In short, all challenged mice demonstrated clear signs of allergic response, demonstrating the ant venom proteins will immunize even at the lowest tested doses, and in the absence of adjuvants upon first exposure. Details are given as per the pertinent sections below.

Ant venom promotes eosinophil recruitment to peritoneal cavity

To test the premise that venom proteins are the causative factors of allergic reactions to fire ant stings, we sensitized mice via venom proteins into the hind footpad. Fourteen days later, the same mice were challenged by a second injection into the peritoneal cavity. The observed increase in peritoneal cellularity in venom-sensitized mice was mainly due to significant eosinophil recruitment. Injection of venom proteins into non-sensitized (naive) mice promotes merely mild, non-significant eosinophil recruitment (Fig. 2). Interestingly, inoculating ovalbumin (OVA) into mice previously sensitized with combined venom proteins + OVA promotes recruitment of eosinophils comparable to positive controls of combined OVA + aluminum hydroxide (as an adjuvant). This result suggests a potential role of venom proteins as adjuvants in allergic sensitization.

Figure 2 Ant venom promotes eosinophil recruitment to peritoneal cavity. Mice were previously injected (sensitized: S) with saline (Sal), OVA + AlOH 3 (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. Full size image

Eotaxin production triggered by ant venom

Peritoneal eotaxin accumulation in mice challenged with venom protein was higher in saline-injected mice (Fig. 3). The increased eotaxin levels of venom-inoculated mice were comparable to concomitant positive controls (see red bar ‘SOACO’ on Fig. 3). Taken together, Figs. 2 and 3 strongly suggest that venom proteins per se can actively promote sensitization-dependent eosinophil recruitment to the peritoneal cavity after a second exposure through the production of eosinophilotactic signals such as eotaxin.

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. Full size image

Dendritic cells activation by ant venom

To investigate a potential role of RIFA venom proteins as adjuvants in immunization, we evaluated murine bone marrow-derived dendritic cells (BMDC) after exposure to venom proteins or alkaloids from the venom (Fig. 4A–C). Dendritic cells are responsible for the induction of adaptive immune responses, and seen as ‘sentinels’ for the immune system25. The venom alkaloids proved extremely cytotoxic even at 10 µg/mL (data not shown) indicating these venom components may not be relevant direct immunogenic stimuli for dendritic cells. Meanwhile, the venom proteins elicited an activation response, as shown by the upregulation of MHCII and CD86 (Fig. 4). This demonstrates that the venom proteins can directly activate dendritic cells to promote antigen presentation, of which the increased expression of MHCII is a first marker signal of immunological activation. Following expression of MHCII, secondary signals (costimulatory signals) are needed to elicit a response from lymphocytes, including CD86. The fact that the highest doses of venom generated a weaker response may indicate the presence of intrinsically dynamic components, such as proteinase-regulated zymogens and enzymatic inhibitors. Toxin activation by dilution could be a simple mechanism to achieve high levels of damage towards injected victims whilst ensuring a natural resistance by the ants. In fact at least one phospholipase inhibitor is described from the venom of RIFA26, indicating secreted proteases might be involved in the activation of zymogens.

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. Full size image

In vivo hypersensitivity

The literature reports that the hypersensitive reactions following RIFA stings may include swelling of distal body parts which might lead to a life-threatening throat obstruction in humans6,7, initially signaled by wheezing, difficulty in breathing, and a coarse voice. To test for allergen-triggered swelling, mice were sensitized with an intradermal inoculum of venom proteins into their right hind footpad (sensitization), and 14 days later the opposite hind footpad (left) was challenged with another inoculum. Both footpads were measured every 30 min from the moment of inoculation until up to 2 h after injections.

Naturally, all assayed mice displayed marked swelling in the injected footpad as a result of the inoculum, but the effects become visible as the injected volume is drained. Full drainage of the initial swelling is completed within 2 hours following the injections. After 2 weeks following the first exposure, a second inoculation (i.e. challenge) resulted in persistent footpad swelling of venom-exposed animals comparable to positive controls (OVA + Al 2 (OH) 3 ) (Fig. 5). To address a possible role of venom proteins as adjuvants, mice were sensitized with an association between OVA and venom protein, or just OVA. When further challenged with OVA, only the mice that received OVA + venom proteins presented footpad swelling (Fig. 6). These results establish a sensitization-dependent swelling upon later challenge as an immunogenic response to fire ant venom proteins.

