Th17-Inducing Cytokines IL-6 and IL-23 Are Crucial for Granuloma Formation during Experimental Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), a chronic granulomatous disease caused by the thermally dimorphic fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii, has the highest mortality rate among systemic mycosis. The T helper 1-mediated immunity is primarily responsible for acquired resistance during P. brasiliensis infection, while susceptibility is associated with Th2 occurrence. Th17 is a population of T CD4+ cells that, among several chemokines and cytokines, produces IL-17A and requires the presence of IL-1, IL-6, and TGF-ß for differentiation in mice and IL-23 for its maintenance. Th17 has been described as an arm of the immune system that enhances host protection against several bacterial and fungal infections, as Pneumocystis carinii and Candida albicans. In this study, we aimed to evaluate the Th17 immune response and the role of Th17-associated cytokines (IL-6, IL-23, and IL-17A) during experimental PCM. First, we observed that P. brasiliensis infection [virulent yeast strain 18 of P. brasiliensis (Pb18)] increased the IL-17A production in vitro and all the evaluated Th17-associated cytokines in the lung tissue from C57BL/6 wild-type mice. In addition, the deficiency of IL-6, IL-23, or IL-17 receptor A (IL-17RA) impaired the compact granuloma formation and conferred susceptibility during infection, associated with reduced tumor necrosis factor-α, IFN-γ, and inducible nitric oxide synthase enzyme expression. Our data suggest that IL-6 production by bone marrow-derived macrophages (BMDMs) is important to promote the Th17 differentiation during Pb18 infection. In accordance, the adoptive transfer of BMDMs from C57BL/6 to infected IL-6-/- or IL-17RA-/- mice reduced the fungal burden in the lungs compared to nontransferred mice and reestablished the pulmonary granuloma formation. Taken together, these results suggest that Th17-associated cytokines are involved in the modulation of immune response and granuloma formation during experimental PCM.

Interaction between saliva's adenosine and tick parasitism: effects on feeding and reproduction.

BACKGROUND: It has recently been demonstrated that saliva from Rhipicephalus sanguineus ticks contains adenosine (ADO) and prostaglandin E2 (PGE2), two non-protein molecules that have significant immunomodulatory properties. These molecules can inhibit cytokine production by dendritic cells (DCs), while also reducing the expression of CD40 in these cells. However, more studies are needed for a better understanding of their participation in the feeding of ticks in vivo. This work, therefore, evaluated the importance of ADO during tick infestations. Mice were infested with adult ticks (3 couples/mouse), and their skin was collected at the tick-infested site (3rd and 7th day), and mRNA for receptors of ADO was quantified by real-time PCR. RESULTS: Tick infestation increased by four and two times the expression of the A2b and A3v1 receptors on day 3, respectively, while expression of other ADO receptors was unaltered. In addition, we treated mice (n = 10/group) daily with 8-(p-Sulfophenyl)theophylline, 8-pSPT, 20 mg/kg, i.p.), a non-selective antagonist of ADO receptors, and evaluated the performance of ticks during infestations. Female ticks fed on 8-pSPT-treated mice presented a reduction in their engorgement, weight and hatching rates of egg masses, and survival times of larvae compared to the same parameters presented by ticks in the control group. To investigate if these 8-pSPT-treated mice presented altered immune responses, we performed three tick infestations and collected their lymph node cells to determine the percentages and activation state of DCs and cytokine production by lymphocytes by flow cytometry (Cytometric Bead Array technique, CBA). Our data showed that 8-pSPT-treated mice presented an increase in the percentage of DCs as well as of their stimulatory and co-stimulatory molecules (CD40, CD80 and MHCII). Regarding production of T cell cytokines, we observed a significant increase in the levels of IL-2 and a significant decrease in IL-10, IL-17, TNF-α and IFN-γ cytokines. CONCLUSIONS: These results suggest that ADO produced by ticks helps them feed and reproduce and that this effect may be due to modulation of host DCs and T cells.

IL-18 triggered by the Nlrp3 inflammasome induces host innate resistance in a pulmonary model of fungal infection.

Pathogens are sensed by innate immune receptors that initiate an efficient adaptive immune response upon activation. The elements of the innate immune recognition process for Paracoccidioides brasiliensis include TLR-2, TLR-4, and dectin-1. However, there are additional receptors necessary for the host immune responses to P. brasiliensis. The nucleotide-binding oligomerization domain-like receptor (NLRs), which activate inflammasomes, are candidate receptors that deserve renewed investigation. After pathogen infection, the NLRs form large signaling platforms called inflammasomes, which lead to caspase-1 activation and maturation of proinflammatory cytokines (IL-18 and IL-1ß). In this study, we showed that NLR family pyrin domain-containing 3 (Nlrp3) is required to induce caspase-1 activation and further secretion of IL-1ß and IL-18 by P. brasiliensis-infected macrophages. Additionally, potassium efflux and lysosomal acidification induced by the fungus were important steps in the caspase-1 activation mechanism. Notably, Nlrp3 and caspase-1 knockout mice were more susceptible to infection than were the wild-type animals, suggesting that the Nlrp3-dependent inflammasomes contribute to host protection against P. brasiliensis. This protective effect occurred owing to the inflammatory response mediated by IL-18, as shown by an augmented fungus burden in IL-18 knockout mice. Taken together, our results show that the Nlrp3 inflammasome is essential for resistance against P. brasiliensis because it orchestrates robust caspase-1 activation and triggers an IL-18-dependent proinflammatory response.

