Pentoxifylline reduces inflammation and prevents myocardial perfusion derangements in experimental chronic Chagas' cardiomyopathy.

BACKGROUND: Myocardial perfusion defect (MPD) is common in chronic Chagas cardiomyopathy (CCC) and is associated with inflammation and development of left ventricular systolic dysfunction. We tested the hypothesis that pentoxifylline (PTX) could reduce inflammation and prevent the development of MPD in a model of CCC in hamsters. METHODS AND RESULTS: We investigated with echocardiogram and rest myocardial perfusion scintigraphy at baseline (6-months after T. cruzi infection/saline) and post-treatment (after additional 2-months of PTX/saline administration), female Syrian hamsters assigned to 3 groups: T. cruzi-infected animals treated with PTX (CH + PTX) or saline (CH + SLN); and uninfected control animals (CO). At the baseline, all groups showed similar left ventricular ejection fraction (LVEF) and MPD areas. At post-treatment evaluation, there was a significant increase of MPD in CH + SLN group (0.8 ± 1.6 to 9.4 ± 9.7%), but not in CH + PTX (1.9 ± 3.0% to 2.7 ± 2.7%) that exhibited MPD area similar to CO (0.0 ± 0.0% to 0.0 ± 0.0%). The LVEF decreased in both infected groups. Histological analysis showed a reduced inflammatory infiltrate in CH + PTX group (395.7 ± 88.3 cell/mm2), as compared to CH + SLN (515.1 ± 133.0 cell/mm2), but larger than CO (193.0 ± 25.7 cell/mm2). The fibrosis and TNF-α expression was higher in both infected groups. CONCLUSIONS: The prolonged use of PTX is associated with positive effects, including prevention of MPD development and reduction of inflammation in the chronic hamster model of CCC.

Co-Exposure of Cardiomyocytes to IFN-γ and TNF-α Induces Mitochondrial Dysfunction and Nitro-Oxidative Stress: Implications for the Pathogenesis of Chronic Chagas Disease Cardiomyopathy.

Infection by the protozoan Trypanosoma cruzi causes Chagas disease cardiomyopathy (CCC) and can lead to arrhythmia, heart failure and death. Chagas disease affects 8 million people worldwide, and chronic production of the cytokines IFN-γ and TNF-α by T cells together with mitochondrial dysfunction are important players for the poor prognosis of the disease. Mitochondria occupy 40% of the cardiomyocytes volume and produce 95% of cellular ATP that sustain the life-long cycles of heart contraction. As IFN-γ and TNF-α have been described to affect mitochondrial function, we hypothesized that IFN-γ and TNF-α are involved in the myocardial mitochondrial dysfunction observed in CCC patients. In this study, we quantified markers of mitochondrial dysfunction and nitro-oxidative stress in CCC heart tissue and in IFN-γ/TNF-α-stimulated AC-16 human cardiomyocytes. We found that CCC myocardium displayed increased levels of nitro-oxidative stress and reduced mitochondrial DNA as compared with myocardial tissue from patients with dilated cardiomyopathy (DCM). IFN-γ/TNF-α treatment of AC-16 cardiomyocytes induced increased nitro-oxidative stress and decreased the mitochondrial membrane potential (ΔΨm). We found that the STAT1/NF-κB/NOS2 axis is involved in the IFN-γ/TNF-α-induced decrease of ΔΨm in AC-16 cardiomyocytes. Furthermore, treatment with mitochondria-sparing agonists of AMPK, NRF2 and SIRT1 rescues ΔΨm in IFN-γ/TNF-α-stimulated cells. Proteomic and gene expression analyses revealed that IFN-γ/TNF-α-treated cells corroborate mitochondrial dysfunction, transmembrane potential of mitochondria, altered fatty acid metabolism and cardiac necrosis/cell death. Functional assays conducted on Seahorse respirometer showed that cytokine-stimulated cells display decreased glycolytic and mitochondrial ATP production, dependency of fatty acid oxidation as well as increased proton leak and non-mitochondrial oxygen consumption. Together, our results suggest that IFN-γ and TNF-α cause direct damage to cardiomyocytes' mitochondria by promoting oxidative and nitrosative stress and impairing energy production pathways. We hypothesize that treatment with agonists of AMPK, NRF2 and SIRT1 might be an approach to ameliorate the progression of Chagas disease cardiomyopathy.

