BluePort: a platform to study the eosinophilic response of mice to the bite of a vector of Leishmania parasites, Lutzomyia longipalpis sand flies

Abstract

Background: Visceral Leishmaniasis is a serious human disease transmitted, in the New World, by Lutzomyia longipalpis sand flies. Natural resistance to Leishmania transmission in residents of endemic areas has been attributed to the acquisition of immunity to sand fly salivary proteins. One theoretical way to accelerate the acquisition of this immunity is to increase the density of antigen-presenting cells at the sand fly bite site. Here we describe a novel tissue platform that can be used for this purpose.

Methodology/principal findings: BluePort is a well-vascularized and macrophage-rich compartment induced in the subcutaneous tissue of mice via injection of agarose beads covered with Cibacron blue. We describe the sequence of inflammatory events leading to its formation and how it can be used to study the dermal response to the bite of L. longipalpis sand flies. Results presented indicate that a shift in the inflammatory response, from neutrophilic to eosinophilic, is the main histopathological feature associated with the immunity acquired through repeated exposure to the bite of sand flies, and that the BluePort tissue compartment could be used to accelerate this process. In addition, changes observed inside the BluePort parenchyma indicate that it could be used to study complex immunobiological processes, and to develop ectopic secondary lymphoid structures.

Conclusions/significance: Understanding the characteristics of the dermal response to the bite of sand flies is a critical element of strategies to control leishmaniasis using vaccines that target salivary proteins. Finding that dermal eosinophilia is such a prominent component of the anti-salivary immunity induced by repeated exposure to sand fly bites raises one important consideration: how to avoid the immunological conflict derived from a protective Th2-driven immunity directed to sand fly saliva with a protective Th1-driven immunity directed to the parasite. The BluePort platform is an ideal tool to address experimentally this conundrum.

Figure 1. Acute inflammatory response to CBa-beads.

Histopathological changes observed 6 hours (A and B), 24 hours (C and D), 48 hours (E), 96 hours (F and G) and 15 days (H) after injection of CBa-beads in the subcutaneous tissue of BALB/c mice. Arrows indicate marginalized neutrophils in (A), neutrophils in the space between CBa-beads in (B, C and D), neutrophil extracellular traps (NETs) in (E), and mixed cellular infiltrate in (F-H). Hematoxylin-eosin stain. Magnification: 200x (B, C and E), 400x (A and H) and 1000x (D and G).

Figure 2. Chronic inflammatory response to CBa-beads.

Histopathological changes observed 30 days (A and B), 60 days (C and D), 90 days (E and F) and 120 days (G and H) days after injection of CBa-beads. Arrows indicate endothelial cells in (A), macrophages in (B), neutrophils inside a blood vessel in (C), capsule in (D), multinucleated foreign-body giant cells in (E), mast cells in (F), eosinophils in (G) and lymphocytes in (H). Hematoxylin-eosin stain, 1000x magnification.

Figure 3. Immuno-histochemical analysis of BluePort-resident cells.

Reactivity of antibodies to CD31, marker of endothelial cells, in (A and B), and to F4/80, marker of macrophages, in (C and D). Magnification 200x (A and C) and 1000x (B and D).

Figure 4. Localization and appearance of the BluePort tissue compartment.

Macroscopic appearance of the blue nodule (BluePort) formed one month after injection of CBa-beads in the subcutaneous tissue of mice (A and B). Macroscopic (C) and microscopic appearance (D) of BluePort-associated skin 24 hours after exposure of naïve mice to the bite of L. longipalpis sand flies. Black arrows indicate skin erythema in (C), and vasodilatation of dermal blood vessels in (D). White arrow indicates vasodilatation of blood vessels inside the BluePort parenchyma in (D). Hematoxylin-eosin stain, magnification 100x in (D).

Figure 5. Inflammatory response of naïve and immune mice to sand fly bites on BluePort-associated skin.

Evolution of the inflammatory response on naïve mice (A–D), and mice pre-exposed multiple times to the bite of sand flies (E–H). Images correspond to samples taken 24 hours (A and E), 48 hours (B and F), 72 hours (C and G) and 96 hours (D and H) post-exposure. Black arrows indicate neutrophils and white arrows eosinophils. Hematoxylin-eosin stain, 1000x magnification.

Figure 6. Inflammatory response of naïve and immune mice to sand fly bites on normal skin.

Inflammatory response detected 48 hours after exposure to the bite of L. longipalpis sand flies on naïve mice (A and B) and immune mice (C and D). Black arrows indicate neutrophils and white arrows eosinophils. Hematoxylin-eosin stain. Magnification, 200x in (A and C), 1000X in (B and D).

Figure 7. Inflammatory response in mice exposed twice to sand fly bites on BluePort-associated skin.

Evolution of the inflammatory response in samples collected 24 hours (A, E and I), 48 hours (B, F and J), 72 hours (C, G and K) and 96 hours (D, H and L) after exposure for the second time to the bite of sand flies. Black arrows indicate neutrophils and white arrows indicate eosinophils. Hematoxylin-eosin stain. Magnification: 100x in (A–D), 1000x in dermis (E–H) and hypodermis (I–L).

Figure 8. Histopathological changes in the parenchyma of a BluePort adjacent to sand fly bite-induced dermal eosinophilia.

Images represent inflammatory changes observed 24 hours after exposure, for a second time, to the bite of sand flies on BluePort-associated skin. Arrows indicate: foreign-body giant cells (A), attack and destruction of CBa-beads by neutrophils (B) and macrophages (C), and lymphocytic infiltration of the BluePort parenchyma (D). Hematoxylin-eosin stain, 1000x magnification.

Figure 9. Lectin-blot and immuno-blot analysis of L. longipalpis salivary glycoproteins.

Effect of enzymatic deglycosylation with PNGase F on two sources of sand fly salivary glycoproteins, SGL and HSL. Samples were stained for protein composition profile (Imperial), probed for the presence of N-linked glycans (GNL, AAL) and O-linked glycans (VVL, PNA), or probed for reactivity with IgG antibodies of animals exposed to the bite of sand flies on BluePort-associated skin. Arrows indicate the main antigenic salivary glycoproteins.