New Insights on the Adjuvant Properties of the Leishmania infantum Eukaryotic Initiation Factor

Figure 2

In vivo effect of recombinant LieIF protein on the peritoneal exudate cells. (a) Schematic representation of the experimental protocol. Female BALB/c mice were i.p. injected in the right quadrant with 500 μL of LieIF suspension (10 μg/mouse) in sterile PBS containing equal quantity of OVA protein (10 μg/mouse) or with 500 μL of OVA suspension (10 μg/mouse) in sterile PBS, while mice receiving only PBS were included. 2 and 24 h after injection, the peritoneal exudate cells (PEC) were harvested with 5 mL of ice-cold PBS. (b) Relative expression of TNF-α, IL-1β, and NF-κB2 genes in PEC. 2 h post-immunization, PEC were derived from each experimental group and relative expression of TNF-α, IL-1β, and NF-κB2 genes was determined by real-time PCR, performed with a SYBR Green PCR Master Mix. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used for normalization, and all expression levels were computed via the ΔΔCt method. Results shown are representative of three independent experiments. indicates statistically significant differences compared to PBS-immunized mice while indicates significant differences compared to OVA-immunized group. (c) Recombinant LieIF protein induces the upregulated expression of CD80 co-stimulatory molecule in PEC. 24 h post-immunization, PEC were harvested from each experimental group and cell surface expression of CD80 and CD86 co-stimulatory molecules was assessed by FACS analysis. The results are expressed as median fluorescent intensity (MFI) and as percentage (%) of cells expressing CD80 and CD86 molecules. Data are presented as mean of three independent experiments. The histogram overlay is representative of one experiment. and indicate statistically significant differences as compared to PBS- and OVA-immunized groups, respectively. (d) Recombinant LieIF protein promotes the production of NO by PEC. 24 h post-immunization, PEC were harvested from each experimental group and were further incubated in vitro with LieIF (10 μg/mL), IFN-γ (1 ng/mL), and LPS (1 μg/mL) or with LieIF+IFN-γ and LPS+IFN-γ, for 24 h at 37°C under 5% CO₂ environment. After the incubation period, NO production of each experimental group was determined in supernatants with the Griess reaction. Data are presented as mean of three independent experiments. For each in vivo experimental group, comparisons with cultured PEC that received no stimulation in vitro (light green bars) are indicated with and comparisons with cultured PEC received LPS (red bars) or LPS+IFN-γ (yellow bars) are indicated with ≠. Comparisons among the in vivo experimental groups are indicated with . (e) Recombinant LieIF protein elicits the secretion of MIP-1α by PEC. 24 h post-immunization, PEC were harvested from each experimental group and were further incubated in vitro with LieIF (10 μg/mL), IFN-γ (1 ng/mL), and LPS (1 μg/mL) or with LieIF+IFN-γ and LPS+IFN-γ, for 24 h at 37°C in the presence of 5% CO₂ environment. At the end of incubation period, culture supernatants were collected and MIP-1α levels were determined by ELISA. Data are presented as mean of three independent experiments. For each in vivo experimental group, comparisons with cultured PEC that received no stimulation in vitro (light green bars) are indicated with , comparisons with cultured PEC that received LPS (red bars) or LPS+IFN-γ (yellow bars) are indicated with ≠, and comparisons between cultured PEC that received LieIF (light blue bars) and LieIF+IFN-γ (orange bars) are indicated with ≠≠. Comparisons among the in vivo experimental groups are indicated with .

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