In pulmonary paracoccidioidomycosis IL-10 deficiency leads to increased immunity and regressive infection without enhancing tissue pathology

BACKGROUND: Cellular immunity is the main defense mechanism in paracoccidioidomycosis (PCM), the most important systemic mycosis in Latin America. Th1 immunity and IFN-γ activated macrophages are fundamental to immunoprotection that is antagonized by IL-10, an anti-inflammatory cytokine. Both in human and experimental PCM, several evidences indicate that the suppressive effect of IL-10 causes detrimental effects to infected hosts. Because direct studies have not been performed, this study was aimed to characterize the function of IL-10 in pulmonary PCM. METHODOLOGY/PRINCIPAL FINDINGS: Wild type (WT) and IL-10(-/-) C57BL/6 mice were used to characterize the role of IL-10 in the innate and adaptive immunity against Paracoccidioides brasiliensis (Pb) infection. We verified that Pb-infected peritoneal macrophages from IL-10(-/-) mice presented higher phagocytic and fungicidal activities than WT macrophages, and these activities were associated with elevated production of IFN-γ, TNF-α, nitric oxide (NO) and MCP-1. For in vivo studies, IL-10(-/-) and WT mice were i.t. infected with 1×10(6) Pb yeasts and studied at several post-infection periods. Compared to WT mice, IL-10(-/-) mice showed increased resistance to P. brasiliensis infection as determined by the progressive control of pulmonary fungal loads and total clearance of fungal cells from dissemination organs. This behavior was accompanied by enhanced delayed-type hypersensitivity reactions, precocious humoral immunity and controlled tissue pathology resulting in increased survival times. In addition, IL-10(-/-) mice developed precocious T cell immunity mediated by increased numbers of lung infiltrating effector/memory CD4(+) and CD8(+) T cells. The inflammatory reactions and the production of Th1/Th2/Th17 cytokines were reduced at late phases of infection, paralleling the regressive infection of IL-10(-/-) mice. CONCLUSIONS/SIGNIFICANCE: Our work demonstrates for the first time that IL-10 plays a detrimental effect to pulmonary PCM due to its suppressive effect on the innate and adaptive immunity resulting in progressive infection and precocious mortality of infected hosts.

Whole blood genome-wide gene expression profile in males after prolonged wakefulness and sleep recovery

Pellegrino R, Sunaga DY, Guindalini C, Martins RC, Mazzotti DR, Wei Z, Daye ZJ, Andersen ML, Tufik S. Whole blood genome-wide gene expression profile in males after prolonged wakefulness and sleep recovery. Physiol Genomics 44: 1003-1012, 2012. First published September 4, 2012; doi: 10.1152/physiolgenomics.00058.2012.-Although the specific functions of sleep have not been completely elucidated, the literature has suggested that sleep is essential for proper homeostasis. Sleep loss is associated with changes in behavioral, neurochemical, cellular, and metabolic function as well as impaired immune response. Using high-resolution microarrays we evaluated the gene expression profiles of healthy male volunteers who underwent 60 h of prolonged wakefulness (PW) followed by 12 h of sleep recovery (SR). Peripheral whole blood was collected at 8 am in the morning before the initiation of PW (Baseline), after the second night of PW, and one night after SR. We identified over 500 genes that were differentially expressed. Notably, these genes were related to DNA damage and repair and stress response, as well as diverse immune system responses, such as natural killer pathways including killer cell lectin-like receptors family, as well as granzymes and T-cell receptors, which play important roles in host defense. These results support the idea that sleep loss can lead to alterations in molecular processes that result in perturbation of cellular immunity, induction of inflammatory responses, and homeostatic imbalance. Moreover, expression of multiple genes was downregulated following PW and upregulated after SR compared with PW, suggesting an attempt of the body to re-establish internal homeostasis. In silico validation of alterations in the expression of CETN3, DNAJC, and CEACAM genes confirmed previous findings related to the molecular effects of sleep deprivation. Thus, the present findings confirm that the effects of sleep loss are not restricted to the brain and can occur intensely in peripheral tissues.

