Lutzomyia longipalpis Saliva Induces Heme Oxygenase-1 Expression at Bite Sites

Abstract

Sand flies bite mammalian hosts to obtain a blood meal, driving changes in the host inflammatory response that support the establishment of Leishmania infection. This effect is partially attributed to components of sand fly saliva, which are able to recruit and activate leukocytes. Our group has shown that heme oxygenase-1 (HO-1) favors Leishmania survival in infected cells by reducing inflammatory responses. Here, we show that exposure to sand fly bites is associated with induction of HO-1 in vivo. Histopathological analyses of skin specimens from human volunteers experimentally exposed to sand fly bites revealed that HO-1 and Nrf2 are produced at bite sites in the skin. These results were recapitulated in mice ears injected with a salivary gland sonicate (SGS) or exposed to sand fly bites, indicating that vector saliva may be a key factor in triggering HO-1 expression. Resident skin macrophages were the main source HO-1 at 24-48 h after bites. Additionally, assays in vivo after bites and in vitro after stimulation with saliva both demonstrated that HO-1 production by macrophages was Nrf2-dependent. Collectively, our data demonstrates that vector saliva induces early HO-1 production at the bite sites, representing a major event associated with establishment of naturally-transmitted Leishmania infections.

Keywords: Lutzomyia longipalpis; Nrf2; heme oxygenase-1; macrophages; saliva; sand fly bite; skin.

Figure 1

Experimental exposure to Lutzomyia longiplapis sand flies induces HO-1 and Nrf2 protein expression at bite sites in healthy human volunteers. (A,B,D,E) Immunohistochemistry of paraffin-embedded sections of skin specimens obtained by biopsy of the bite sites 48 h after sand fly exposure. (A) HO-1 staining in sand fly-exposed skin, 400X. (B) Higher magnification from image (A) (1000X). (C) Nrf-2 staining in sand fly-exposed skin, 400X. (D) Higher magnification from image (C) (1000X). (E) HO-1 staining in unbitten skin (1000X). (F) Nrf-2 staining in unbitten skin (1000X). Bar, 20 μm. Red arrows point to cells positively stained with HO-1 or Nrf2. Digital images 400 × (lower magnification) or 1000X (higher magnification) were captured using a Nikon E600 microscope and an Olympus Q-Color 1 digital camera with the Image Pro Plus software. (G) Plasma HO-1 levels in individuals (n = 7) before and after nine exposures to sand fly bites were measured by ELISA. Plasma samples were collected 48 h after each exposure to sand fly bites. Each line represents one individual volunteer. Data represent individual values and were compared using the Kruskal-Wallis test (p = 0.08).

Figure 2

Bites of Lutzomyia longipalpis sand flies induce Nfr2-dependent HO-1 protein expression by resident macrophages in mice. Ears of C57BL/6 mice were exposed to bites of 20 uninfected L. longipalpis sand flies (UB, uinfected bite) and tissue was processed for total protein extraction before or after sorting by flow cytometry. (A,B) Western blots of total protein extracts from mice ears. Hsp90 was used as a loading control. (A) kinetics of HO-1 expression following sand fly bites. Total protein extract from a mouse spleen was used as a positive control. (B) HO-1 expression in wild-type, Nrf2 knockout (Nrf2^−/−) and IL-10 knockout (IL-10^−/−) mice 24 h after exposure to sand fly bites. (C,D) Ear cells recovered 24 h after sand fly bites were stained with antibodies for population sorting by flow cytometry prior to total protein extraction. (C) Gating strategy for sorting populations of non-myeloid cells (I), neutrophils (II), resident macrophages (III) and inflammatory monocytes (IV). (D) Western blot of total protein extract from populations sorted according to (C). Naïve total ear cells were used as a negative control (CT). Histone H3 was used as a loading control. Blots are representative of two to three independent experiments.

Figure 3

Lutzomyia longipalpis saliva activates ARE, nuclear translocation of Nrf2, and HO-1 expression in human macrophages. (A) THP-1 derived macrophages were plated on coverslips and stimulated with either the equivalent of one pair of salivary gland sonicate (SGS) per well or 30 μM CoPP (cobalt protoporphyrin IX) for 24 h, then stained for HO-1. Cells were counter-stained with 1 μg/mL of DAPI to visualize the nucleus. Orange and Blue colors indicate positive staining for HO-1 and nuclei, respectively. Bar, 20 μm. Digital images (400 × original magnification) were captured on a Nikon E600 microscope coupled to an Olympus Q-Color 1 digital camera, and visualized using Image Pro Plus software. (B,C) THP-1 macrophages were treated with one pair SGS per well, followed by extraction of total protein, and detection by Western blot. (B) HO-1. (C) Nrf-2. β-actin and Lamin B were used as loading controls. (D) RAW 264.7 cells were transiently transfected with p3xARE- or pNrf2-promoterluciferase reporter plasmids constructs and treated with 30 μm CoPP or 10 mM DL-sulforaphane (SFN) or one pair SGS per well for 24 h post-transfection. Whole-cell lysates were analyzed for luciferase activity of p3xARE and Nrf2. Data are representative of three independent experiments.

Figure 4

Lutzomyia longipalpis saliva induces HO-1 protein expression at bite sites in mice. C57BL/6 mice were injected intradermally in the ear with the equivalent of 1 pair of L. longipalpis salivary gland sonicate (SGS). (A) Western blot of whole-tissue extract 24 h after sand fly exposure. β-actin was used as a loading control. (B) Optical density of the bands in (A) was quantified using ImageJ software. Data are presented as the ratio of HO-1 to β-actin. Data are representative of two independent experiments.