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Characterization of cell subpopulations in liver tissues from DENV-3 fatal cases.. Sections were stained with (a) H.E. or (b, d, f) incubated with specific antibodies in immunohistochemical assays. (a) Liver of a representative dengue case showing diffuse mononuclear infiltrates around the portal space; (b) detection of hyperplasic Kupffer cells (CD68+) observed mainly in sinusoidal capillaries of the dengue cases; (c) quantification of CD68+ cells in dengue cases and controls (non-dengue cases); (d) detection of CD4+ cells manly in portal space of the dengue cases; (e) quantification of CD4+ cells in controls and dengue cases; (f) CD8+ cells detected mainly in portal space; (g) quantification of CD8+ cells. MI:mononuclear cell infiltrates. Asterisks indicate differences that are statistically significant between dengue cases and control groups, (*p < 0.05). Staining controls are shown in S1 Fig.
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Histopathological studies of all samples showed diffuse mononuclear cell infiltrates, mainly around the portal space (Fig 1 panel a). Detection of CD68+ cells revealed the presence of hyperplasic Kupffer cells and/or circulating macrophages (Fig 1 panel b), although quantification analysis did not show statistical difference when comparing dengue to control samples (Fig 1 panel c). Yet, we detected numerous CD4+ (Fig 1 panel d), and CD8+ (Fig 1 panel f) T cells placed mainly in sinusoidal capillaries. Quantification of these cell populations showed a significant increase in the number of T lymphocytes present in liver samples of dengue cases when compared to controls (about 5 and 3 fold, respectively:Fig 1 panels e and g).
Detection of cytokine-producing cells in liver tissues.. (a) Detection of TNF-alpha in macrophages and Kupffer cells in sinusoidal capillaries; (b and c) detection of INF-alpha in Kupffer cells and lymphocytes; (d) production of anti-inflammatory cytokines (IL-10 in monocytes and lymphocytes circulating in sinusoidal capillaries and TGFbeta detected in macrophages inside the portal space; (h) TGF-beta-expressing macrophages and Kupffer cells in sinusoidal capillaries; (i and j) detection of RANTES in endothelium and Kupffer cells; (e, f, g, k and l) quantification of the number of cells expressing these cytokines in the hepatic tissue. Monocytes (Mo); Macrophages (Mphi); Kupffer cells (KC); lymphocyte (Ly) and sinusoidal endothelium cells (E). Asterisks indicate differences that are statistically significant between groups (*p < 0.05). Staining controls are shown in S1 Fig.
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IFN-gamma was found mostly in lymphocytes, Kupffer cells and monocytes (Fig 2 panels b and c). In sinusoidal capillaries, we also detected groups of cells with an anti-inflammatory profile, such as IL-10-expressing monocytes and lymphocytes (Fig 2 panel d) and TGF-beta-expressing macrophages and Kupffer cells (Fig 2 panel h). The chemokine RANTES/CCL5 was detected mainly in endothelium and Kupffer cells (Fig 2 panels i and j). Quantification of cells producing the inflammatory cytokines TNF-alpha, IFN-gamma and RANTES/CCL5 revealed a significant increase (4-, 4.5- and 3-fold, respectively) in dengue group compared to control (Fig 2 panels e, f and l), while the number of cells expressing the anti-inflammatory cytokines IL-10 and TGF-beta did not change significantly in either group (Fig 2 panels g and k).
Characterization of mononuclear cell subpopulations in lung tissues collected from DENV-3 fatal cases.. Lung tissue sections stained with (a) H.E. or (b, d and f) incubated with specific antibodies in immunohistochemistry assays. (a) Mononuclear infiltrates located in interstitial septa; (b) macrophages (CD68+ cells) observed in alveolar capillaries; (d and f) CD4+ and CD8+ T lymphocytes respectively, found in interstitial infiltrates septa; (c, e and g) quantification of cell subpopulations in the lung tissue. Macrophage (CD68); Mononuclear infiltrate (MI); edema (E); alveolar macrophage (AM); (CD4+) lymphocytes; (CD8+) lymphocytes. Asterisks indicate differences that are statistically significant between analyzed groups (*p < 0.05). Staining controls are shown in S2 Fig.