STING Signaling Drives Production of Innate Cytokines, Generation of CD8+ T Cells and Enhanced Protection Against Trypanosoma cruzi Infection.

A variety of signaling pathways are involved in the induction of innate cytokines and CD8+ T cells, which are major players in protection against acute Trypanosoma cruzi infection. Previous data have demonstrated that a TBK-1/IRF3-dependent signaling pathway promotes IFN-ß production in response to Trypanosoma cruzi, but the role for STING, a main interactor of these proteins, remained to be addressed. Here, we demonstrated that STING signaling is required for production of IFN-ß, IL-6, and IL-12 in response to Trypanosoma cruzi infection and that STING absence negatively impacts activation of IRF-dependent pathways in response to the parasite. We reported no significant activation of IRF-dependent pathways and cytokine expression in RAW264.7 macrophages in response to heat-killed trypomastigotes. In addition, we showed that STING is essential for T. cruzi DNA-mediated induction of IFN-ß, IL-6, and IL-12 gene expression in RAW264.7 macrophages. We demonstrated that STING-knockout mice have significantly higher parasitemia from days 5 to 8 of infection and higher heart parasitism at day 13 after infection. Although we observed similar heart inflammatory infiltrates at day 13 after infection, IFN-ß, IL-12, CXCL9, IFN-γ, and perforin gene expression were lower in the absence of STING. We also showed an inverse correlation between parasite DNA and the expression of CXCL9, IFN-γ, and perforin genes in the hearts of infected animals at day 13 after infection. Finally, we reported that STING signaling is required for splenic IFN-ß and IL-6 expression early after infection and that STING deficiency results in lower numbers of splenic parasite-specific IFN-γ and IFN-γ/perforin-producing CD8+ T cells, indicating a pivotal role for STING signaling in immunity to Trypanosoma cruzi.

Host dysbiosis negatively impacts IL-9-producing T-cell differentiation and antitumour immunity.

BACKGROUND: Host-microbiota interactions shape T-cell differentiation and promote tumour immunity. Although IL-9-producing T cells have been described as potent antitumour effectors, their role in microbiota-mediated tumour control remains unclear. METHODS: We analysed the impact of the intestinal microbiota on the differentiation of colonic lamina propria IL-9-producing T cells in germ-free and dysbiotic mice. Systemic effects of the intestinal microbiota on IL-9-producing T cells and the antitumour role of IL-9 were analysed in a model of melanoma-challenged dysbiotic mice. RESULTS: We show that germ-free mice have lower frequency of colonic lamina propria IL-9-producing T cells when compared with conventional mice, and that intestinal microbiota reconstitution restores cell frequencies. Long-term antibiotic treatment promotes host dysbiosis, diminishes intestinal IL-4 and TGF-ß gene expression, decreases the frequency of colonic lamina propria IL-9-producing T cells, increases the susceptibility to tumour development and reduces the frequency of IL-9-producing T cells in the tumour microenvironment. Faecal transplant restores intestinal microbiota diversity, and the frequency of IL-9-producing T cells in the lungs of dysbiotic animals, restraining tumour burden. Finally, recombinant IL-9 injection enhances tumour control in dysbiotic mice. CONCLUSIONS: Host-microbiota interactions are required for adequate differentiation and antitumour function of IL-9-producing T cells.

Chronic exposure to diesel particles worsened emphysema and increased M2-like phenotype macrophages in a PPE-induced model.

