Aedes aegypti saliva impairs M1-associated proinflammatory phenotype without promoting or affecting M2 polarization of murine macrophages

Abstract

Background: During the feeding process, the mouthparts of hematophagous mosquitoes break the skin barrier and probe the host tissue to find the blood. The saliva inoculated in this microenvironment modulates host hemostasis, inflammation and adaptive immune responses. However, the mechanisms involved in these biological activities remain poorly understood and few studies explored the potential roles of mosquito saliva on the individual cellular components of the immune system. Here, we report the immunomodulatory activities of Aedes aegypti salivary cocktail on murine peritoneal macrophages.

Results: The salivary gland extract (SGE) of Ae. aegypti inhibited the production of nitric oxide and inflammatory cytokines such as interleukin-6 (IL-6) and IL-12, as well as the expression of inducible nitric oxide synthase and NF-κB by murine macrophages stimulated by lipopolysaccharide (LPS) plus interferon-γ (IFN-γ). The spare respiratory capacity, the phagocytic and microbicidal activities of these macrophages were also reduced by Ae. aegypti SGE. These phenotypic changes are consistent with SGE suppressing the proinflammatory program of M1 macrophages. On the other hand, Ae. aegypti SGE did not influence M2-associated markers (urea production, arginase-1 and mannose receptor-1 expression), either in macrophages alternatively activated by IL-4 or in those classically activated by LPS plus IFN-γ. In addition, Ae. aegypti SGE did not display any cytokine-binding activity, nor did it affect macrophage viability, thus excluding supposed experimental artifacts.

Conclusions: Given the importance of macrophages in a number of biological processes, our findings help to enlighten how vector saliva modulates vertebrate host immunity.

Keywords: Aedes aegypti; Inflammation; M1/M2 polarization; Macrophages; Saliva; microbicidal activity.

Fig. 1

Aedes aegypti SGE inhibits NO production and iNOS expression by M1-polarized murine macrophages. Thioglycolate-elicited peritoneal macrophages were collected and cultured as described in “Methods”. Cells were preincubated with complete medium (control group) or with SGE (final concentration indicated in the figure; otherwise 40 µg/ml) for 1 h and stimulated or not with LPS plus IFN-γ (final concentration: 10 ng/ml of each). NO production was indirectly estimated after 48 h in culture supernatants by Griess reaction (a). iNOS expression was evaluated after 24 h in cell lysates by Western blot (b). The relative expression of iNOS was determined by densitometry and the results were presented as percentage in relation to the control group (considered as 100%) (c). Results are expressed as the mean ± SEM. *P < 0.05 versus control group (cells incubated with medium only); ^#P < 0.05 versus “LPS + IFN-γ” group

Fig. 2

Aedes aegypti SGE affects viability of murine lymphocytes but not of macrophages. Thioglycolate-elicited peritoneal macrophages and total spleen cells were collected and cultured as described in “Methods”. Cells were maintained into culture tubes and incubated with complete medium (control group) or with SGE (final concentration: 10 and 40 µg/ml) for 4 h. Annexin V staining was evaluated by flow cytometry in CD3⁺ cells (T lymphocytes) (a), CD19⁺ cells (B lymphocytes) (b), and CD11b⁺F4/80⁺ cells (macrophages) (c). In another set of experiments, cells were preincubated with complete medium (control group) or with SGE (final concentration: 10 and 40 µg/ml) for 1 h and stimulated with Con A (0.5 μg/ml final concentration) for spleen cell cultures (d) or LPS plus IFN-γ (10 ng/ml of each, final concentration) for macrophage cultures (e). Twenty-five microliters of 0.01% resazurin were added to the cultures and after 48 h, cell viability was evaluated by reading the cultures absorbance at 570 and 600 nm. Results are expressed as the mean ± SEM. *P < 0.05 versus control group (cells incubated with medium only); ^#P < 0.05 versus “SGE 10” group

