Targeting the Annexin A1-FPR2/ALX pathway for host-directed therapy in dengue disease.

Host immune responses contribute to dengue's pathogenesis and severity, yet the possibility that failure in endogenous inflammation resolution pathways could characterise the disease has not been contemplated. The pro-resolving protein Annexin A1 (AnxA1) is known to counterbalance overexuberant inflammation and mast cell (MC) activation. We hypothesised that inadequate AnxA1 engagement underlies the cytokine storm and vascular pathologies associated with dengue disease. Levels of AnxA1 were examined in the plasma of dengue patients and infected mice. Immunocompetent, interferon (alpha and beta) receptor one knockout (KO), AnxA1 KO, and formyl peptide receptor 2 (FPR2) KO mice were infected with dengue virus (DENV) and treated with the AnxA1 mimetic peptide Ac2-26 for analysis. In addition, the effect of Ac2-26 on DENV-induced MC degranulation was assessed in vitro and in vivo. We observed that circulating levels of AnxA1 were reduced in dengue patients and DENV-infected mice. Whilst the absence of AnxA1 or its receptor FPR2 aggravated illness in infected mice, treatment with AnxA1 agonistic peptide attenuated disease manifestationsatteanuated the symptoms of the disease. Both clinical outcomes were attributed to modulation of DENV-mediated viral load-independent MC degranulation. We have thereby identified that altered levels of the pro-resolving mediator AnxA1 are of pathological relevance in DENV infection, suggesting FPR2/ALX agonists as a therapeutic target for dengue disease.

Interleukin-32γ in the Control of Acute Experimental Chagas Disease.

Chagas disease (CD) is an important parasitic disease caused by Trypanosoma cruzi. Interleukin-32 (IL-32) plays an important role in inflammation and in the development of Th1/Th17 acquired immune responses. We evaluated the influence of IL-32γ on the immune response profile, pathogenesis of myocarditis in acute experimental CD, and control of the disease. For this, C57BL/6 wild-type (WT) and IL-32γTg mice were infected subcutaneously with 1,000 forms of Colombian strain of T. cruzi. In the histopathological analyzes, T. cruzi nests, myocarditis, and collagen were quantified in cardiac tissue. Cytokine productions (IL-32, IFN-γ, TNF-α, IL-10, and IL-17) were measured in cardiac homogenate by ELISA. The IL-32γTg mice showed a better control of parasitemia and T. cruzi nests in the heart than WT mice. Infected-WT and -IL-32γTg mice showed similar levels of IFN-γ, TNF-α, and IL-17, but IL-10 was significantly higher expressed in IL-32γTg than in WT mice. The cytokine profile found in IL-32γTg animals contributed to body weight maintenance, parasitemia control, and survival. Our results indicate that the presence of human IL-32γ in mice infected with the Colombian strain of T. cruzi is important for infection control during the acute phase of Chagas disease.

In-depth characterization of congenital Zika syndrome in immunocompetent mice: Antibody-dependent enhancement and an antiviral peptide therapy.

BACKGROUND: Zika virus (ZIKV) infection during pregnancy may cause major congenital defects, including microcephaly, ocular, articular and muscle abnormalities, which are collectively defined as Congenital Zika Syndrome. Here, we performed an in-depth characterization of the effects of congenital ZIKV infection (CZI) in immunocompetent mice. METHODS: Pregnant dams were inoculated with ZIKV on embryonic day 5.5 in the presence or absence of a sub-neutralizing dose of a pan-flavivirus monoclonal antibody (4G2) to evaluate the potential role of antibody-dependent enhancement phenomenon (ADE) during short and long outcomes of CZI. FINDINGS: ZIKV infection induced maternal immune activation (MIA), which was associated with occurrence of foetal abnormalities and death. Therapeutic administration of AH-D antiviral peptide during the early stages of pregnancy prevented ZIKV replication and death of offspring. In the post-natal period, CZI was associated with a decrease in whole brain volume, ophthalmologic abnormalities, changes in testicular morphology, and disruption in bone microarchitecture. Some alterations were enhanced in the presence of 4G2 antibody. INTERPRETATION: Our results reveal that early maternal ZIKV infection causes several birth defects in immunocompetent mice, which can be potentiated by ADE phenomenon and are associated with MIA. Additionally, antiviral treatment with AH-D peptide may be beneficial during early maternal ZIKV infection. FUND: This work was supported by the Brazilian National Science Council (CNPq, Brazil), Minas Gerais Foundation for Science (FAPEMIG), Funding Authority for Studies and Projects (FINEP), Coordination of Superior Level Staff Improvement (CAPES), National Research Foundation of Singapore and Centre for Precision Biology at Nanyang Technological University.