Different degrees of malnutrition and immunological alterations according to the aetiology of cirrhosis: a prospective and sequential study

Abstract Objectives In this work we investigated how immunological dysfunction and malnutrition interact in alcoholic and viral aetiologies of cirrhosis. Methods To investigate the matter, 77 cirrhotic patients divided in three aetiologies [Alcohol, HCV and Alcohol + HCV) and 32 controls were prospectivelly and sequentially studied. Parameters of humoral immunity (Components 3 and 4 of seric complement and immunoglobulins A M, G and E) and of cellular immunity (total leukocytes and lymphocytes in peripheral blood, T lymphocytes subpopulations, CD4+ and CD8+, CD4+/CD8+ ratio and intradermic tests of delayed hypersensitivity), as well as nutrititional parameters: anthropometric measures, serum albumin and transferrin were evaluated. Results Multiple statistical comparisons showed that IgM was higher in HCV group; IgG was significantly elevated in both HCV and Alcohol + HCV, whereas for the Alcohol group, IgE was found at higher titles. The analysis of T- lymphocytes subpopulations showed no aetiologic differences, but intradermic tests of delayed hypersensitivity did show greater frequency of anergy in the Alcohol group. For anthropometric parameters, the Alcohol +HCV group displayed the lowest triceps skinfold whereas creatinine – height index evaluation was more preserved in the HCV group. Body mass index, arm muscle area and arm fat area showed that differently from alcohol group, the HCV group was similar to control. Conclusion Significant differences were found among the main aetiologies of cirrhosis concerning immunological alterations and nutritional status: better nutrition and worse immunology for HCV and vice-versa for alcohol.

DNA vaccine containing the mycobacterial hsp65 gene prevented insulitis in MLD-STZ diabetes

Abstract Background Our group previously demonstrated that a DNA plasmid encoding the mycobacterial 65-kDa heat shock protein (DNA-HSP65) displayed prophylactic and therapeutic effect in a mice model for tuberculosis. This protection was attributed to induction of a strong cellular immunity against HSP65. As specific immunity to HSP60 family has been detected in arthritis, multiple sclerosis and diabetes, the vaccination procedure with DNA-HSP65 could induce a cross-reactive immune response that could trigger or worsen these autoimmune diseases. Methods In this investigation was evaluated the effect of a previous vaccination with DNA-HSP65 on diabetes development induced by Streptozotocin (STZ). C57BL/6 mice received three vaccine doses or the corresponding empty vector and were then injected with multiple low doses of STZ. Results DNA-HSP65 vaccination protected mice from STZ induced insulitis and this was associated with higher production of IL-10 in spleen and also in the islets. This protective effect was also concomitant with the appearance of a regulatory cell population in the spleen and a decreased infiltration of the islets by T CD8+ lymphocytes. The vector (DNAv) also determined immunomodulation but its protective effect against insulitis was very discrete. Conclusion The data presented in this study encourages a further investigation in the regulatory potential of the DNA-HSP65 construct. Our findings have important implications for the development of new immune therapy strategies to combat autoimmune diseases.

Lymphocyte transformation assay for C neoformans antigen is not reliable for detecting cellular impairment in patients with Neurocryptococcosis

Background: Cryptococcus neoformans causes meningitis and disseminated infection in healthy individuals, but more commonly in hosts with defective immune responses. Cell-mediated immunity is an important component of the immune response to a great variety of infections, including yeast infections. We aimed to evaluate a specific lymphocyte transformation assay to Cryptococcus neoformans in order to identify immunodeficiency associated to neurocryptococcosis (NCC) as primary cause of the mycosis. Methods: Healthy volunteers, poultry growers, and HIV-seronegative patients with neurocryptococcosis were tested for cellular immune response. Cryptococcal meningitis was diagnosed by India ink staining of cerebrospinal fluid and cryptococcal antigen test (Immunomycol-Inc, SP, Brazil). Isolated peripheral blood mononuclear cells were stimulated with C. neoformans antigen, C. albicans antigen, and pokeweed mitogen. The amount of H-3-thymidine incorporated was assessed, and the results were expressed as stimulation index (SI) and log SI, sensitivity, specificity, and cut-off value (receiver operating characteristics curve). We applied unpaired Student t tests to compare data and considered significant differences for p<0.05. Results: The lymphotoxin alpha showed a low capacity with all the stimuli for classifying patients as responders and non-responders. Lymphotoxin alpha stimulated by heated-killed antigen from patients with neurocryptococcosis was not affected by TCD4+ cell count, and the intensity of response did not correlate with the clinical evolution of neurocryptococcosis. Conclusion: Response to lymphocyte transformation assay should be analyzed based on a normal range and using more than one stimulator. The use of a cut-off value to classify patients with neurocryptococcosis is inadequate. Statistical analysis should be based on the log transformation of SI. A more purified antigen for evaluating specific response to C. neoformans is needed.