Chronic exposure to ambient levels of air pollution induces respiratory illness exacerbation by increasing inflammatory responses and apoptotic cells in pulmonary tissues. The ineffective phagocytosis of these apoptotic cells (efferocytosis) by macrophages has been considered an important factor in these pathological mechanisms. Depending on microenvironmental stimuli, macrophages can assume different phenotypes with different functional actions. M1 macrophages are recognized by their proinflammatory activity, whereas M2 macrophages play pivotal roles in responding to microorganisms and in efferocytosis to avoid the progression of inflammatory conditions. To verify how exposure to air pollutants interferes with macrophage polarization in emphysema development, we evaluated the different macrophage phenotypes in a PPE- induced model with the exposure to diesel exhaust particles. C57BL/6 mice received intranasal instillation of porcine pancreatic elastase (PPE) to induce emphysema, and the control groups received saline. Both groups were exposed to diesel exhaust particles or filtered air for 60 days according to the groups. We observed that both the diesel and PPE groups had an increase in alveolar enlargement, collagen and elastic fibers in the parenchyma and the number of macrophages, lymphocytes and epithelial cells in BAL, and these responses were exacerbated in animals that received PPE instillation prior to exposure to diesel exhaust particles. The same response pattern was found inCaspase-3 positive cell analysis, attesting to an increase in cell apoptosis, which is in agreement with the increase in M2 phenotype markers, measured by RT-PCR and flow cytometry analysis. We did not verify differences among the groups for the M1 phenotype. In conclusion, our results showed that both chronic exposure to diesel exhaust particles and PPE instillation induced inflammatory conditions, cell apoptosis and emphysema development, as well as an increase in M2 phenotype macrophages, and the combination of these two factors exacerbated these responses. The predominance of the M2-like phenotype likely occurred due to the increased demand for efferocytosis. However, M2 macrophage activity was ineffective, resulting in emphysema development and worsening of symptoms.

Dectin-1 Activation Exacerbates Obesity and Insulin Resistance in the Absence of MyD88.

The underlying mechanism by which MyD88 regulates the development of obesity, metainflammation, and insulin resistance (IR) remains unknown. Global deletion of MyD88 in high-fat diet (HFD)-fed mice resulted in increased weight gain, impaired glucose homeostasis, elevated Dectin-1 expression in adipose tissue (AT), and proinflammatory CD11c+ AT macrophages (ATMs). Dectin-1 KO mice were protected from diet-induced obesity (DIO) and IR and had reduced CD11c+ AT macrophages. Dectin-1 antagonist improved glucose homeostasis and decreased CD11c+ AT macrophages in chow- and HFD-fed MyD88 KO mice. Dectin-1 agonist worsened glucose homeostasis in MyD88 KO mice. Dectin-1 expression is increased in AT from obese individuals. Together, our data indicate that Dectin-1 regulates AT inflammation by promoting CD11c+ AT macrophages in the absence of MyD88 and identify a role for Dectin-1 in chronic inflammatory states, such as obesity. This suggests that Dectin-1 may have therapeutic implications as a biomarker for metabolic dysregulation in humans.

Acute lung injury is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile.

Nicotinic α-7 acetylcholine receptor (nAChRα7) is a critical regulator of cholinergic anti-inflammatory actions in several diseases, including acute respiratory distress syndrome (ARDS). Given the potential importance of α7nAChR as a therapeutic target, we evaluated whether PNU-282987, an α7nAChR agonist, is effective in protecting the lung against inflammation. We performed intratracheal instillation of LPS to generate acute lung injury (ALI) in C57BL/6 mice. PNU-282987 treatment, either before or after ALI induction, reduced neutrophil recruitment and IL-1ß, TNF-α, IL-6, keratinocyte chemoattractant (KC), and IL-10 cytokine levels in the bronchoalveolar lavage fluid (P < 0.05). In addition, lung NF-κB phosphorylation decreased, along with collagen fiber deposition and the number of matrix metalloproteinase-9+ and -2+ cells, whereas the number of tissue inhibitor of metalloproteinase-1+ cells increased (P < 0.05). PNU-282987 treatment also reduced lung mRNA levels and the frequency of M1 macrophages, whereas cells expressing the M2-related markers CD206 and IL-10 increased, suggesting changes in the macrophage profile. Finally, PNU-282987 improved lung function in LPS-treated animals. The collective results suggest that PNU-282987, an agonist of α7nAChR, reduces LPS-induced experimental ALI, thus supporting the notion that drugs that act on α7nAChRs should be explored for ARDS treatment in humans.-Pinheiro, N. M., Santana, F. P. R., Almeida, R. R., Guerreiro, M., Martins, M. A., Caperuto, L. C., Câmara, N. O. S., Wensing, L. A., Prado, V. F., Tibério, I. F. L. C., Prado, M. A. M., Prado, C. M. Acute lung injury is reduced by the α7nAChR agonist PNU-282987 through changes in the macrophage profile.

