Extracellular matrix alterations in experimental Leishmania amazonensis infection in susceptible and resistant mice

Abstract

Leishmania is inoculated, by the bite of an infected sandfly, into the skin of the host, where the promastigotes are phagocyted by dermal macrophages. The dermal region comprises cells and abundant extracellular matrix. Studies show that matrix metalloproteinases play an important role in host defense responses against pathogens in mammals and that their activities lead to the production of antimicrobial peptides. The aim of this study is to evaluate the changes in the distribution of fibronectin and laminin as well as in the elastic system fibres during the course of infection caused by Leishmania amazonensis in mice with distinct genetic backgrounds of susceptibility to this parasite. The results showed that BALB/c presented an enhancement of fibronectin during the course of infection when compared to their control group while the infected or non-infected C3H.He showed a decrease of this protein at end of the experiment. Laminin, on the other hand, remained unaltered in both strains. Also in both BALB/c and C3H.He mice the elastic and elaunin fibres remained unchanged while the oxytalan fibres decreased along the experiment. Ninety days after the infection C3H.He mice had recovered their capacity to produce oxytalan fibres.

Figure 1

Footpad swelling in BALB/c and C3H.He mouse strains infected by 10⁴ L. amazonensis amastigotes (H21 MHOM/BR/76/MA-76) in the left hind footpad. Each point represents the average increase in footpad thickness ± SEM (n = 5).

Figure 2

Histopathological analysis of skin fragments from left footpad of BALB/c (A, B, C and D) and C3H.He (E, F, G and H) mock-infected mice, 30 days after mock infection. A. Normal morphology of footpad is observed. The integrity of the epidermis and dermis, the presence of adipocytes and ducts of regular glands can be seen (HE). B. Elastic (thin arrow), elaunin (thick arrow) and oxytalan (arrow) fibres with normal morphology and location are also observed (Weigert). C: Picrosirius red in non-polarized light showing the regular arrangement of the skin showing epidermis, dermis and hypodermis. D. Picrosirius red in polarized light shows the normal arrangement of type I collagen (orange and yellow) and type III (green). E. Normal morphology of footpad is observed. The organization of the epidermis and dermis were maintained, the presence of adipocytes and ducts of regular glands can also be seen (HE). F. Elastic fibres (thin arrow), elaunin fibres (thick arrow) with normal morphology and location are noted (Weigert). G. Picrosirius red in non-polarized light showing the regular organization of the skin showing epidermis, dermis and hypodermis. H. Picrosirius red polarized light shows the normal organization of type I collagen (orange and yellow) and type III (green).

Figure 3

Histopathological analysis of footpad skin from mice subcutaneously infected with 10⁴ amastigotes of L. amazonensis. A. BALB/c mice 30 days after infection. Intense inflammatory infiltrate in the reticular dermis (***), with numerous vacuolated macrophages (arrowheads) and polymorphonuclear cells, and slight dissociation of the extracellular matrix fibres (plain arrow) (HE). B. C3H.He mice 30 dpi. Intact papillar dermis and moderate inflammatory infiltrate (***) rich in neutrophils and parasitized macrophages in the reticular dermis (HE). C and D: BALB/c mice 30 days after infection. Picrosirius red in non-polarized (C) and polarized light (D). The image shows type I collagen (red) and a significant production of collagen type III (green) between inflammatory cells present in the reticular dermis. E. BALB/c mice 30 dpi. Window shows elastic (thin arrow) and elaunin fibres (thick arrow) preserved. F. C3H.He mice 60 days after infection. Integrity of elastic (thin arrow) and elaunin (thick arrow) and absence of oxytalan fibres (Weigert). G and H: C3H.He mice 60 days after infection. Picrosirius red in non-polarized (G) and polarized light (H). Attempt to repair infected dermis, with presence of collagens type I (red), III and I neoformed (yellow).

Figure 4

Histopathological analysis of footpad skin from mice subcutaneously infected with 10⁴ amastigotes of L. amazonensis. A and B: C3H.He mice 90 days after infection. A: Proliferation and arrangement of collagen fibre (plain arrow) compatible to dermis restoration (Masson); B: Dermo-epidermic junction shows preserved elastic (thin arrow) and elaunin fibres (thick arrow) (Weigert); C: BALB/c mice 120 days after infection. Diffuse inflammatory infiltrate (***) composed of PMN and macrophages heavily parasitized (arrowhead); D: C3H.He mice 120 dpi. (HE). It was observed an absence of lesions and regeneration of tissue architecture.

Figure 5

Immunohistochemical analysis of skin fragments from the left hind footpad from mice subcutaneously infected with 10⁴ L. amazonensis amastigotes. Fibronectin is labeled in green (FITC) and L. amazonensis amastigotes in red (Cy3): A: BALB/c infected mice and B: C3H.He infected mice 30 days after infection; C: BALB/c infected mice and D: C3H.He infected mice 120 days after infection.