Taenia crassiceps antigens induce a Th2 immune response and attenuate injuries experimentally induced by neurotoxoplasmosis in BALB/c mice.

Toxoplasma gondii is a pathogenic agent responsible for causing both systemic and local disease which elicits a typically pro-inflammatory, Th1 immune response. Taenia crassiceps antigen induces a Th2 immune response that immunomodulates Th1 based infections. Therefore the aim of this study was to evaluate whether T. crassiceps cysticerci antigens are able to modulate the inflammatory response triggered in experimental neurotoxoplasmosis (NT). BALB/c mice were inoculated with T. gondii cysts and/or cysticerci antigens and euthanized at 60 and 90days after inoculation (DAI). The histopathology of the brains and cytokines produced by spleen cells culture were performed. The animals from the NT group, 90DAI (NT90), presented greater intensity of lesions such as vasculitis, meningitis and microgliosis and cytokines from Th1 profile characterized by high levels of IFN-gamma. While in the T. crassiceps antigens group, 60DAI, there were more discrete lesions and high levels of IL-4, a Th2 cytokine. In the NT co-inoculated with cysticerci antigens group the parenchyma lesions were more discrete with lower levels of IFN-gamma and higher levels of IL-4 when compared to NT90. Therefore the inoculation of T. crassiceps antigens attenuated the brain lesions caused by T. gondii inducing a Th2 immune response.

IFN-gamma role in granuloma formation in experimental subcutaneous cysticercosis.

Cysticercosis is an infection caused by the metacestode larval stage of Taenia parasites in tissues and elicits a host-parasite reaction in which the immune response may be decisive in the disease development. The aim of this study was to evaluate the role of IFNγ (IFN-gamma) in the experimental model of subcutaneous infection with Taenia crassiceps (T. crassiceps) cysticerci using IFNγ knockout mice. Male C57BL/6 and C57BL/6 KO IFNγ mice 8-12 weeks of age were inoculated with T. crassiceps cysticerci into the subcutaneous tissue of the dorsum. At 7 and 30 (acute phase), 60 and 90 (chronic phase) days post infection, animals from each group had their blood and the subcutaneous tissues collected for serologic and pathological studies. IFNγ and IL-4 were dosed and the histopathological analysis was performed. In the presence of IFNγ there was the establishment of a mixed Th1/Th2 systemic immune profile. This profile also locally induced the granuloma formation which was constituted by cells that played important roles in the parasitary destruction and that were likely associated to the Th1 axis of mixed immune response. On the other hand, the absence of IFNγ appears to favor the parasitary growth which may be related to the development of a systemic Th2 immune response. This profile influenced the granuloma formation with immunoregulatory properties and appears to be important in the collagen synthesis.

Leishmania (Viannia) braziliensis amastigotes from patients with mucosal leishmaniasis have increased ability to disseminate and are controlled by nitric oxide at the early stage of murine infection.

Mucosal leishmaniasis (ML) caused by Leishmania (Vianna) braziliensis usually appears after the healing of the primary lesion when amastigotes disseminate from the infection site to the mucosal area. Here, we investigated murine infection with amastigotes obtained from patients with ML or localized cutaneous leishmaniasis (LCL). Amastigotes were used to infect wild type, IFN-γ KO and inducible nitric oxide synthase (iNOS) KO mice. Amastigotes from patients with LCL induced lesions that appeared earlier in IFN-γ KO than parasites from ML. The lesion after infection with ML appeared early in iNOS KO than in IFN-γ KO mice and in iNOS KO mice parasites from ML and LCL cause similar lesions at the initial phase of infection, while parasites from ML induced greater lesions than the ones from LCL at the late phase. A greater number of parasites were observed in spleen of IFN-γ KO and iNOS KO mice infected with amastigotes from patients with ML than those with LCL. Parasites from ML infect a lower percentage of macrophages and are killed independent on IFN-γ and dependent on NO. The data suggest that amastigotes responsible for mucosal lesion in humans develop slowly on the initial phase of infection due to high susceptibility to NO and they have an increased ability to disseminate.

