Bruno B. Andrade

Host-directed therapy of tuberculosis based on interleukin-1 and type I interferon crosstalk

Tuberculosis remains second only to HIV/AIDS as the leading cause of mortality worldwide due to a single infectious agent. Despite chemotherapy, the global tuberculosis epidemic has intensified because of HIV co-infection, the lack of an effective vaccine and the emergence of multi-drug-resistant bacteria. Alternative host-directed strategies could be exploited to improve treatment efficacy and outcome, contain drug-resistant strains and reduce disease severity and mortality. The innate inflammatory response elicited by Mycobacterium tuberculosis (Mtb) represents a logical host target. Here we demonstrate that interleukin-1 (IL-1) confers host resistance through the induction of eicosanoids that limit excessive type I interferon (IFN) production and foster bacterial containment. We further show that, in infected mice and patients, reduced IL-1 responses and/or excessive type I IFN induction are linked to an eicosanoid imbalance associated with disease exacerbation. Host-directed immunotherapy with clinically approved drugs that augment prostaglandin E2 levels in these settings prevented acute mortality of Mtb-infected mice. Thus, IL-1 and type I IFNs represent two major counter-regulatory classes of inflammatory cytokines that control the outcome of Mtb infection and are functionally linked via eicosanoids. Our findings establish proof of concept for host-directed treatment strategies that manipulate the host eicosanoid network and represent feasible alternatives to conventional chemotherapy.

Innate and adaptive interferons suppress IL-1α and IL-1β production by distinct pulmonary myeloid subsets during Mycobacterium tuberculosis infection.

Interleukin-1 (IL-1) receptor signaling is necessary for control of Mycobacterium tuberculosis (Mtb) infection, yet the role of its two ligands, IL-1α and IL-1β, and their regulation in vivo are poorly understood. Here, we showed that both IL-1α and IL-1β are critically required for host resistance and identified two multifunctional inflammatory monocyte-macrophage and DC populations that coexpressed both IL-1 species at the single-cell level in lungs of Mtb-infected mice. Moreover, we demonstrated that interferons (IFNs) played important roles in regulating IL-1 production by these cells in vivo. Type I interferons inhibited IL-1 production by both subsets whereas CD4(+) T cell-derived IFN-γ selectively suppressed monocyte-macrophages. These data provide a cellular basis for both the anti-inflammatory effects of IFNs and probacterial functions of type I IFNs during Mtb infection and reveal differential regulation of IL-1 production by distinct cell populations as an additional layer of complexity in the activity of IL-1 in vivo.

Severe Plasmodium vivax malaria exhibits marked inflammatory imbalance

BackgroundDespite clinical descriptions of severe vivax malaria cases having been reported, data regarding immunological and inflammatory patterns are scarce. In this report, the inflammatory and immunological status of both mild and severe vivax malaria cases are compared in order to explore immunopathological events in this disease.Methods and ResultsActive and passive malaria case detections were performed during 2007 in Buritis, Rondônia, in the Brazilian Amazon. A total of 219 participants enrolled the study. Study individuals were classified according to the presence of Plasmodium vivax infection within four groups: non-infected (n = 90), asymptomatic (n = 60), mild (n = 50) and severe vivax infection (n = 19). A diagnosis of malaria was made by microscopy and molecular assays. Since at present no clear criteria define severe vivax malaria, this study adapted the consensual criteria from falciparum malaria. Patients with severe P. vivax infection were younger, had lived for shorter time in the endemic area, and recalled having experienced less previous malaria episodes than individuals with no malaria infection and with mild or asymptomatic infection. Strong linear trends were identified regarding increasing plasma levels of C reactive protein (CRP), serum creatinine, bilirubins and the graduation of disease severity. Plasma levels of tumour necrosis factor (TNF), interferon-gamma(IFN-gamma) and also IFN-gamma/interleukin-10 ratios were increased and exhibited a linear trend with gradual augmentation of disease severity. Both laboratory parameters of organ dysfunction and inflammatory cytokines were reduced during anti-parasite therapy in those patients with severe disease.ConclusionDifferent clinical presentations of vivax malaria infection present strong association with activation of pro-inflammatory responses and cytokine imbalance. These findings are of utmost importance to improve current knowledge about physiopathological concepts of this serious widespread disease.

Immune reconstitution inflammatory syndrome: the trouble with immunity when you had none

Some individuals who are infected with HIV rapidly deteriorate shortly after starting antiretroviral therapy, despite effective viral suppression. This reaction, referred to as immune reconstitution inflammatory syndrome (IRIS), is characterized by tissue-destructive inflammation and arises as CD4+ T cells re-emerge. It has been proposed that IRIS is caused by a dysregulation of the expanding population of CD4+ T cells specific for a co-infecting opportunistic pathogen. Here, we argue that IRIS instead results from hyper-responsiveness of the innate immune system to T cell help, a mechanism that may be shared by the many manifestations of IRIS that occur following the reversal of other types of immunosuppression in pathogen-infected hosts.

