High Matrix Metalloproteinase Activity Is a Hallmark of Periapical Granulomas

Introduction: The inability to distinguish periapical cysts from granulomas before performing root canal treatment leads to uncertainty in treatment outcomes because cysts have lower healing rates. Searching for differential expression of molecules within cysts or granulomas could provide information with regard to the identity of the lesion or suggest mechanistic differences that may form the basis for future therapeutic intervention. Thus, we investigated whether granulomas and cysts exhibit differential expression of extracellular matrix (ECM) molecules. Methods: Human periapical granulomas, periapical cysts, and healthy periodontal ligament tissues were used to investigate the differential expression of ECM molecules by microarray analysis. Because matrix metalloproteinases (MMP) showed the highest differential expression in the microarray analysis, MMPs were further examined by in situ zymography and immunohistochemistry. Data were analyzed by using one-way analysis of variance followed by the Tu-key test. Results: We observed that cysts and granulomas differentially expressed several ECM molecules, especially those from the MMP family. Compared with cysts, granulomas exhibited higher MMP enzymatic activity in areas stained for MMP-9. These areas were composed of polymorphonuclear cells (PMNs) in contrast to cysts. Similarly, MMP-13 was expressed by a greater number of cells in granulomas compared with cysts. Conclusion: Our findings indicate that high enzymatic MIMP activity in PMNs together with MMP-9 and MMP-13 stained cells could be a molecular signature of granulomas unlike periapical cysts. (J Endod 2009;35:1234-1242)

Matrix metalloproteinases with gelatinolytic activity induced by Paracoccidioides brasiliensis infection

P>Matrix metalloproteinases (MMPs) modulate extracellular matrix turnover, inflammation and immunity. We studied MMP-9 and MMP-2 in experimental paracoccidioidomycosis. At 15 and 120 days after infection (DAI) with virulent Paracoccidioides brasiliensis, MMP-9 was positive by immunohistochemistry in multinucleated giant cells, in mononuclear cells with macrophage and lymphocyte morphologies and also in fungal cells in the lesions of susceptible and resistant mice. Using gelatin zymography, pro- and active MMP-9 and active MMP-2 were detected in all infected mice, but not in controls. Gelatinolytic activity was not observed in P. brasiliensis extracts. Semiquantitative analysis of gelatinolytic activities revealed weak or absent MMP-2 and strong MMP-9 activity in both mouse strains at 15 DAI, declining at 120 DAI. Avirulent P. brasiliensis-infected mice had residual lesions with MMP-9-positive pseudoxantomatous macrophages, but no gelatinase activity at 120 DAI. Our findings demonstrate the induction of MMPs, particularly MMP-9, in experimental paracoccidioidomycosis, suggesting a possible influence in the pattern of granulomas and in fungal dissemination.

Nitric oxide participation in granulomatous response induced by Paracoccidioides brasiliensis infection in mice

The role of nitric oxide (NO) in granulomas of Paracoccidioides brasiliensis-infected inducible NO synthase-deficient C57BL/6 mice (iNOS KO) and their wild-type counterparts and its association with osteopontin (OPN) and matrix metalloproteinases (MMPs) was studied. At 15 days after infection (DAI), iNOS KO mice showed compact and necrotic granulomas with OPN+ macrophages and multinucleated giant cells, whereas wild-type mice developed loose granulomas with many fungi and OPN+ cells distributed throughout the tissue. In addition, high OPN levels and fungal load were observed in iNOS KO mice. Both experimental groups had MMP-9 activity. At 120 DAI, iNOS KO had smaller granulomas with OPN+ cells, lower OPN levels, lower fungal load and decreased MMP-9 activity compared with wild-type mice. These findings suggest that NO has an important role in granuloma modulation, by controlling OPN and MMP production, as well as by inducing loose granulomas formation and fungal dissemination, resulting, at later phases, in progression of paracoccidioidomycosis.

Osteopontin involvement in granuloma formation and in the severity of Paracoccidioides brasiliensis infection

The participation of osteopontin (OPN) in Paracoccidioides brasiliensis infected mice, its association to granulomatogenesis, severity of infection, pattern of lesions, nitric oxide (NO) levels and fungal load were evaluated in this investigation. Immunohistochemistry analysis showed marked OPN staining in extracellular matrix and in macrophages and multinucleated giant cells at the center of lesions, suggesting a possible role of OPN in the distribution of these cells within the granulomas. At 15 days post-infection with a virulent P. brasiliensis isolate, OPN(+) cells were more numerous and intensely immunostained in the loose granulomas of susceptible mice than in those of resistant mice. In addition, high fungal loads and low NO levels were observed in susceptible mice. At 120 days after infection, resistant mice had increased total OPN levels (ELISA) and OPN positivity in compact granulomas, higher NO levels and lower fungal loads than susceptible mice. Residual lesions associated with low OPN levels, high NO and control of fungal dissemination were observed in both mouse strains at 120 days post-infection with the slightly virulent fungal isolate. Therefore, OPN could be associated with higher severity of the disease in an early phase of infection and with a degree of control of the progressive infection.

MyD88 Signaling Is Required for Efficient Innate and Adaptive Immune Responses to Paracoccidioides brasiliensis Infection

The mechanisms that govern the initial interaction between Paracoccidioides brasiliensis, a primary dimorphic fungal pathogen, and cells of the innate immunity need to be clarified. Our previous studies showed that Toll-like receptor 2 (TLR2) and TLR4 regulate the initial interaction of fungal cells with macrophages and the pattern of adaptive immunity that further develops. The aim of the present investigation was to assess the role of MyD88, an adaptor molecule used by TLRs to activate genes of the inflammatory response in pulmonary paracoccidioidomycosis. Studies were performed with normal and MyD88(-/-) C57BL/6 mice intratracheally infected with P. brasiliensis yeast cells. MyD88(-/-) macrophages displayed impaired interaction with fungal yeast cells and produced low levels of IL-12, MCP-1, and nitric oxide, thus allowing increased fungal growth. Compared with wild-type (WT) mice, MyD88(-/-) mice developed a more severe infection of the lungs and had marked dissemination of fungal cells to the liver and spleen. MyD88(-/-) mice presented low levels of Th1, Th2, and Th17 cytokines, suppressed lymphoproliferation, and impaired influx of inflammatory cells to the lungs, and this group of cells comprised lower numbers of neutrophils, activated macrophages, and T cells. Nonorganized, coalescent granulomas, which contained high numbers of fungal cells, characterized the severe lesions of MyD88(-/-) mice; the lesions replaced extensive areas of several organs. Therefore, MyD88(-/-) mice were unable to control fungal growth and showed a significantly decreased survival time. In conclusion, our findings demonstrate that MyD88 signaling is important in the activation of fungicidal mechanisms and the induction of protective innate and adaptive immune responses against P. brasiliensis.