Essential role of leukotriene B4 on Leishmania (Viannia) braziliensis killing by human macrophages.

Although Leishmania (Viannia) braziliensis is the most prevalent species that cause American tegumentary leishmaniasis (ATL), the immune response against this parasite has been poorly investigated. Upon activation, macrophages produce a series of pro-inflammatory molecules, including the lipid mediator leukotriene B4 (LTB4). LTB4 has been shown to enhance several macrophage functions, but its role in human macrophages is less known. Here, we investigated the role of LTB4 on human monocyte-derived macrophages infected with human isolate of L. (V.) braziliensis (IMG3). It was found that human macrophages produce LTB4 upon infection with Leishmania, which by autocrine or paracrine activation of its high affinity receptor BLT1, potentiates macrophage leishmanicidal activity. This LTB4 effect is mediated by increased secretion of reactive oxygen species (ROS). Moreover, Leishmania infection decreased the expression of BLT1, leading to the speculation that this could represent a parasite escape mechanism to establish a chronic inflammatory infection. Therefore, our data suggest that LTB4 could be used in therapeutic strategies to control Leishmania infection.

Arginase activity is associated with fibrosis in experimental infection with Taenia crassiceps, but does not play a major role in resistance to infection.

Murine infection with Taenia crassiceps cysticerci is used as an experimental model for human and animal cysticercosis. In this infection parasites can be found associated with an inflammatory infiltrate enriched with macrophages. Experimental evidence exists supporting a role for either NO-producing classically activated (CAMΦ) or arginase- and CD301-expressing alternatively activated macrophages (AAMΦ) in T. crassiceps resistance. In both cell types, arginine is utilized as an important mediator in macrophage effector functions. To investigate whether there is an association between arginine availability, susceptibility to T. crassiceps and other parameters such as fibrosis, BALB/c mice were infected intraperitoneally with cysticerci and treated daily with the arginase inhibitor nor-NOHA or supplemented with l-arginine and followed for eight weeks. The numbers and developmental stages of parasites were evaluated as well as the presence of CD301+ AAMΦ, arginase activity and collagen deposition in the peritoneal membrane. Treatment with the arginase inhibitor or supplementation with l-arginine did not change the parasitic load or profile of the infection. However, the arginase inhibitor significantly decreased the deposition of collagen. These results suggest that arginase activity does not interfere with parasite control during experimental infection with T. crassiceps, but it is important for fibrosis in cysticercosis.

Ecto-nucleotidase activities of promastigotes from Leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome.

BACKGROUND: Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. CONCLUSIONS/SIGNIFICANCE: Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.

Improvements in obtaining New World Leishmania sp from mucosal lesions: notes on isolating and stocking parasites.