IL-10-Producing Regulatory B Cells Are Decreased in Patients with Common Variable Immunodeficiency.

Common variable immunodeficiency (CVID) is the most prevalent symptomatic primary immunodeficiency in adults. CVID patients often present changes in the frequency and function of B lymphocytes, reduced number of Treg cells, chronic immune activation, recurrent infections, high incidence of autoimmunity and increased risk for malignancies. We hypothesized that the frequency of B10 cells would be diminished in CVID patients because these cells play an important role in the development of Treg cells and in the control of T cell activation and autoimmunity. Therefore, we evaluated the frequency of B10 cells in CVID patients and correlated it with different clinical and immunological characteristics of this disease. Forty-two CVID patients and 17 healthy controls were recruited for this study. Cryopreserved PBMCs were used for analysis of T cell activation, frequency of Treg cells and characterization of B10 cells by flow cytometry. IL-10 production by sorted B cells culture and plasma sCD14 were determined by ELISA. We found that CVID patients presented decreased frequency of IL-10-producing CD24hiCD38hi B cells in different cell culture conditions and decreased frequency of IL-10-producing CD24hiCD27+ B cells stimulated with CpG+PIB. Moreover, we found that CVID patients presented lower secretion of IL-10 by sorting-purified B cells when compared to healthy controls. The frequency of B10 cells had no correlation with autoimmunity, immune activation and Treg cells in CVID patients. This work suggests that CVID patients have a compromised regulatory B cell compartment which is not correlated with clinical and immunological characteristics presented by these individuals.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Co-administration of plasmid-encoded granulocyte-macrophage colony-stimulating factor increases human immunodeficiency virus-1 DNA vaccine-induced polyfunctional CD4+ T-cell responses.

T-cell based vaccines against human immunodeficiency virus (HIV) generate specific responses that may limit both transmission and disease progression by controlling viral load. Broad, polyfunctional, and cytotoxic CD4+T-cell responses have been associated with control of simian immunodeficiency virus/HIV-1 replication, supporting the inclusion of CD4+ T-cell epitopes in vaccine formulations. Plasmid-encoded granulocyte-macrophage colony-stimulating factor (pGM-CSF) co-administration has been shown to induce potent CD4+ T-cell responses and to promote accelerated priming and increased migration of antigen-specific CD4+ T-cells. However, no study has shown whether co-immunisation with pGM-CSF enhances the number of vaccine-induced polyfunctional CD4+ T-cells. Our group has previously developed a DNA vaccine encoding conserved, multiple human leukocyte antigen (HLA)-DR binding HIV-1 subtype B peptides, which elicited broad, polyfunctional and long-lived CD4+ T-cell responses. Here, we show that pGM-CSF co-immunisation improved both magnitude and quality of vaccine-induced T-cell responses, particularly by increasing proliferating CD4+ T-cells that produce simultaneously interferon-γ, tumour necrosis factor-α and interleukin-2. Thus, we believe that the use of pGM-CSF may be helpful for vaccine strategies focused on the activation of anti-HIV CD4+ T-cell immunity.

Modulating APOBEC expression enhances DNA vaccine immunogenicity.

DNA vaccines have failed to induce satisfactory immune responses in humans. Several mechanisms of double-stranded DNA (dsDNA) sensing have been described, and modulate DNA vaccine immunogenicity at many levels. We hypothesized that the immunogenicity of DNA vaccines in humans is suppressed by APOBEC (apolipoprotein B (APOB) mRNA-editing, catalytic polypeptide)-mediated plasmid degradation. We showed that plasmid sensing via STING (stimulator of interferon (IFN) genes) and TBK-1 (TANK-binding kinase 1) leads to IFN-ß induction, which results in APOBEC3A mRNA upregulation through a mechanism involving protein kinase C signaling. We also showed that murine APOBEC2 expression in HEK293T cells led to a 10-fold reduction in intracellular plasmid levels and plasmid-encoded mRNA, and a 2.6-fold reduction in GFP-expressing cells. A bicistronic DNA vaccine expressing an immunogen and an APOBEC2-specific shRNA efficiently silenced APOBEC2 both in vitro and in vivo, increasing the frequency of induced IFN-γ-secreting T cells. Our study brings new insights into the intracellular machinery involved in dsDNA sensing and how to modulate it to improve DNA vaccine immunogenicity in humans.

