Figure 2. L. donovani-infected sand fly bites activate the NLRP3 inflammasome in neutrophils.

(A, C–G and I) Mice ears processed 6h after exposure to 20 infected (IS) or uninfected (US) sand flies.

(A) Ex-vivo IL1β protein levels from ear lysates measured by ELISA. Data are representative of two independent experiments (n ≥ 7 mice ears per condition). Dotted line represents the mean + SEM of endogenous IL1β levels in naïve samples.

(B) Mice ear sections stained with either anti-IL1β antibody targeting the mature protein or its isotype control (CTL) at 3–18h after IS or US. Pictures are representative of two independent experiments (n = 2 mice ears per condition per timepoint). Scale bars indicate 20µm. Arrows indicate IL1β secreting cells.

(C–E) Cells recovered from mice ears after IS were stained for flow cytometry. Representative plots of IL1β⁺ cells (C) back-gated (red) onto LY6G⁺ / CD11b⁺ neutrophils (D). (E) The number of IL1β⁺ neutrophils per 10⁵ ear cells from two independent experiments (n = 6 mice ears per condition).

(F) Representative Western blot of NLRP3, Caspase 1 and IL1β protein levels after IS and US (n = 4 mice ears per condition).

(G) Representative Western blot of NLRP3 protein levels after immunoprecipitation using anti-ASC antibody (n = 2 mice ears per condition).

(H) NLRP3 and IFNβ mRNA expression determined by qPCR at 3–18h after IS or US. Data are pooled from three independent experiments (n ≥ 7 mice ears per condition per timepoint).

(I) Mice ear sections stained with TUNEL. Pictures are representative of two independent experiments (n = 3 mice ears per condition). Boxed area is magnified to highlight apoptotic cells (arrow). Scale bars indicate 20µm.

(J) Mature IL1β and IL-10 cytokine levels in cell culture supernatant after in vitro stimulation of bone marrow-derived macrophages with L. donovani (Ld) stationary parasites in the presence or absence of lipopolysaccharide (LPS). Med, medium. Data are representative of two to three independent experiments (n = 2 replicates per condition).

Statistical significance *P < 0.05, **P < 0.01, ***P < 0.001 calculated by the unpaired two-tailed t test (A and H), the Mann-Whitney test (E), and the one-way ANOVA followed by a Holm-Sidak multiple comparisons test (J). Error bars indicate the mean ± SEM.

See also Table S1.