Tegumentary leishmaniasis is an endemic protozoan disease that, in Brazil, is caused by parasites from Viannia or Leishmania complex. The clinical forms of cutaneous disease comprise localized, disseminated, mucosal or mucocutaneous, and diffuse leishmaniasis. Viannia complex parasites are not easy to isolate from patient lesions, especially from mucosal lesions, and they are difficult to culture. The aim of the present study was to compare the efficiency of ex vivo (culture) and in vivo (IFNγ-deficient mice) parasite isolation methods to improve the isolation rate and storage of stocks of New World Leishmania sp that cause cutaneous leishmaniasis (CL) or mucosal leishmaniasis (ML). Biopsy fragments from cutaneous or mucosal lesions were inoculated into culture medium or mouse footpads. We evaluated 114 samples (86 CL, 28 ML) using both methods independently. Samples from CL patients had a higher isolation rate in ex vivo cultures than in mice (34.1% vs. 18.7%, P<0.05). Nevertheless, almost twice the number of isolates from ML lesions was isolated using the mouse model compared to ex vivo cultures (mouse, 6/25; culture, 3/27). The overall rates of isolation were 40.2% for CL samples and 29.6% for ML samples. Of the 43 isolations, we successfully stocked 35 isolates (81.4%; 27 CL, 8 ML). Contaminations were more frequently detected in cultures of ML than CL lesions. For comparison, the use of both methods simultaneously was performed in 74 samples of CL and 25 samples of ML, and similar results were obtained. Of the eight ML isolates, five were isolated only in mice, indicating the advantage of using the in vivo method to obtain ML parasites. All parasites obtained from in vivo isolation were cryopreserved, whereas only 68% of ex vivo isolations from CL lesions were stocked. In conclusion, the use of genetically modified mice can improve the isolation of parasites from ML. Isolation and stocking of New World Leishmania parasites, especially those from ML that are almost absent in laboratory stocks, are critical for evaluating parasite genetic diversity as well as studying host-parasite interactions to identify biological markers of Leishmania. In this paper, we also discuss some of the difficulties associated with isolating and stocking parasites.

Energetic metabolism of axenic promastigotes of Leishmania (Viannia) braziliensis.

Leishmania spp are protozoans capable of carbohydrates degradation and as energy source they can use glucose, aminoacids or lipids from the environment. The products of the metabolic pathways such as organic acids may be used as an index of their energetic metabolic profile. Therefore, in this study a metabolic profile comparison was made between promastigotes from one reference strain (MHOM/BR/1975/M2903) and two different isolates of Leishmania (Viannia) braziliensis (MHOM/BR/2003/IMG3 and MHOM/BR/2005/RPL5). The parasites culture was performed in complete Grace's culture media seeded in 24-well plates at 26°C. During the growth curve performance samples were collected from the logarithmic and stationary phases of culture and therefore analyzed by high performance liquid chromatography (HPLC) and spectrophotometry assays to determine the concentrations of glucose, lactate, citrate, α-ketoglutarate, succinate, fumarate, malate, oxaloacetate and ß-hydroxybutirate which are indicative of the energetic pathways. It was possible to detect an increase in the glucose from the stationary phase from the M2903 strain when compared to the logarithmic phase while in the IMG3 and RPL5 isolates there was a decrease (p<0.05). The spectrophotometric and chromatographic results indicated that the logarithmic phase which presents higher energy consumption due to the intense replication rate have the energetic pathways intensified. It was also possible to note some metabolic differences between the analyzed parasites which may indicate possible adaptations of the parasite when facing different environmental and physiological conditions during its life cycle and that these differences may help in the understanding of the diversity of the host-parasite relationship from Leishmania parasites.

Morphological alterations and growth inhibition of Leishmania (L.)amazonensis promastigotes exposed to zidovudine (AZT).

Leishmania parasites cause a worldwide public health disease and its treatment is still based on pentavalent antimonials which present financial and toxicologic limitations. Some nucleosidic derivatives have demonstrated anti-leishmanial properties and this study aims to evaluate the in vitro morphologic alterations and growth inhibition of Leishmania (L.) amazonensis promastigotes exposed to zidovudine at several concentrations. The citotoxicity of zidovudine (AZT) to macrophages was determined by an MTT assay. After which the promastigotes were exposed to concentrations of AZT, ranging from 1 to 50 µM. The evaluation of survival and morphometry alterations were performed in two distinct phases of in vitro growth, on the third and sixth days, representing the logarithmic and stationary phases, respectively. Slides with the promastigotes were photographed and analyzed using Image J. A significant reduction of parasite number in the logarithmic phase of in vitro growth was observed when the parasites were submitted to 20, 30, 40, and 50 µM of AZT. Morphometric alterations were observed such as an increase in width of the body, cytoplasmic granulations and vacuolizations. These data indicate the toxicity of AZT which prevents the parasite's multiplication, indicating a promising use of AZT as an anti-leishmania drug.

Mycoplasmal lipid-associated membrane proteins and Mycoplasma arthritidis mitogen recognition by serum antibodies from patients with rheumatoid arthritis.

