The contribution of Toll-like receptor 2 to the innate recognition of a Leishmania infantum silent information regulator 2 protein

Abstract

We have characterized a Leishmania protein belonging to the silent information regulator 2 (SIR2) family [SIR2 related protein 1 (SIR2RP1)] that might play an immunoregulatory role during infection through its capacity to trigger B-cell effector functions. We report here that SIR2RP1 leads to the proliferation of activated B cells, causing increased expression of major histocompatibility complex (MHC) II and the costimulatory molecules CD40 and CD86, which are critical ligands for T-cell cross-talk during the development of adaptive immune responses. In contrast, B cells isolated from Toll-like receptor 2 (TLR2) knockout mice were unable to respond to the SIR2RP1 stimulus. Similarly, SIR2RP1 induced the maturation of dendritic cells (DCs) in a TLR2-dependent manner with the secretion of pro-inflammatory cytokines [interleukin (IL)-12 and tumour necrosis factor (TNF)-alpha] and enhanced the costimulatory properties of DCs. Nevertheless, immunization assays demonstrated that TLR2-deficient mice were able to mount a specific humoral response to SIR2RP1. Interestingly, further investigations showed that macrophages were activated by SIR2RP1 even in the absence of TLR2. Therefore, a different type of interplay between SIR2RP1 and the major antigen-presenting cells in vivo could explain the immune response observed in TLR2-deficient mice. Together, these results demonstrate that TLR2 signalling contributes to SIR2RP1 recognition by innate immune host cells.

Figure 1

The effect of SIR2RP1 on mRNA expression of Toll-like receptor and downstream signaling molecules. (a) 2·5 × 10⁵ spleen isolated B-cells were cultured with or without 2 μg of SIR2RP1 for 20 hr. Total RNA was extracted from the B cells and reverse transcribed to cDNA. The cDNA sample was used as a template for 25–30 cycles of PCR using primers for TLR1, TLR2, TLR4, TLR6, MyD88 and NF-κB. GAPDH was used as control housekeeping gene to assure the samples homogeneity. Ethidium bromide-stained PCR products were photographed, and the images were digitized and analyzed. (b) The PCR products were quantified and expressed as the ratio of each product regarding the GAPDH band density (mRNA expression units). The data represent the mean ± standard deviation of three independent experiments. *P < 0·05.

Figure 2

Cell proliferation responses upon SIR2RP1 treatment. Total spleen cells (a) or purified spleen B-cells (b) 2·5 × 10⁵ cells per well from BALB/c, C57BL/6 WT and C57BL/6 TLR2^(−/−) mice were incubated with increasing concentrations of SIR2RP1 (0·02, 0·2, 2, 10 μg per well) for 72 hr. The corresponding stimulation index was determined by dividing the c.p.m. value for a given set of SIR2RP1-stimulated cells by the c.p.m. of the associated SIR2RP1-untreated cell (c) Proliferative response was also assessed using a LPS stimulus (d) Proliferation was assessed by [³H]-thymidine as described in Material and Methods section. Data are presented as mean c.p.m. and standard deviations subtracted from background values from unstimulated cells. The data are representative of three independent experiments.

Figure 3

TLR2-dependent secretion of interleukins by B cells. Purified spleen B-cells derived from BALB/c (black bars), C57BL/6 WT (white bars) or C57BL/6 TLR2^(−/−) (dashed bars) mice were cultured in 96-well plates at 1 × 10⁶/ml in the absence or the presence of LPS (1 μg/ml) or SIR2RP1 (1 or 10 μg/ml). After 72 hr of incubation, the levels of TNF-α (a) and IL-12p40 (b) were measured in the culture supernatants by ELISA. The results shown are one representative of two different experiments that yielded similar results. *P < 0·05, **P < 0·01.

Figure 4

Immunization of C57BL/6 TLR2^(−/−) mice with SIR2RP1 induced a decrease of IgG1/IgG2a ratio. Groups of 3 mice were immunized i.p. with SIR2RP1. Fifteen days after the last immunization, the levels of SIR2RP1-specific IgG (a) IgG1 (b) and IgG2 (c) antibodies were quantified in the sera of C57BL/6 WT, C57BL/6 TLR2^(−/−), and BALB/c mice by ELISA-limiting dilution. The ratio between SIR2RP1-specific IgG1 and IgG2a levels is shown for 1/200 and 1/800 sera dilutions (d) The asterisk indicates a statistically significant difference (P < 0·05) between C57BL/6 WT or BALB/c mice and TLR2-deficient C57BL/6 mice. The data represent the mean and the standard deviation of three animals analyzed individually and representative of two independent experiments.

Figure 6

Different outcome in the SIR2RP1-APC interaction. BM-Dendritic cells and BM-Macrophage derived from C57BL/6 WT (black bars) or C57BL/6 TLR2^(−/−) (white bars) mice were cultured in 24-well plates at 1 × 10⁶/ml in the absence or the presence of LPS (1 μg/ml) or SIR2RP1 (1, 5 or 10 μg/ml). After 24 hr the levels of IL-10, IL-12p40, IL-12p70 and TNF-α were measured in the culture supernatants by ELISA. The results shown are one representative of three different experiments that yielded similar results. N.D. – below the detection limit; For BM-dendritic cells and BM-Macrophage, *P < 0·05, **P < 0·01 between SIR2RP1 stimulated and unstimulated C57Bl/6 WT or C57BL/6 TLR2^(−/−) mice.

Figure 5

SIR2RP1 do not stimulate TLR2-deficient BM-DC cells. C57BL/6 WT and C57BL/6 TLR2^(−/−) mice BM-DC were incubated for 24 hr with medium (thin solid line), 10 μg/ml of SIR2RP1 (thick solid line) or 1 μg/ml of LPS (dotted line) and the surface expression of CD40, CD80, CD86 and class II MHC was determined by flow cytometry analysis. The shaded line represents the correspondent isotypic control. Results are representative of one of three independent experiments.

Figure 7

Allostimulatory capacity of BM-DC cultured with SIR2RP1. Immature DCs generated from C57Bl/6 WT or C57BL/6 TLR2^(−/−) mice were stimulated with SIR2RP1 (10 μg/ml) or LPS (1 μg/ml). After 4 days, DC were treated with mitomycin C, washed, and then cultured with freshly isolated allogeneic T cells from BALB/c mice. Proliferation of T cells in the allogeneic MLR was measured by [³H]-thymidine incorporation on the final 16 hr of the culture period of 5 days. Results are expressed as mean c.p.m. of quadruplicate values. *P < 0·05 and **P < 0·01 compared to the unstimulated control.