Toll-like receptor 9 signaling in dendritic cells regulates neutrophil recruitment to inflammatory foci following Leishmania infantum infection

Abstract

Leishmania infantum is a protozoan parasite that causes visceral leishmaniasis (VL). This infection triggers dendritic cell (DC) activation through the recognition of microbial products by Toll-like receptors (TLRs). Among the TLRs, TLR9 is required for DC activation by different Leishmania species. We demonstrated that TLR9 is upregulated in vitro and in vivo during infection. We show that C57BL/6 mice deficient in TLR9 expression (TLR9(-/-) mice) are more susceptible to infection and display higher parasite numbers in the spleen and liver. The increased susceptibility of TLR9(-/-) mice was due to the impaired recruitment of neutrophils to the infection foci associated with reduced levels of neutrophil chemoattractants released by DCs in the target organs. Moreover, both Th1 and Th17 cells were also committed in TLR9(-/-) mice. TLR9-dependent neutrophil recruitment is mediated via the MyD88 signaling pathway but is TIR domain-containing adapter-inducing interferon beta (TRIF) independent. Furthermore, L. infantum failed to activate both plasmacytoid and myeloid DCs from TLR9(-/-) mice, which presented reduced surface costimulatory molecule expression and chemokine release. Interestingly, neutrophil chemotaxis was affected both in vitro and in vivo when DCs were derived from TLR9(-/-) mice. Our results suggest that TLR9 plays a critical role in neutrophil recruitment during the protective response against L. infantum infection that could be associated with DC activation.

FIG 1

L. infantum infection induces TLR9 expression in DCs. (A and B) TLR9 expression levels in WT BMDCs infected with L. infantum (1:5 ratio) or medium for 24 h (A) and splenic DCs from WT mice at 6 wpi and uninfected (NI) control mice (B) were determined in vitro. The percentages and integrated mean fluorescence intensity (iMFI) of CD11c^high cells expressing TLR9 were determined by flow cytometry. (C) tlr9 mRNA expression levels in the spleens and livers of WT mice at 4 and 6 wpi were determined by real-time PCR and normalized to the level of the constitutively expressed HPRT gene. mRNA expression levels were calculated as the fold change compared to values for uninfected littermate controls. Data are expressed as the means ± SEM. Representative data from two independent experiments performed in quadruplicate (A) are shown (n = 4 to 5 [B and C]). *, P < 0.05 (determined by Student's t test [A and B] or two-way ANOVA with Tukey's post hoc test [C]) relative to medium (A) or uninfected controls (B and C).

FIG 2

TLR9 participates in the control of L. infantum infection. Parasite burdens in the spleens (A) and livers (B) of WT and TLR9^−/− mice at 4 and 6 wpi were determined by an LDA. Data are expressed as the means ± SEM and are representative of data from three independent experiments performed separately (n = 4 to 5). *, P < 0.05 (by Student's t test) relative to the WT group.

FIG 3

TLR9 promotes neutrophil recruitment to infection foci. (A and B) Dot plots representing neutrophils in the spleens of WT and TLR9^−/− mice at 4 wpi (A) and 6 wpi (B) as determined by flow cytometry. (C and D) Bar graphs displaying the percentages (C) and the absolute numbers (D) of cells characterized as a LY6G⁺ MHC-II⁻ population in spleens of WT and TLR9^−/− mice at 4 and 6 wpi or uninfected mice (NI). (E) Kinetics of neutrophil migration to the liver assessed by immunohistochemistry with anti-mouse antibody 7/4 in tissues of WT and TLR9^−/− mice at different times postinfection. WT and TLR9^−/− mice were intraperitoneally infected with 1 × 10⁷ cells/ml of L. infantum. (F) PECs were harvested 24 h after parasite infection, and the neutrophil population was phenotyped as being LY6G⁺ MHC-II⁻ by flow cytometry. (G) Parasite burden in the spleen and liver after neutrophil depletion in infected WT mice. Data are expressed as the means ± SEM. Representative data from three (A to D) or two (E to G) independent experiments are shown (n = 4 to 5). *, P < 0.05 (determined by Student's t test [E to G] or two-way ANOVA with Tukey's post hoc test [C and D]) compared with the WT (A to F) or IgG (G) group. #, P < 0.05 (by Student's test) compared to naive WT mice; &, P < 0.05 (by Student's test) compared with naive TLR9^−/− mice.

FIG 4

Neutrophil activation is not altered in TLR9^−/− mice infected with L. infantum. CD11b^high and CXCR2 expression as well as CD62L shedding were used to characterize the neutrophil activation profile in vitro following L. infantum infection. (A to C) Bone marrow-derived neutrophils from WT and TLR9^−/− mice were cultured at a ratio of 5:1 (Leishmania parasites to neutrophils) with Leishmania or medium for 12 h for in vitro activation assays. (D to F) For in vivo activation assays, blood neutrophils from WT and TLR9^−/− mice at 6 wpi were used. The LY6G⁺ MHC-II⁻ population was analyzed by flow cytometry. Histograms represent data for infected and noninfected (NI) WT and TLR9^−/− mice. MFI, mean fluorescence intensity. (G) A migration assay was performed by using WT and TLR9^−/− neutrophils with MIP-2 (30 ng/ml) or medium as a chemotactic factor for 1 h. Data are expressed as the means ± SEM. Representative data from two (A to F) and three (G) independent experiments performed in quadruplicate (A to C) and quintuplicate (G) are shown (n = 4 to 5 [D to F]). #, P < 0.05 (by two-way ANOVA with Tukey's post hoc test) compared with medium.

