Sand Fly Salivary Proteins Induce Strong Cellular Immunity in a Natural Reservoir of Visceral Leishmaniasis with Adverse Consequences for Leishmania
Figure 5
Bites of Lu. longipalpis sand flies induce a strong focal and systemic adaptive cellular immune response in dogs immunized with LJL143 or LJM17.
(A–C) Dogs were exposed to uninfected and infected sand flies for 10 min one month after the final immunization with either LJM17, LJL143 or the empty plasmid (control). (A–C) Skin biopsies (6mm) obtained from bite sites 48 h post challenge with 20 and five uninfected and 10 infected sand flies were cut in half and processed for histology and RNA extraction. (A) Representative H&E staining and immunohistochemical labeling of T cells (anti-CD3) and macrophages (Mac387) at the bite sites of 20 uninfected sand flies in LJL143- and LJM17-immunized and control dogs. (B) Reverse-transcriptase quantitative PCR showing the expression levels of IFN-γ, IL-12, IL-4 and TGF-β at the bite sites of 20 or five uninfected sand flies in LJL143- and LJM17-immunized and control dogs (for control dogs RNA was combined from sites of 20 and 5 uninfected sand fly bites). (C) Same as (B) using 10 infected sand flies. Histological sections from bite sites of five uninfected and 10 infected sand flies are provided as Figure S1 and Figure S2, respectively. (D–E) PBMC from LJL143- and LJM17-immunized and control dogs obtained one week after exposure to sand flies. (D) Frequency and mean fluorescence intensity (MFI) of CD3+ T cells following stimulation with medium, rLJL143 or rLJM17. (E) Frequency of CD4+ and CD8+ T cells expressing IFN-γ in PBMC from LJL143- and LJM17-immunized dogs. Error bars represent means±S.E. * P<0.05, ** P<0.01.