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. 2018 Sep 26;8(1):14427.
doi: 10.1038/s41598-018-32327-z.

The allergic response mediated by fire ant venom proteins

Daniel Zamith-Miranda  1 , Eduardo G P Fox  2 , Ana Paula Monteiro  1 , Diogo Gama  3 , Luiz E Poublan  1 , Almair Ferreira de Araujo  1 , Maria F C Araujo  4 , Georgia C Atella  4 , Ednildo A Machado  3 , Bruno L Diaz  5
Affiliations

The allergic response mediated by fire ant venom proteins

Daniel Zamith-Miranda et al. Sci Rep. .

Abstract

Fire ants are widely studied, invasive and venomous arthropod pests. There is significant biomedical interest in immunotherapy against fire ant stings. However, mainly due to practical reasons, the physiological effects of envenomation has remained poorly characterized. The present study takes advantage of a recently-described venom protein extract to delineate the immunological pathways underlying the allergic reaction to fire ant venom toxins. Mice were injected with controlled doses of venom protein extract. Following sensitization and a second exposure, a marked footpad swelling was observed. Based on eosinophil recruitment and production of Th2 cytokines, we hereby establish that fire ant proteins per se can lead to an allergic response, which casts a new light into the mechanism of action of these toxins.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Hypothesized allergic reaction following a fire ant sting. Upon a first exposure, (left panel) peripheral resident cells recruit blood leukocytes that will differentiate into macrophages and dendritic cells. These phagocytes will endocytose and process the venom’s antigens. Dendritic cells exposed to venom components will be activated and mature, thus promoting their ability to trigger the adaptive immune response. In this scheme, a dendritic cell migrates into a lymph node to present fire ant venom-derived peptides via MHCII to a naive T cell in the presence of the costimulatory molecule CD86. Upon activation, T cells become Th2 cells and proliferate under secretion of IL-4, and which will eventually activate an antigen-compatible B lymphocyte. Activated lymphocytes undergo clonal expansion inside the lymph node. A key event at the immunization stage takes place within ca. 7–10 days: activated B cells mature into IgE-secretory plasma cells, and the secreted allergen-specific IgE bind to peripheral mast-cells’ FcεRI (this immunization event is marked with an *). Upon a second exposure (right panel), antigens will trigger a specific, amplified reaction: (i) mast cells carrying antigen-specific IgE are activated to secrete inflammatory factors (not tested in the present study); (ii) specific lymphocytes secrete IgE and IL-4 promoting further cell activation; (iii) the amplified reaction increases edema and promote intense recruiting of eosinophils.
Figure 2
Figure 2
Ant venom promotes eosinophil recruitment to peritoneal cavity. Mice were previously injected (sensitized: S) with saline (Sal), OVA + AlOH3 (SOACO), ant venom (10 μg = S10; 100 μg = S100), or OVA + 10 μg ant venom (SOVCO). After 2 weeks, mice were submitted to an intraperitoneal challenge (Challenge: C) with saline, OVA (CO), or 10 μg ant venom extract (C10). Twenty-four hours after the challenge, the peritoneal cells were harvested and the eosinophils were counted. Measurements are expressed as the percentage of eosinophils among the peritoneal cell population. Boxplot of pooled internal replicates from three independent experiments, where the vertical lines are upper and lower limits, and the internal line are median values. Different letters indicate statistical significance (p < 0.05) between groups as compared by Kruskal-Wallis followed by Dunn’s Multiple Comparisons Test.
Figure 3
Figure 3
Fire ant venom induces eotaxin production in the peritoneal cavity. Mice were previously immunized with saline, ovalbumin + Al(OH)3 (SOACO), or 100 μg ant venom extract (S100C10). After 2 weeks, mice were submitted to an intraperitoneal challenge with saline, ovalbumin, or 10 μg ant venom extract, respectively. Within 24 h after the challenge, mice peritoneal exudates were harvested and eotaxin quantification was performed by ELISA. Vertical axis numbers are calculated concentrations of eotaxin in pg/mL. Bars represent means from pooled replicates deriving from two independent experiments; dots are raw values. Treatment S100C10 was statistically different from Saline control by nonparametric Wilcoxon-Mann-Whitney test at alpha = 0.05.
Figure 4
Figure 4
Ant venom activates dendritic cells in vitro. BMDCs were stimulated with zymosan (1:5) or with different concentrations of fire ant venom extract. After a 24 h incubation period, the expression of MHCII (A), CD86 (B), and MHCII + CD86 (C) were evaluated. Bars represent means and errors of three independent experiments. Different letters indicate statistical significance (p-value < 0.05) between groups as compared by Kruskal-Wallis followed by Dunn’s Multiple Comparisons Test.
Figure 5
Figure 5
Footpad swelling after ant venom protein-fraction challenge. Mice were previously sensitized with saline, ovalbumin (OVA) + AlOH3 (SOACO), or fire ant venom extract (S10C10). After 2 weeks mice were respectively challenged with another exposure to saline, OVA or ant venom extract, and their footpad swelling was measured every 30 mins for 120 minutes. Lines represent means of the obtained swelling measures and the shaded area are standard errors from three independent experiments (N = 5 mice per group from 3 independent experiments).
Figure 6
Figure 6
Adjuvant function of fire ant venom proteins. Mice were previously immunized with saline, ovalbumin + ant venom extract (SOVCO), or ovalbumin (SOCO). After 2 weeks the mice were challenged with new injections of saline or ovalbumin; footpad swelling was measured within every 30 mins for 120 minutes. Lines represent means; shaded area are standard errors measured at each analysis time point (N = 5 mice per group from 3 independent experiments).
Figure 7
Figure 7
Fire ant venom induces lymph node response. Mice were previously immunized with either OVA + AlOH3 (SOA) or with 10 µg (S10) or 100 µg (S100) of of ant venom proteins extract. Two weeks after first exposure, the draining (popliteal) lymph nodes (LN) were isolated and cell totals were determined. The box plot represents upper and lower limits and quartiles from six independent experimental results; the internal line is the median. Different letters indicate statistical significance (p < 0.05) between groups as compared by Kruskal-Wallis followed by Dunn’s Multiple Comparisons Test.
Figure 8
Figure 8
Ant venom induces cytokine response in lymph node cells. Mice were previously immunized with saline, ovalbumin + AlOH3 (SOA) or with 10 µg (S10) or 100 µg (S100) of ant venom proteins. After 2 weeks, popliteal lymph nodes were dissected and macerated for cell extraction, followed by stimulation with α-CD3. The cytokine IL-4 was quantified from lymph nodes supernatants after 24 h (left side) and 48 h (righthand side) of incubation. Bars are mirrored for size comparison representing the means (n = 2 independent experiments) while the whiskers are the interval towards the maximum value; nd = not detectable.
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