Expression of apoptosis mediators p53 and caspase 3, 7, and 9 in chronic rhinosinusitis with nasal polyposis.

BACKGROUND: The causal factor for the perpetuation of the inflammatory process in chronic rhinosinusitis with nasal polyps (CRSwNPs) has been extensively studied. However, little is known about the influence of cell death in this disease. Thus, the molecular assessment of mechanisms involved in apoptosis might shed light on the pathogenesis of CRSwNPs. This study was designed to evaluate the gene expression of different apoptotic factors in patients with NPs compared with control patients. METHODS: The mRNA expression of the apoptosis mediators caspase 3, 7, and 9 and of p53 protein was analyzed using quantitative reverse transcription-polymerase chain reaction in 25 NPs and 18 control samples. RESULTS: We observed significantly lower expression of p53 and caspase 3 and 9 in patients with CRSwNPs compared with the controls, whereas caspase 7 expression was not significantly different from the controls. CONCLUSION: The reduced expression of these apoptosis factors in CRSwNPs might be related to higher proliferation and the perpetuation of inflammatory cells hindering the control of the disease. A better understanding of the possible influence of apoptosis factors on CRSwNPs could provide rationale for future therapies.

CD44+/CD24- cells and lymph node metastasis in stage I and II invasive ductal carcinoma of the breast.

The presence of tumor-initiating cells (CD44(+)/CD24(-)) in solid tumors has been reported as a possible cause of cancer metastasis and treatment failure. Nevertheless, little is know about the presence of CD44(+)/CD24(-) cells within the primary tumor and metastasis. The proportion of CD44(+)/CD24(-) cells was analyzed in 40 samples and in 10 lymph node metastases using flow cytometry phenotyping. Anti-human CD326 (EpCam; FITC), anti-human CD227 (MUC-1; FITC), anti-human CD44 (APC), and anti-human CD24 (PE), anti-ABCG2 (PE), and anti-CXCR4 (PeCy7) were used for phenotype analysis. The mean patient age was 60.5 years (range, 33-87 years); mean primary tumor size (pT) was 1.8 cm (0.5-3.5 cm). The Wilcoxon or Kruskal-Wallis test was used for univariate analyses. Logistic regression was used for multivariate analysis. The median percentage of CD44(+)/CD24(-) cells within primary invasive ductal carcinomas (IDC) was 2.7% (range, 0.2-71.2). In lymph node metastases, we observed a mean of 6.1% (range, 0.07-53.7). The percentage of CD44(+)/CD24(-) cells in IDCs was not associated with age, pT, tumor grade and HER2. We observed a significantly enrichment of CD44(+)/CD24(-) and ABCG2(+) cells in ESA(+) cell population in patients with positive lymph nodes (P = 0.02 and P = 0.04, respectively). Our data suggest that metastatic dissemination is associated with an increase in tumor-initiating cells in stage I and II breast cancer.

CCR2 receptor is essential to activate microbicidal mechanisms to control Toxoplasma gondii infection in the central nervous system.

Chemokines comprise a structurally related family of cytokines that regulate leukocyte trafficking. Because infection with Toxoplasma gondii can induce an important inflammatory reaction that, if left uncontrolled, can lead to death, we investigated the role of the chemokine receptor CCR2 in T. gondii infection. We orally infected CCR2(-/-) mice with five ME-49 T. gondii cysts and monitored morbidity, survival, and immune response thereafter. The CCR2(-/-) mice displayed higher susceptibility to infection as all mice died on day 28 after infection. Despite similar Th1 responses, a more evident anti-inflammatory response was induced in the peripheral organs of CCR2(-/-) mice compared with wild-type C57BL/6 mice. Additionally, CCR2(-/-) mice presented greater parasitism and a milder inflammatory reaction in their peripheral organs with lesser CD4(+) and MAC-1(+) and greater CD8(+) cell migration. The parasite load decreased in these organs in CCR2(-/-) mice but remained uncontrolled in the central nervous system. Additionally, we observed down-regulated inducible nitric oxide synthase expression in peripheral organs from CCR2(-/-) mice that was associated with a small nitric oxide production by spleen macrophages. In conclusion, in the absence of CCR2, another mechanism is activated to control tissue parasitism in peripheral organs. Nevertheless, CCR2 is essential for the activation of microbicidal mediators that control T. gondii replication in the central nervous system.

Increased activities of cardiac matrix metalloproteinases matrix metalloproteinase (MMP)-2 and MMP-9 are associated with mortality during the acute phase of experimental Trypanosoma cruzi infection.

The strong inflammatory reaction that occurs in the heart during the acute phase of Trypanosoma cruzi infection is modulated by cytokines and chemokines produced by leukocytes and cardiomyocytes. Matrix metalloproteinases (MMPs) have recently emerged as modulators of cardiovascular inflammation. In the present study we investigated the role of MMP-2 and MMP-9 in T. cruzi-induced myocarditis, by use of immunohistochemical analysis, gelatin zymography, enzyme-linked immunosorbent assay, and real-time polymerase chain reaction to analyze the cardiac tissues of T. cruzi-infected C57BL/6 mice. Increased transcripts levels, immunoreactivity, and enzymatic activity for MMP-2 and MMP-9 were observed by day 14 after infection. Mice treated with an MMP inhibitor showed significantly decreased heart inflammation, delayed peak in parasitemia, and improved survival rates, compared with the control group. Reduced levels of cardiac tumor necrosis factor-alpha, interferon-gamma, serum nitrite, and serum nitrate were also observed in the treated group. These results suggest an important role for MMPs in the induction of T. cruzi-induced acute myocarditis.