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After analysis of lung sections of dengue cases we observed a diffuse mononuclear infiltrate in alveolar septa and edema areas (Fig 3 panel a), thus, indicating that this tissue could also be targeted by immune mechanisms triggered by infection. T cells (Fig 3 panel f) mainly in the blood vessels. The quantification analysis revealed a 4-fold increase of lymphocytes in the dengue group when compared to non-dengue samples (Fig 3 panels e and g), whereas the number of CD68+ was not statistically different from the controls (Fig 3 panel c).
Cytokine-producing cells profile in lung tissues of dengue fatal cases.. Immunohistochemical analysis of lung sections collected from dengue fatal cases exhibited (a) TNF-alpha and (b) INF-gamma production in alveolar macrophages; (c) IL-10 detected in monocytes/macrophages and lymphocytes in alveolar septa; (g) TGF-beta produced by numerous macrophages and lymphocytes in the septa; (h) RANTES detected in endothelium cells and several macrophages in pulmonary tissue; (d-f, i and j) quantification of cytokine-expressing cells in the lung sections. Monocyte (Mo); Alveolar macrophage (AM); Endothelium (E); Lymphocytes (Ly). Asterisks indicate differences that are statistically significant between dengue cases and controls (*p < 0.05). Staining controls are shown in S2 Fig.
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TNF-alpha, which is an important cytokine related to dengue pathogenesis, was detected mainly in alveolar macrophages. (Fig 4 panel a). Increased numbers of TNF-alpha- (Fig 4 panel d), IFN-gamma- (Fig 4 panels b and e), IL-10- (Fig 4 panels c and f) and TGF-beta- (Fig 4 panels g and i) expressing cells such as macrophages and lymphocytes were characterized in the tissues of dengue cases, when compared to controls. Vascular permeability impairment was also addressed in dengue cases by the presence of several RANTES-expressing endothelial cells and alveolar macrophages in the perivascular space (Fig 4 panel h). The quantification of these subpopulations was found to be increased in dengue cases when compared to controls (Fig 4 panel j).
Cytokine-producing cells in renal tissues of dengue cases.. (a) TNF-alpha detected in monocytes located in blood vessels; (b) Production of INF-gamma observed in circulating macrophages and lymphocytes located in the interstitial space; (c) IL-10 found in endothelial cells of glomerulus; (g) TGF-beta production by lymphocytes present mainly inside renal glomeruli; (h) RANTES detected in macrophages and lymphocytes in the interstitial renal space; (d-f, i and j) quantification of the number of cells expressing the above cytokines. Macrophages (Mphi); Lymphocytes (Ly). Asterisks indicate differences that are statistically significant between analyzed groups (*p < 0.05). Staining controls are shown in S3 Fig.
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The evaluation of cytokine profile in renal samples revealed TNF-alpha being produced mainly by monocytes/macrophages present in the medullar region (Fig 6 panel a). IFN-gamma-producing cells, such as macrophages, were found in blood vessels located also in the medullar region (Fig 6 panel b). The anti-inflammatory cytokines IL-10 and TGF-gamma were observed mostly in lymphocytes within the renal glomerulus (Fig 6 panel c) and macrophages (Fig 6 panel g), while RANTES production was detected mainly in macrophages only (Fig 6 panel h). The number of cells producing of TNF-alpha and IFN-gamma in dengue cases was 3- and 5-fold higher, respectively, after comparison with non-dengue cases (Fig 6 panels d and e). Concerning the number of cells expressing TGF-beta (Fig 6 panel i), IL-10 (Fig 6 panel f) and RANTES (Fig 6 panel j), in dengue cases we noted increments of about 2.5-, 3- and 13-fold, respectively.