immunotherapy in a variety of disease states, including cancer and a range of infections. In this review, we discuss the design of synthetic glycolipid activators for iNKT cells, their impact on adaptive immune responses and their use to modulate iNKT cell responses to improve immunity against infections and cancer. Current challenges in translating results from preclinical animal studies to humans are also discussed.

Haem-oxygenase-1 (HO-1) is an enzyme responsible for the degradation of haem that can suppress inflammation, through the production of carbon monoxide (CO). It has been shown in several experimental models that genetic and pharmacological induction of HO-1, as well as non-toxic administration of CO, can reduce inflammatory diseases, such as endotoxic shock, type 1 diabetes and graft rejection. Recently, it was shown that the HO-1/CO system can alter the function of antigen-presenting cells (APCs) and reduce T-cell priming, which can be beneficial during immune-driven inflammatory diseases. The molecular mechanisms by which the HO-1 and CO reduce both APC- and T-cell-driven immunity are just beginning to be elucidated. In this article we discuss recent findings related to the immune regulatory capacity of HO-1 and CO at the level of recognition of pathogen-associated molecular patterns and T-cell priming by APCs. Finally, we propose a possible regulatory role for HO-1 and CO over the recently described mitochondria-dependent immunity. These concepts could contribute to the design of new therapeutic tools for inflammation-based diseases. Fil: Riquelme, Sebastián A.. Pontificia Universidad Católica de Chile; Chile Fil: Carreño, Leandro J.. Pontificia Universidad Católica de Chile; Chile Fil: Espinoza, Janyra A.. Pontificia Universidad Católica de Chile; Chile Fil: Mackern Oberti, Juan Pablo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Medicina y Biología Experimental de Cuyo; Argentina. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas; Argentina Fil: Alvarez Lobos, Manuel M.. Pontificia Universidad Católica de Chile; Chile Fil: Riedel, Claudia. Universidad Andrés Bello; Chile Fil: Bueno, Susan M.. Pontificia Universidad Católica de Chile; Chile Fil: Kalergis, Alexis M.. Pontificia Universidad Católica de Chile; Chile

RESUMEN: Aunque se ha logrado un conocimiento amplio acerca de las células T asesinas naturales (iNKT), aún no existe consenso sobre sus mecanismos de activación. Dichas células reconocen diferentes antígenos glicolipídicos presentados por medio de la molécula CD1d, los cuales pueden ser endógenos, exógenos derivados de organismos como bacterias y sintéticos desarrollados para aplicaciones clínicas. Existe mucho interés en entender cómo estas distintas variantes glicolipídicas inducen diferentes tipos de polarización, pero ha sido muy difícil llegar a un consenso, debido a que la respuesta depende de varios factores como la naturaleza, la internalización y el procesamiento de los glicolípidos. Además, la activación de las células iNKT la determinan el tipo y estado de activación de la célula presentadora de antígeno, las moléculas coestimuladoras, los mecanismos de transactivación y la localización de los complejos CD1d-glicolípido en distintas microrregiones de la membrana plasmática, como las balsas lipídicas. Esta revisión explora la evidencia sobre los factores que afectan la activación de las células iNKT con el fin de entender su potencial inmunomodulador. ABSTRACT: A great amount of knowledge on natural killer T cells (iNKTs) is now available, but a consensus about their activation mechanisms has not been reached. These cells recognize different glycolipid antigens through the CD1d molecule. Such antigens may be endogenous, derived from bacteria (foreign) and synthetic, the latter have been developed for clinical applications. There exists much interest in understanding how these different glycolipid compounds induce different types of polarization, but it has been difficult to reach a consensus due to the fact that responses depend on different factors such as: the nature of the molecule, the internalization process and the presentation of the glycolipids. Moreover, activation of iNKT cells is determined by the type and state of the antigen presenting cell, the co-stimulatory molecules, the transactivation mechanisms and the location of the glycolipid-CD1d complexes on the plasma membrane, such as the lipid rafts. This review explores the evidence about the factors that affect activation of iNKT cells in order to understand their immune-modulatory potential.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple functional alterations affecting immune cells, such as B cells, T cells, dendritic cells (DCs) and monocytes. During SLE, the immunogenicity of monocytes and DCs is significantly up-regulated, promoting the activation of self-reactive T cells. Accordingly, it is important to understand the contribution of these cells to the pathogenesis of SLE and the mechanisms responsible for their altered functionality during disease. One of the key enzymes that control monocyte and DC function is haem oxygenase-1 (HO-1), which catalyses the degradation of the haem group into biliverdin, carbon monoxide and free iron. These products possess immunosuppressive and anti-inflammatory capacities. The main goal of this work was to determine HO-1 expression in monocytes and DCs from patients with SLE and healthy controls. Hence, peripheral blood mononuclear cells were obtained from 43 patients with SLE and 30 healthy controls. CD14(+) monocytes and CD4(+) T cells were sorted by FACS and HO-1 expression was measured by RT-PCR. In addition, HO-1 protein expression was determined by FACS. HO-1 levels in monocytes were significantly reduced in patients with SLE compared with healthy controls. These results were confirmed by flow cytometry. No differences were observed in other cell types, such as DCs or CD4(+) T cells, although decreased MHC-II levels were observed in DCs from patients with SLE. In conclusion, we found a significant decrease in HO-1 expression, specifically in monocytes from patients with SLE, suggesting that an imbalance of monocyte function could be partly the result of a decrease in HO-1 expression.

