Morphophysiological changes in the splenic extracellular matrix of Leishmania infantum-naturally infected dogs is associated with alterations in lymphoid niches and the CD4+ T cell frequency in spleens

Abstract

The spleen is one of the main affected organs in canine visceral leishmaniasis (CVL). Disorganization of the splenic white pulp (SWP) has been associated with immunosuppression and disease progression. This study aims to assess structural and cellular changes in the splenic extracellular matrix of dogs with CVL, correlating these changes with the parasite load and clinical signs. Splenic fragments were collected from 41 naturally infected animals for parasite load quantification by quantitative PCR, histopathological analysis and immunohistochemistry for CD3+, CD4+, and CD8+ T cells; CD21+ B cells; Ki-67+, IFN-γ+, and IL-10+ cells; and the MMP-9 and ADAM-10 enzymes. Laminin, collagen and fibronectin deposition were also evaluated. The animals were grouped according to the level of SWP organization. SWP disorganization was accompanied by a reduction in the quantity of lymphoid follicles/mm2 (p > 0.0001). Animals with moderate to intense SWP disorganization showed more clinical signs (p = 0.021), higher laminin (p = 0.045) and collagen deposition (p = 0.036), higher MMP-9 expression (p = 0.035) and lower numbers of CD4+ T cells (p = 0.027) in the spleen than the animals with organized SWP. These data suggest that splenic structure and function are drastically altered and compromised during CVL.

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.