Role of Sand Fly Saliva in Human and Experimental Leishmaniasis: Current Insights

Leishmaniases are wide spread diseases transmitted to their vertebrate host by infected sand fly. The saliva from these arthropods contains a vast repertoire of pharmacologically active molecules that hampers the host’s haemostatic, inflammatory and immune responses. The early interactions between Leishmania and the host’s immune response are closely linked to disease evolution or protection against the protozoan, and the ectoparasite saliva contributes directly to these interactions. Current studies have depicted these features, and these relations are being widely explored. There are concrete indications that the host response against sand fly saliva influences disease outcome in leishmaniasis. Additionally, there are demonstrations that immunization with whole sand fly saliva, or its components, leads to protection against leishmaniasis in different host species. The combination of these evidences opens up optimistic perspectives for improving vaccine development against Leishmania infection.

Human anti-saliva immune response following experimental exposure to the visceral leishmaniasis vector, Lutzomyia longipalpis

Experiments in animals verified that phlebotomine saliva enhances Leishmania infection, and vaccination with saliva prevents disease. We have shown that individuals from an endemic area of visceral leishmaniasis displayed robust antibody responses to saliva from the vector Lutzomyia longipalpis, which correlated with anti‐parasite cell‐mediated immunity. Here, we explored human anti‐saliva responses following exposure to sand flies, using an in vivo bite model in which normal volunteers were exposed four times to 30 laboratory‐reared Lu. longipalpis. Following the third exposure, normal volunteers developed diverse dermatological reactions at the site of insect bite. Serum from normal volunteers displayed high levels of anti‐salivary gland sonicate IgG1, IgG4 and IgE as well as several salivary gland proteins. Furthermore, following in vitro stimulation with salivary gland sonicate, there was an increased frequency of CD4+CD25+ and CD8+CD25+ T cells as well as IFN‐γ and IL‐10 synthesis. Strikingly, 1 year after the first exposure, PBMC from the volunteers displayed recall IFN‐γ responses that correlated with a significant reduction in infection rates using a macrophage‐lymphocyte autologous culture. Together, these data suggest that human immunization against sand fly saliva is feasible and recall responses are obtained even 1 year after exposure, opening perspectives for vaccination in man.

Metabolic Adaptation to Tissue Iron Overload Confers Tolerance to Malaria

Disease tolerance is a defense strategy that limits the fitness costs of infection irrespectively of pathogen burden. While restricting iron (Fe) availability to pathogens is perceived as a host defense strategy, the resulting tissue Fe overload can be cytotoxic and promote tissue damage to exacerbate disease severity. Examining this interplay during malaria, the disease caused by Plasmodium infection, we find that expression of the Fe sequestering protein ferritin H chain (FtH) in mice, and ferritin in humans, is associated with reduced tissue damage irrespectively of pathogen burden. FtH protection relies on its ferroxidase activity, which prevents labile Fe from sustaining proapoptotic c-Jun N-terminal kinase (JNK) activation. FtH expression is inhibited by JNK activation, promoting tissue Fe overload, tissue damage, and malaria severity. Mimicking FtHs antioxidant effect or inhibiting JNK activation pharmacologically confers therapeutic tolerance to malaria in mice. Thus, FtH provides metabolic adaptation to tissue Fe overload, conferring tolerance to malaria.

Using recombinant proteins from Lutzomyia longipalpis saliva to estimate human vector exposure in visceral Leishmaniasis endemic areas.

Background Leishmania is transmitted by female sand flies and deposited together with saliva, which contains a vast repertoire of pharmacologically active molecules that contribute to the establishment of the infection. The exposure to vector saliva induces an immune response against its components that can be used as a marker of exposure to the vector. Performing large-scale serological studies to detect vector exposure has been limited by the difficulty in obtaining sand fly saliva. Here, we validate the use of two sand fly salivary recombinant proteins as markers for vector exposure. Methodology/principal findings ELISA was used to screen human sera, collected in an area endemic for visceral leishmaniasis, against the salivary gland sonicate (SGS) or two recombinant proteins (rLJM11 and rLJM17) from Lutzomyia longipalpis saliva. Antibody levels before and after SGS seroconversion (n = 26) were compared using the Wilcoxon signed rank paired test. Human sera from an area endemic for VL which recognize Lu. longipalpis saliva in ELISA also recognize a combination of rLJM17 and rLJM11. We then extended the analysis to include 40 sera from individuals who were seropositive and 40 seronegative to Lu. longipalpis SGS. Each recombinant protein was able to detect anti-saliva seroconversion, whereas the two proteins combined increased the detection significantly. Additionally, we evaluated the specificity of the anti-Lu. longipalpis response by testing 40 sera positive to Lutzomyia intermedia SGS, and very limited (2/40) cross-reactivity was observed. Receiver-operator characteristics (ROC) curve analysis was used to identify the effectiveness of these proteins for the prediction of anti-SGS positivity. These ROC curves evidenced the superior performance of rLJM17+rLJM11. Predicted threshold levels were confirmed for rLJM17+rLJM11 using a large panel of 1,077 serum samples. Conclusion Our results show the possibility of substituting Lu. longipalpis SGS for two recombinant proteins, LJM17 and LJM11, in order to probe for vector exposure in individuals residing in endemic areas.