Respiratory syncytial virus (RSV) is the leading cause for childhood hospitalization and respiratory distress, being recognized as a major health and economic burden worldwide. RSV can exploit host immunity and cause a strong inflammatory response that leads to lung damage and virus dissemination. Unfortunately, the immune response elicited by RSV normally fails to protect against subsequent exposures to the virus. Despite intense research during the 50 years after the discovery of RSV, scientists are just beginning to understand the mechanisms contributing to pathology and to the inadequate immune response shown by susceptible individuals. Here, we discuss some of the most important advances made in this field that could lead to the development of new prophylactic tools.

Salmonella enterica serovar Typhimurium can enter non-phagocytic cells, such as intestinal epithelial cells, by virtue of a Type Three Secretion System (TTSS) encoded in the Salmonella Pathogenicity Island 1 (SPI-1), which translocates bacterial effector molecules into the host cell. Salmonella can also be taken up by dendritic cells (DCs). Although the role of SPI-1 in non-phagocytic cell invasion is well established, its contribution to invasion of phagocytic cells has not been evaluated. Here, we have tested the invasive capacity of a S. Typhimurium strain lacking a key component of its TTSS-1 (DeltaInvC) leading to defective translocation of SPI-1-encoded effectors. Whereas this mutant Salmonella strain was impaired for invasion of non-phagocytic cells, it was taken up by DCs at a significantly higher rate than wild-type Salmonella. Similar to wild-type Salmonella, the DeltaInvC mutant strain retained the capacity to avoid antigen presentation to T cells. However, mice infected with the DeltaInvC mutant strain showed higher survival rate and reduced organ colonization. Our data suggest that, besides promoting phagocytosis by non-phagocytic cells, SPI-1 modulates the number of bacteria that enters DCs. The SPI-1 could be considered not only as an inducer of epithelial cell invasion but as a controller of DC entry.

Effector CTL contribute to tumoral immunity by killing tumor cells through secretion of cytotoxic granules and cytokines. Activation of CTL requires specific recognition of cognate peptide-MHC-I (pMHC) complexes on the tumor cell surface by the CTL TCR. It has been suggested that the half-life (t(1/2)) of the TCR/pMHC interaction modulates the activation of naive CD8(+) T cells; however, it remains unknown whether CTL effector function can also be regulated by the TCR/pMHC t(1/2). Here, we have studied CTL activity in response to tumor cells loaded with pMHC that bind the TCR with different t(1/2). We observed that the TCR/pMHC t(1/2) can differentially regulate CTL effector function during the interaction with tumor cells and defines the nature of anti-tumoral CTL responses in vivo. Although prolonged TCR/pMHC t(1/2) promoted only partial expression of cytotoxic molecules, short t(1/2) induced partial polarization of lytic machinery toward target cells. In contrast, intermediate TCR/pMHC t(1/2) induced strong expression of cytotoxic molecules, efficient polarization of lytic machinery and subsequent release of toxic granules by CTL that killed tumor cells. Consistently, efficient in vivo CTL-mediated tumor clearance was only observed for tumors expressing intermediate t(1/2) pMHC ligands. These data suggest that there is an optimal TCR/pMHC t(1/2) for efficient CTL activity.

Infection with Salmonella enterica serovar Typhimurium (S. Typhimurium) causes a severe and lethal systemic disease in mice, characterized by poor activation of the adaptive immune response against Salmonella-derived antigens. Recently, we and others have reported that this feature relies on the ability of S. Typhimurium to survive within murine dendritic cells (DCs) and avoid the presentation of bacteria-derived antigens to T cells. In contrast, here we show that infection of murine DCs with either S. Typhi or S. Enteritidis, two serovars adapted to different hosts, leads to an efficient T-cell activation both in vitro and in vivo. Accordingly, S. Typhi and S. Enteritidis failed to replicate within murine DCs and were quickly degraded, allowing T-cell activation. In contrast, human DCs were found to be permissive for survival and proliferation of S. Typhi, but not for S. Typhimurium or S. Enteritidis. Our data suggest that Salmonella host restriction is characterized by the ability of these bacteria to survive within DCs and avoid activation of the adaptive immune response in their specific hosts.