Fig. 3

Macrophage spare respiratory capacity is decreased in the presence of Ae. aegypti SGE. Thioglycolate-elicited peritoneal macrophages were collected and cultured in Seahorse plates as described in Methods. Mitochondrial function was compared in basal conditions and after the sequential addition of oligomycin (1 μg/ml), CCCP (4.5 μM) and rotenone/antimycin A (1 μM each) on a Seahorse flux analyzer. Basal and maximal OCR are expressed as the mean ± SEM. *P < 0.05 versus control group (cells incubated with medium only)

Fig. 4

Aedes aegypti SGE differentially modulates inflammatory and anti-inflammatory cytokine production, as well as NF-κB expression, by M1-polarized murine macrophages. Thioglycolate-elicited peritoneal macrophages were collected and cultured as described in “Methods”. Cells were preincubated with complete medium (control group) or with SGE (final concentration: 40 µg/ml) for 1 h and then stimulated or not with LPS plus IFN-γ (final concentration: 10 ng/ml of each). Cell culture supernatants were collected after 6 h of culture for TNF-α (c) or after 48 h for IL-6 (a), IL-12 (b) and IL-10 (d) determinations by ELISA. Phosphorylated NF-κB (pNF-κB) expression was evaluated after 30 min in cell lysates by Western blot (e). The relative expression of pNF-κB was determined by densitometry and the results were presented as percentage in relation to the control group (considered as 100%) (f). Results are expressed as the mean ± SEM. *P < 0.05 versus control group (cells incubated with medium only); ^#P < 0.05 versus “LPS + IFN-γ” group

Fig. 5

Aedes aegypti SGE does not bind murine IL-6 or IL-12. Different concentrations of Ae. aegypti SGE (1, 5 and 10 µg/ml) were preincubated with a serial dilution of mouse recombinant IL-6 or IL-12 for 15 minutes at 37 °C. Samples were then transferred to ELISA plates coated with anti-IL-6 or anti-IL-12 capture monoclonal antibody. For each case, a control group (a serial dilution of recombinant cytokine diluted in buffer only) was assayed under the same conditions (a, c). ELISA plate wells were coated with Ae. aegypti SGE (10 µg/ml), anti-IL-6 or anti-IL-12 antibody and incubated with serial dilutions of recombinant IL-6 or IL-12 (b, d). The detection of the cytokines was performed as described in “Methods”. Results are expressed as the mean ± SEM

Fig. 6

Aedes aegypti SGE impairs M1 but not M2 polarization. Thioglycolate-elicited peritoneal macrophages were collected and cultured as described in “Methods”. Cells were preincubated with complete medium (control group) or with SGE (final concentration: 40 µg/ml) for 1 h and maintained in medium (M0 condition), stimulated with LPS plus IFN-γ (final concentration: 10 ng/ml of each) for M1 polarization or with IL-4 (final concentration: 20 ng/ml) for M2 polarization. After 48 h, culture supernatants were collected for NO determination by Griess reaction (a) and the cell lysate were prepared for urea determination as a product of the arginase activity (b). Arginase-1 (c) and mannose receptor-1 (MRC-1) (e) expression were evaluated by Western blot. The relative expression of arginase-1 and MRC-1 were determined by densitometry and the results were presented as percentage in relation to the control group (considered as 100%) (d and f, respectively). Results are expressed as the mean ± SEM. *P < 0.05 versus respective control group (cells incubated with medium or SGE only)

Fig. 7

Aedes aegypti SGE impairs bacterial internalization and microbicidal activity by murine macrophages. Thioglycolate-elicited peritoneal macrophages were collected and cultured as described in “Methods”. Cells were preincubated with complete medium (control group) or with SGE (final concentration: 40 μg/ml) for 1 h and stimulated with green fluorescent E. coli at a multiplicity of infection of 10. Bacterial uptake was evaluated after 1 h by flow cytometry and represented as density plots (a) and as median fluorescence intensity (MFI) (b) in F4/80⁺ cells (macrophages); the bacterial killing was estimated after 5 h by determination of colony-forming units (c). Results are expressed as the mean ± SEM. *P < 0.05 versus E. coli group