FIG 5

TLR9 is required for DC maturation and production of neutrophil chemotactic factors. (A) Spleen cells from WT and TLR9^−/− mice were collected at 6 wpi and in vitro stimulated with L. infantum antigen (50 μg/ml) or medium. The levels of KC and MIP-2 in culture supernatants were then measured by an ELISA. (B) WT and TLR9^−/− BMDCs were infected with L. infantum (5:1) or medium for 24 h. BMDCs were harvested, and costimulatory molecule expression was evaluated by flow cytometry. (C) Levels of surface markers on DCs from infected WT and TLR9^−/− mice at 6 wpi were determined. All analyses were performed on the CD11c^high population. (D and E) KC and MIP-2 levels in the supernatants from WT and TLR9^−/− BMDCs cultured with medium, L. infantum (D), or CpG for 24 h (E) were measured by an ELISA. Data are expressed as the means ± SEM. Shown are data from one representative of two (E) and three (A to D) independent experiments performed in quadruplicate (B, D, and E) (n = 4 to 5 [A and C]). #, P < 0.05 (by two-way ANOVA with Tukey's post hoc test) compared with medium (A, B, D, and E); *, P < 0.05 (by two-way ANOVA with Tukey's post hoc test [B to E] or Student's test [A, C]) compared with the WT group.

FIG 6

TLR9 is important for neutrophil recruitment mediated by the production of chemoattractants by DCs. (A) In vitro migration assays using a Boyden chamber were performed with WT bone marrow-derived neutrophils, using supernatants from WT or TLR9^−/− BMDCs cultured with L. infantum or medium as a chemoattractant. MIP-2 (30 ng/ml) or medium was used as the positive or negative control, respectively. (B) Air pouches were created on the dorsal side of WT mice injected with the supernatant from WT or TLR9^−/− BMDCs cultured with L. infantum or medium. LPS (1 μg/pouch) or medium was used as the positive or negative control, respectively. (C) Filtered supernatants from WT or TLR9^−/− BMDCs cultured with L. infantum or medium were injected into air pouches. Exudate cells were harvested and stained for neutrophil phenotype. Data are expressed as the means ± SEM. Shown are representative data from three (A and B) independent experiments performed in quintuplicate (A) (n = 5 [B and C]). *, P < 0.05 (by two-way ANOVA with Tukey's post hoc test) for TLR9^−/− mice compared with WT mice.

FIG 7

TLR9-dependent neutrophil recruitment is mediated by the MyD88 signaling pathway. (A) Parasite burdens in spleens and livers from WT, MyD88^−/−, and TRIF^−/− mice were determined at 6 wpi by an LDA. (B) Numbers of neutrophils in spleens of WT, MyD88^−/−, and TRIF^−/− mice at 6 wpi were determined by flow cytometry. (C) Spleen cells from WT, MyD88^−/−, and TRIF^−/− mice at 6 wpi were in vitro stimulated with L. infantum antigen (50 μg/ml) or medium, and the levels of KC and MIP-2 in culture supernatants were measured by an ELISA. Representative data from three independent experiments are shown (n = 5). #, P < 0.05 compared with the NI group; *, P < 0.05 for MyD88^−/− mice compared with WT mice; &, P < 0.05 for MyD88^−/− mice compared with TRIF^−/− mice (determined by two-way ANOVA with Tukey's post hoc test).

FIG 8

Activation and neutrophil chemoattractant expression in pDCs and mDCs are dependent on TLR9 signaling during L. infantum infection. (A and B) Spleen cells from uninfected WT or TLR9^−/− mice at 6 wpi were isolated and harvested for flow cytometry analysis. Dot plots represent the frequencies of CD11c⁺ B220⁺ and CD11c⁺ B220⁻ cells (A), and graphs represent the absolute numbers of these cells (B). (C to F) Costimulatory molecule expression was evaluated by gating the CD11c⁺ B220⁺ (C and D) or CD11c⁺ B220⁻ (E and F) population. The histograms represent data from infected WT and TLR9^−/− mice (C and E). (G and H) KC and MIP-2 mRNA expression levels in sorted CD11c⁺ B220⁺ or CD11c⁺ B220⁻ cells from WT and TLR9^−/− mice at 6 wpi were determined by real-time PCR and normalized to the expression level of the constitutively expressed GAPDH gene. Data are expressed as the means ± SEM. Representative data from two independent experiments (A to F) are shown (n = 4 to 5 [A to H]). #, P < 0.05 compared with the NI group; *, P < 0.05 compared with infected WT mice (two-way ANOVA with Tukey's post hoc test [B, D, and F] or Student's t test [G and H]).