Polymorphic toxin systems: Comprehensive characterization of trafficking modes, processing, mechanisms of action, immunity and ecology using comparative genomics

Background: Proteinaceous toxins are observed across all levels of inter-organismal and intra-genomic conflicts. These include recently discovered prokaryotic polymorphic toxin systems implicated in intra-specific conflicts. They are characterized by a remarkable diversity of C-terminal toxin domains generated by recombination with standalone toxin-coding cassettes. Prior analysis revealed a striking diversity of nuclease and deaminase domains among the toxin modules. We systematically investigated polymorphic toxin systems using comparative genomics, sequence and structure analysis. Results: Polymorphic toxin systems are distributed across all major bacterial lineages and are delivered by at least eight distinct secretory systems. In addition to type-II, these include type-V, VI, VII (ESX), and the poorly characterized "Photorhabdus virulence cassettes (PVC)", PrsW-dependent and MuF phage-capsid-like systems. We present evidence that trafficking of these toxins is often accompanied by autoproteolytic processing catalyzed by HINT, ZU5, PrsW, caspase-like, papain-like, and a novel metallopeptidase associated with the PVC system. We identified over 150 distinct toxin domains in these systems. These span an extraordinary catalytic spectrum to include 23 distinct clades of peptidases, numerous previously unrecognized versions of nucleases and deaminases, ADP-ribosyltransferases, ADP ribosyl cyclases, RelA/SpoT-like nucleotidyltransferases, glycosyltranferases and other enzymes predicted to modify lipids and carbohydrates, and a pore-forming toxin domain. Several of these toxin domains are shared with host-directed effectors of pathogenic bacteria. Over 90 families of immunity proteins might neutralize anywhere between a single to at least 27 distinct types of toxin domains. In some organisms multiple tandem immunity genes or immunity protein domains are organized into polyimmunity loci or polyimmunity proteins. Gene-neighborhood-analysis of polymorphic toxin systems predicts the presence of novel trafficking-related components, and also the organizational logic that allows toxin diversification through recombination. Domain architecture and protein-length analysis revealed that these toxins might be deployed as secreted factors, through directed injection, or via inter-cellular contact facilitated by filamentous structures formed by RHS/YD, filamentous hemagglutinin and other repeats. Phyletic pattern and life-style analysis indicate that polymorphic toxins and polyimmunity loci participate in cooperative behavior and facultative 'cheating' in several ecosystems such as the human oral cavity and soil. Multiple domains from these systems have also been repeatedly transferred to eukaryotes and their viruses, such as the nucleo-cytoplasmic large DNA viruses. Conclusions: Along with a comprehensive inventory of toxins and immunity proteins, we present several testable predictions regarding active sites and catalytic mechanisms of toxins, their processing and trafficking and their role in intra-specific and inter-specific interactions between bacteria. These systems provide insights regarding the emergence of key systems at different points in eukaryotic evolution, such as ADP ribosylation, interaction of myosin VI with cargo proteins, mediation of apoptosis, hyphal heteroincompatibility, hedgehog signaling, arthropod toxins, cell-cell interaction molecules like teneurins and different signaling messengers.

