Characterization of the early inflammatory infiltrate at the feeding site of infected sand flies in mice protected from vector-transmitted Leishmania major by exposure to uninfected bites

Abstract

Background: Mice exposed to sand fly saliva are protected against vector-transmitted Leishmania major. Although protection has been related to IFN-γ producing T cells, the early inflammatory response orchestrating this outcome has not been defined.

Methodology/principal findings: Mice exposed to uninfected P. duboscqi bites and naïve mice were challenged with L. major-infected flies to characterize their early immune response at the bite site. Mostly, chemokine and cytokine transcript expression post-infected bites was amplified in exposed compared to naïve mice. In exposed mice, induced chemokines were mostly involved in leukocyte recruitment and T cell and NK cell activation; IL-4 was expressed at 6 h followed by IFN-γ and iNOS2 as well as IL-5 and IL-10 expression. In naïve animals, the transcript expression following Leishmania-infected sand fly bites was suppressed. Expression profiles translated to an earlier and significantly larger recruitment of leukocytes including neutrophils, macrophages, Gr+ monocytes, NK cells and CD4+ T cells to the bite site of exposed compared to naïve mice post-infected bites. Additionally, up to 48 hours post-infected bites the number of IFN-γ-producing CD4+ T cells and NK cells arriving at the bite site was significantly higher in exposed compared to naïve mice. Thereafter, NK cells become cytolytic and persist at the bite site up to a week post-bite.

Conclusion/significance: The quiet environment induced by a Leishmania-infected sand fly bite in naïve mice was significantly altered in animals previously exposed to saliva of uninfected flies. We propose that the enhanced recruitment of Gr+ monocytes, NK cells and CD4 Th1 cells observed at the bite site of exposed mice creates an inhospitable environment that counters the establishment of L. major infection.

Figure 1. Mice exposed to uninfected P. duboscqi bites are protected against vector-transmitted L. major.

(A) Representative parasite load and percent metacyclics in the gut of infected P. duboscqi sand flies the day of transmission. The mean ± SEM are shown. (B–C) Mice exposed to uninfected bites in the right ear (▪) or are naïve () were challenged with 10 L. major-infected P. duboscqi sand flies in the left ear two weeks after the last exposure. (B) Weekly measurement of ear lesions after transmission. The mean ± SEM for 10 mice per group are shown. (C) The number of parasites per ear for five mice at four weeks post-transmission determined by limiting dilution assay. The bar represents the mean parasite burden per ear. Data are representative of three independent experiments. *p<0.02; **p = 0.008.

Figure 2. The expression of inflammatory transcripts in naïve and sand fly-exposed mice following vector-transmitted L. major.

Mice exposed to uninfected bites in the right ear or naïve mice were challenged with 10 L. major-infected P. duboscqi sand flies (infected) or 10 uninfected sandflies (uninfected) in the left ear. Six and 12 hours following sand fly transmission, RNA was extracted from the challenged ear of 5 naïve or exposed mice and pooled. (A) Picture of a representative array after RNA hybridization and chemiluminescent detection. Spots were identified using the gene array map and the expressed transcripts were normalized to housekeeping genes. Results are expressed as fold increase in the intensity of the captured signal over the control group. The values are depicted as heat maps using a color code to indicate relative levels of chemokine (B) and cytokine (C) expression. Validation of mRNA expression by quantitative Real Time PCR is shown for IL-4 and CXCL13 at 6 h (D) and IFN-γ and CCL25 at 12 h (E) after transmission. Only genes showing at least a four-fold or higher change in expression in at least three out of four independent experiments are shown. Values represent relative transcript expression for 10 mice per group. *p<0.05, **p≤0.0001.

Figure 3. Characterization of the early leukocyte infiltrate following vector-transmission of L. major.

Ear cells were recovered from naïve and exposed mice at 6 h, 24 h, 48 h and one week after transmission. At the indicated times after challenge, cells were stained ex vivo and analyzed by FACS for surface expression of TCR-β, CD4, NK1.1, F4/80, CD11b, and Ly-6G to identify specific leukocyte populations. (A) Absolute number of leukocytes per ear; (B) The gating strategy used to identify cells (NK1.1^posTCR-β^neg); CD4⁺ T cells (TCR-β^posCD4^pos), neutrophils (F4/80^neg CD11b^highLy6G^high), Gr1⁺ inflammatory monocytes (F4/80^posCD11b^highLy6G^high) and macrophages (F4/80^highLy6G^negCD11b^high); (C) Absolute number of neutrophils, NK cells, Gr1⁺ monocytes, macrophages and CD4⁺ T cells per ear. The numbers shown represent mean ± SEM from 3 independent experiments. Five mice were used per group per experiment. *p<0.05.

Figure 4. IFN- γ expression by T cells and NK cells following infected sand fly bites.

Ear cells were recovered from naïve and exposed mice at 6 h, 24 h, 48 h and one week after transmission and cultured for 16 h. Brefeldin, PMA and ionomycin were added during the last four hours. The percent and absolute number of CD4+ T cells (A) or NK cells (B) expressing IFN-γ. Percentages shown are representative of three independent experiments; absolute numbers shown represent the mean ± SEM from three independent experiments. Five mice were used per group per experiment. *p<0.05.

Figure 5. Mature NK cells expressing Granzyme B persist in exposed mice following infected sand fly bites.

Ear cells were recovered from naïve and exposed mice at 6 h, 24 h, 48 h and one week after transmission and cultured for 16 h. Brefeldin, PMA and ionomycin were added during the last four hours. (A) The gating strategy used to identify NK cells. (B) The percent and absolute number of NK cells producing granzyme B in the NK1.1^posCD11b^posCD27^neg population. Percentages shown are representative of three independent experiments; absolute numbers shown represent the mean ± SEM from 3 independent experiments. Five mice were used per group per experiment. *p<0.05.