Broad and cross-clade CD4+ T-cell responses elicited by a DNA vaccine encoding highly conserved and promiscuous HIV-1 M-group consensus peptides.

T-cell based vaccine approaches have emerged to counteract HIV-1/AIDS. Broad, polyfunctional and cytotoxic CD4(+) T-cell responses have been associated with control of HIV-1 replication, which supports the inclusion of CD4(+) T-cell epitopes in vaccines. A successful HIV-1 vaccine should also be designed to overcome viral genetic diversity and be able to confer immunity in a high proportion of immunized individuals from a diverse HLA-bearing population. In this study, we rationally designed a multiepitopic DNA vaccine in order to elicit broad and cross-clade CD4(+) T-cell responses against highly conserved and promiscuous peptides from the HIV-1 M-group consensus sequence. We identified 27 conserved, multiple HLA-DR-binding peptides in the HIV-1 M-group consensus sequences of Gag, Pol, Nef, Vif, Vpr, Rev and Vpu using the TEPITOPE algorithm. The peptides bound in vitro to an average of 12 out of the 17 tested HLA-DR molecules and also to several molecules such as HLA-DP, -DQ and murine IA(b) and IA(d). Sixteen out of the 27 peptides were recognized by PBMC from patients infected with different HIV-1 variants and 72% of such patients recognized at least 1 peptide. Immunization with a DNA vaccine (HIVBr27) encoding the identified peptides elicited IFN-γ secretion against 11 out of the 27 peptides in BALB/c mice; CD4(+) and CD8(+) T-cell proliferation was observed against 8 and 6 peptides, respectively. HIVBr27 immunization elicited cross-clade T-cell responses against several HIV-1 peptide variants. Polyfunctional CD4(+) and CD8(+) T cells, able to simultaneously proliferate and produce IFN-γ and TNF-α, were also observed. This vaccine concept may cope with HIV-1 genetic diversity as well as provide increased population coverage, which are desirable features for an efficacious strategy against HIV-1/AIDS.

A Recombinant Adenovirus Encoding Multiple HIV-1 Epitopes Induces Stronger CD4+ T cell Responses than a DNA Vaccine in Mice.

T-cell based vaccines against SIV/HIV may reduce both transmission and disease progression by inducing broad and functionally relevant T cell responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency and virus replication. We have previously shown that a DNA vaccine (HIVBr18), encoding 18 HIV CD4 epitopes capable of binding to multiple HLA class II molecules was able to elicit broad, polyfunctional, and long-lived CD4+ and CD8+ T cell responses in BALB/c and multiple HLA class II transgenic mice. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that could be recognized across diverse common HLA class II alleles, this vaccine concept may cope with HIV-1 genetic variability and increase population coverage. Given the low immunogenicity of DNA vaccines in clinical trials, we tested the ability of a recombinant adenovirus serotype 5 encoding the 18 HIV epitopes (Ad5-HIVBr18) to increase specific cellular immune responses. We assessed the breadth and magnitude of HIV-specific proliferative and cytokine responses of CD4+ and CD8+ T cells induced by Ad5-HIVBr18 using different vaccination regimens/routes and compared to DNA immunization. Immunization with Ad5-HIVBr18 induced significantly higher specific CD4+ and CD8+ T cell proliferation, IFN-γ and TNF-α production than HIVBr18. The subcutaneous route of Ad5-HIVBr18 administration was associated with the highest responses. Ad5-HIVBr18 induced higher proliferative and cytokine responses than HIVBr18 up to 28 weeks post-immunization. Our results indicate that a vaccine based on an adenovirus vector encoding the HIVBr18 epitopes shows superior immunogenicity as compared to its DNA counterpart. These results support the possible testing of a vaccine encoding HIVBr18 in non-human primates and future clinical trials.