Saliva from Lutzomyia longipalpis Induces CC Chemokine Ligand 2/Monocyte Chemoattractant Protein-1 Expression and Macrophage Recruitment

Saliva of bloodfeeding arthropods has been incriminated in facilitating the establishment of parasite in their host. We report on the leukocyte chemoattractive effect of salivary gland homogenate (SGH) from Lutzomyia longipalpis on saliva-induced inflammation in an air pouch model. SGH (0.5 pair/animal) was inoculated in the air pouch formed in the back of BALB/c or C57BL/6 mice. L. longipalpis SGH induced a significant influx of macrophages in BALB/c but not in C57BL/6 mice. SGH-induced cell recruitment reached a peak at 12 h after inoculation and was higher than that induced by the LPS control. This differential cell recruitment in BALB/c mice was directly correlated to an increase in CCL2/MCP-1 expression in the air pouch lining tissue. In fact, treatment with bindarit, an inhibitor of CCL2/MCP-1 synthesis, and also with a specific anti-MCP-1 mAb resulted in drastic reduction of macrophage recruitment and inhibition of CCL2/MCP-1 expression in the lining tissue. CCL2/MCP-1 production was also seen in vitro when J774 murine macrophages were exposed to L. longipalpis SGH. The SGH effect was abrogated by preincubation with serum containing anti-SGH IgG Abs as well as in mice previously sensitized with L. longipalpis bites. Interestingly, the combination of SGH with Leishmania chagasi induced an increased recruitment of neutrophils and macrophages when compared with L. chagasi alone. Taken together these results suggest that SGH not only induces the recruitment of a greater number of macrophages by enhancing CCL2/MCP-1 production but also synergizes with L. chagasi to recruit more inflammatory cells to the site of inoculation.

Towards a precise test for malaria diagnosis in the Brazilian Amazon: comparison among field microscopy, a rapid diagnostic test, nested PCR, and a computational expert system based on artificial neural networks

BackgroundAccurate malaria diagnosis is mandatory for the treatment and management of severe cases. Moreover, individuals with asymptomatic malaria are not usually screened by health care facilities, which further complicates disease control efforts. The present study compared the performances of a malaria rapid diagnosis test (RDT), the thick blood smear method and nested PCR for the diagnosis of symptomatic malaria in the Brazilian Amazon. In addition, an innovative computational approach was tested for the diagnosis of asymptomatic malaria.MethodsThe study was divided in two parts. For the first part, passive case detection was performed in 311 individuals with malaria-related symptoms from a recently urbanized community in the Brazilian Amazon. A cross-sectional investigation compared the diagnostic performance of the RDT Optimal-IT, nested PCR and light microscopy. The second part of the study involved active case detection of asymptomatic malaria in 380 individuals from riverine communities in Rondônia, Brazil. The performances of microscopy, nested PCR and an expert computational system based on artificial neural networks (MalDANN) using epidemiological data were compared.ResultsNested PCR was shown to be the gold standard for diagnosis of both symptomatic and asymptomatic malaria because it detected the major number of cases and presented the maximum specificity. Surprisingly, the RDT was superior to microscopy in the diagnosis of cases with low parasitaemia. Nevertheless, RDT could not discriminate the Plasmodium species in 12 cases of mixed infections (Plasmodium vivax + Plasmodium falciparum). Moreover, the microscopy presented low performance in the detection of asymptomatic cases (61.25% of correct diagnoses). The MalDANN system using epidemiological data was worse that the light microscopy (56% of correct diagnoses). However, when information regarding plasma levels of interleukin-10 and interferon-gamma were inputted, the MalDANN performance sensibly increased (80% correct diagnoses).ConclusionsAn RDT for malaria diagnosis may find a promising use in the Brazilian Amazon integrating a rational diagnostic approach. Despite the low performance of the MalDANN test using solely epidemiological data, an approach based on neural networks may be feasible in cases where simpler methods for discriminating individuals below and above threshold cytokine levels are available.