Mycoplasmal lipid-associated membrane proteins (LAMPs) and Mycoplasma arthritidis mitogen (MAM superantigen) are potent stimulators of the immune system. The objective of this work was to detect antibodies to MAM and LAMPs of Mycoplasma hominis and M. fermentans in the sera of patients affected by rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) to identify mycoplasmal products that can be involved in the etiopathogenesis of these autoimmune diseases. Serum samples from female RA and SLE patients and controls, recombinant MAM, and LAMPs of M. hominis PG21 and M. fermentans PG18 were used in Western blot assays. A similar frequency of sera from patients and controls reactive to MAM was detected. A larger number of M. hominis and M. fermentans LAMPs were recognized by sera from RA patients than controls, but no differences were detected between sera from SLE patients and controls. Among the LAMPs recognized by IgG antibodies from RA patients, proteins of molecular masses in a range of <49 and ≥20 KDa (M. hominis) and <102 and ≥58 KDa (M. fermentans) were the most reactive. These preliminary results demonstrate the strong reactivity of antibodies of RA patients with some M. hominis and M. fermentans LAMPs. These LAMPs could be investigated as mycoplasmal antigens that can take part in the induction or amplification of human autoimmune responses.

Increase of NK cells and proinflammatory monocytes are associated with the clinical improvement of diffuse cutaneous leishmaniasis after immunochemotherapy with BCG/Leishmania antigens.

Diffuse cutaneous leishmaniasis (DCL) is characterized by disseminated lesions and the absence of a specific cellular immune response. Here, the immunochemotherapy outcome of a patient with DCL from Amazonian Brazil infected with Leishmania (Leishmania) amazonensis is presented. After several unsuccessful chemotherapy treatment regimens and many relapses, a monthly immunotherapy scheme of L. amazonensis PH8 plus L. (Viannia) braziliensis M2903 monovalent vaccines associated with Bacillus Calmette-Guerin (BCG) was established, one round of which also included an M2903 vaccine associated with intermittent antimonial treatment. Temporary healing of all lesions was achieved, although Leishmania skin tests were negative and interferon gamma was not detected in mononuclear cell cultures stimulated with Leishmania antigens. The frequencies of CD16 (+)CD56(+) NK cells (approximately 2x) and CD14 (+)CD16(+) proinflammatory monocytes (approximately 8x) increased in peripheral blood, and CD56 (+) lymphocytes were found infiltrating the lesions. An association between the increase of the frequency of innate immune system cells and the healing of lesions is shown, suggesting that this protocol of immunotherapy reduced the parasite load and activated NK cells and monocytes.

Macrophages at intermediate stage of maturation produce high levels of IL-12 p40 upon stimulation with Leishmania.

IL-12 is one of the main cytokines driving the immune response to a resistant phenotype in leishmaniasis and in several other diseases involving intracellular microbes. In this study, we investigated IL-12 production by mononuclear phagocytes at several developmental stages when stimulated with Leishmania major, L. amazonensis or L. chagasi. Bone marrow cells were cultured for 4-6 days in vitro in the presence of M-CSF, GM-CSF or IL-3. After density separation, only cells banding at the 40-50% Percoll interface, but not those at 20-40% or 50-80% interfaces, produced large amounts of IL-12 p40 when stimulated with LPS or live Leishmania promastigotes. However, only low levels of IL-12 p70 were produced under these conditions. The high IL-12 p40-producing cells could be similarly derived from mouse strains with different susceptibility to Leishmania. Quantitative analysis of monocyte/macrophage lineage marker expression, in combination with positive and negative selection, led to the conclusion that the high IL-12 p40-producing cells are macrophages at an intermediate stage of maturation between immature and fully differentiated cells, expressing ER-HR3 but only low levels of the mature markers, scavenger receptor and CD11b/Mac-1. They do not express any of the precursor markers CD31/ER-MP12, Ly-6C/ER-MP20 or ER-MP58. Because recruitment of monocytes to an infection site and its draining lymph node is a general phenomenon, the notion that, developing from these monocytes, a population of mononuclear phagocytes at an intermediate maturation stage has the capacity to synthesize large amounts of IL-12 p40 has significant bearing on our understanding of immune regulation in leishmaniasis and also in infections by other pathogens.