Cellular and humoral immune responses against the Plasmodium vivax MSP-119 malaria vaccine candidate in individuals living in an endemic area in north-eastern Amazon region of Brazil

Abstract Background Plasmodium vivax merozoite surface protein-1 (MSP-1) is an antigen considered to be one of the leading malaria vaccine candidates. PvMSP-1 is highly immunogenic and evidences suggest that it is target for protective immunity against asexual blood stages of malaria parasites. Thus, this study aims to evaluate the acquired cellular and antibody immune responses against PvMSP-1 in individuals naturally exposed to malaria infections in a malaria-endemic area in the north-eastern Amazon region of Brazil. Methods The study was carried out in Paragominas, Pará State, in the Brazilian Amazon. Blood samples were collected from 35 individuals with uncomplicated malaria. Peripheral blood mononuclear cells were isolated and the cellular proliferation and activation was analysed in presence of 19 kDa fragment of MSP-1 (PvMSP-119) and Plasmodium falciparum PSS1 crude antigen. Antibodies IgE, IgM, IgG and IgG subclass and the levels of TNF, IFN-γ and IL-10 were measured by enzyme-linked immunosorbent assay. Results The prevalence of activated CD4+ was greater than CD8+ T cells, in both ex-vivo and in 96 h culture in presence of PvMSP-119 and PSS1 antigen. A low proliferative response against PvMSP-119 and PSS1 crude antigen after 96 h culture was observed. High plasmatic levels of IFN-γ and IL-10 as well as lower TNF levels were also detected in malaria patients. However, in the 96 h supernatant culture, the dynamics of cytokine responses differed from those depicted on plasma assays; in presence of PvMSP-119 stimulus, higher levels of TNF were noted in supernatant 96 h culture of malaria patient’s cells while low levels of IFN-γ and IL-10 were verified. High frequency of malaria patients presenting antibodies against PvMSP-119 was evidenced, regardless class or IgG subclass.PvMSP-119-induced antibodies were predominantly on non-cytophilic subclasses. Conclusions The results presented here shows that PvMSP-119 was able to induce a high cellular activation, leading to production of TNF and emphasizes the high immunogenicity of PvMSP-119 in naturally exposed individuals and, therefore, its potential as a malaria vaccine candidate.

Targeting the non-structural protein 1 from dengue virus to a dendritic cell population confers protective immunity to lethal virus challenge

Dengue is the most prevalent arboviral infection, affecting millions of people every year. Attempts to control such infection are being made, and the development of a vaccine is a World Health Organization priority. Among the proteins being tested as vaccine candidates in preclinical settings is the non-structural protein 1 (NS1). In the present study, we tested the immune responses generated by targeting the NS1 protein to two different dendritic cell populations. Dendritic cells (DCs) are important antigen presenting cells, and targeting proteins to maturing DCs has proved to be an efficient means of immunization. Antigen targeting is accomplished by the use of a monoclonal antibody (mAb) directed against a DC cell surface receptor fused to the protein of interest. We used two mAbs (αDEC205 and αDCIR2) to target two distinct DC populations, expressing either DEC205 or DCIR2 endocytic receptors, respectively, in mice. The fusion mAbs were successfully produced, bound to their respective receptors, and were used to immunize BALB/c mice in the presence of polyriboinosinic: polyribocytidylic acid (poly (I:C)), as a DC maturation stimulus. We observed induction of strong anti-NS1 antibody responses and similar antigen binding affinity irrespectively of the DC population targeted. Nevertheless, the IgG1/IgG2a ratios were different between mouse groups immunized with αDEC-NS1 and αDCIR2-NS1 mAbs. When we tested the induction of cellular immune responses, the number of IFN-γ producing cells was higher in αDEC-NS1 immunized animals. In addition, mice immunized with the αDEC-NS1 mAb were significantly protected from a lethal intracranial challenge with the DENV2 NGC strain when compared to mice immunized with αDCIR2-NS1 mAb. Protection was partially mediated by CD4(+) and CD8(+) T cells as depletion of these populations reduced both survival and morbidity signs. We conclude that targeting the NS1 protein to the DEC205(+) DC population with poly (I:C) opens perspectives for dengue vaccine development.

Endotoxin exposure during sensitization to Blomia tropicalis allergens shifts TH2 immunity towards a TH17-mediated airway neutrophilic inflammation: role of TLR4 and TLR2

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation.