Fig 1.
Degrees of disorganization of the splenic white pulp in dogs naturally infected with Leishmania infantum.
(A) Organized splenic white pulp: the white pulp has a distinct periarteriolar lymphocyte sheath, germinal center, mantle zone and marginal zone; (B) slightly disorganized splenic white pulp: the white pulp shows a decrease in the definition of any white pulp region; (C) moderately disorganized splenic white pulp: the white pulp is evident, but its regions are poorly defined or indistinct; (D) intensely disorganized white pulp; the follicular structure is barely distinct from the red pulp or T cell areas. Scale bar = 100 μm. Hematoxylin and eosin. (E) Lymphoid follicle number in the splenic white pulp. (F) The clinical scores of the evaluated dogs. Mann-Whitney test. OR-SD = animals with organized to slightly disorganized splenic white pulp. MD-ID = animals with moderately to intensely disorganized splenic white pulp.
Fig 2.
The extracellular matrix components in the spleens of naturally infected dogs with L. infantum.
(A) Negative control (suppression of primary antibody). (B-D) Fibronectin deposition. Note that the deposition is well distributed throughout the splenic tissue and within the follicle (D). (E) Negative control (suppression of primary antibody). (F-G) The deposition of laminin is homogenous throughout the red pulp; however, laminin expression is delimited around the marginal zone of the white pulp (H). (I) Negative control. (J-L) Collagen deposition in the red pulp and (M) in one granuloma. A, B, D, F, H, I, J and M show the animals with organized splenic white pulp. C, E, G and L show the animals with disorganized splenic white pulp. Magnification: (A-H) 100x; Scale bar: 100 μm and (I-M) 400x; Scale bar: 25 μm. A-H: Immunohistochemistry—aminoethyl carbazole. I-M: Picrosirius red. (N) fibronectin deposition/area; (O) laminin deposition/area; (P) collagen deposition/area. Mann-Whitney test. OR-SD = animals with organized to slightly disorganized splenic white pulp. MD-ID = animals with moderately to intensely disorganized splenic white pulp.
Fig 3.
Deposition of MMP-9 and ADAM-10 during the extracellular matrix remodeling process.
The dogs with disorganized splenic white pulp showed higher MMP-9, but not ADAM-10, expression than the dogs with organized splenic white pulp. (A-B) Negative controls (suppression of primary antibody) (C) Immunostaining of MMP-9. (D) Immunostaining of ADAM-10. Both immunostaining procedures were performed in the red pulp. (E) MMP-9 expression. (F) ADAM-10 expression. Immunohistochemistry of the red pulp—aminoethyl carbazole. Magnification: 400x, Scar bar: 25 μm. OR-SD = animals with organized to slightly disorganized splenic white pulp. MD-ID = animals with moderately to intensely disorganized splenic white pulp.
Fig 4.
CD4+, CD8+ and Ki-67 + expression and cytokines/mm2 splenic tissue of dogs naturally infected with L. infantum.
(A-B) Negative control (suppression of primary antibody). Immunostaining of (C-D) CD3+, (E) CD4+, (F) CD8+, (G) CD21+, (H) Ki-67+ proliferating, (I) IFN-ɣ+ and (J) IL-10+ cells. Magnification in A and C: 100x; Magnification in B and D-J: 400x. Scale bar = 25 μm. (L) CD4+ T cells/mm2. (M) CD8+ T cells/mm2. (N) CD21+ B cells/mm2. (O) Expression of IFN-ɣ/mm2; (P) IL-10/mm2; (Q) KI-67+ cells/mm2. Mann-Whitney test. OR-SD = animals with organized to slightly disorganized splenic white pulp. MD-ID = animals with moderately to intensely disorganized splenic white pulp.
Fig 5.
Association between the histopathological analysis and parasite burden.
(A) Lymphoid follicles, (B) laminin deposition, (C) fibronectin deposition, (D) collagen deposition, (E) MMP-9 expression, (F) ADAM-10 expression, (G) CD4+ cells, (H) CD8+ cells, (I) CD21+ cells and (J) Ki-67+ cells. P-value < 0.05 was considered significant. Mann-Whitney test. Low/OR = low parasite burden and organized to slightly disorganized splenic white pulp. Low/DS = low parasite burden and moderately to intensely disorganized splenic white pulp. High/DS = high parasite burden and moderately to intensely disorganized splenic white pulp.