Figure 5 Footpad swelling after ant venom protein-fraction challenge. Mice were previously sensitized with saline, ovalbumin (OVA) + AlOH 3 (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). Full size image

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). Full size image

Draining lymph node response

We assessed the hyperplasia of the draining lymph node (popliteal) 14 days after sensitization (Fig. 7). The lymph nodes of positive control mice (OVA + Al(OH) 3 ) became greatly swollen two weeks after the sensitization. Venom-sensitized mice showed marked but less intense lymph node hyperplasia when sensitized with venom proteins. These lymph nodes were dissected out of the euthanized mice to evaluate the number of cells and the production of IL-4, the major Th2 cytokine, in order to test whether venom proteins induced priming of T cells in vivo. As indicated by the size of the respective lymph nodes, venom protein-sensitized mice presented an increase in the total cell number per lymph node when compared to the negative controls. Interestingly, lymph node hyperplasia from mice sensitized with the highest dose of venom proteins (100 μg) was not statistically different from the negative control, although a clear trend is apparent (Fig. 7). The increased cell number is indicative of lymphocyte proliferation triggered by venom proteins exposure, although this proliferative activity was less intense than in the positive control (OVA + Al(OH) 3 ). However, when stimulated in vitro for cytokine production, cells from the draining lymph nodes of venom-sensitized mice produced significant amounts of IL-4 (Fig. 8), comparable to OVA + Al(OH) 3 sensitized positive controls, with an apparent tendency to greater production after 48 h (see whiskers on right hand side of axis on Fig. 8).

Figure 7 Fire ant venom induces lymph node response. Mice were previously immunized with either OVA + AlOH 3 (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. Full size image

Figure 8 Ant venom induces cytokine response in lymph node cells. Mice were previously immunized with saline, ovalbumin + AlOH 3 (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. Full size image

Venom proteins as adjuvant

At least four different allergens are described from RIFA venom proteins13. However, the venom components driving the adaptive immune response have not been empirically identified. Crude fire ant venom is mainly composed of alkaloids, which are cytotoxic and insoluble13,26 and cause local tissue damage. Tissue damage is accompanied by a steep inflammatory response, typically followed by later pustule formation. Given the local inflammatory reaction and edema that follows fire ants stings, it is tempting to hypothesize that alkaloids would play a necessary biological role as adjuvants for ensuring the adaptive response (as in Fig. 1) to the few accompanying venom antigens, roughly akin to the role of aluminum adjuvants in injected vaccines27,28. However, venom alkaloids herein proved to be highly toxic to dendritic cells, while the proteins were sufficient to promote antigen presentation (increased MHC-II) and co-stimulatory (increased CD86) capacity (Fig. 4). To test this hypothesis, we injected naive mice with a mixture of venom proteins and ovalbumin in the absence of an aluminum adjuvant. Ovalbumin is innocuous when injected alone, but becomes a potent allergen when administered in combination with an adjuvant like aluminum hydroxide (Fig. 5), thus widely employed as a control in immunology experiments.

Mice previously sensitized with 100 μg of ovalbumin in the presence of 10 µg of fire ant venom protein extract (SOVCO) produced a significant increase in footpad volume when later challenged with ovalbumin, as compared to challenged mice previously sensitized with ovalbumin alone (SOCO) (Fig. 6). The amount of swelling was similar to OVA + Al(OH) 3 sensitized mice (SOACO) (Fig. 5). A similar adjuvant effect was observed when the OVA challenge injection was performed into the peritoneal cavity, where eosinophil influx was determined 24 h later (SOCO: 5.24 ± 1.53%; SOVCO: 12.15 ± 4.55%, p < 0.05). Since the extraction of venom proteins is not specifically demonstrated to eliminate potential microbial contaminants (e.g. LPS) such compounds could be involved in the observed effects.

To assess this possibility, we designed additional assays with heat-treated venom. Injection of venom induced significant eosinophil recruitment only when animals were previously exposed to the venom, indicating that neither contaminants nor the venom extract itself will trigger eosinophilic response without a previous sensitization (Fig. 2). Still, a possible role of contaminants in sensitization remained. To address this point, mice were sensitized with 100 µg of OVA combined with 10 µg of previously heat inactivated (100 °C/60 min) venom extract proteins (SOVhiCO).When SOVhiCO mice were challenged with OVA they elicited comparable amounts of eosinophils in the peritoneal cavity as mice sensitized with OVA alone (SOCO) (Fig. S2). Thus, heat exposure completely eliminated the adjuvant property of the venom extract, suggesting that it is fundamentally enzymatic, given that bacterial contaminants such as LPS are thermo-resistant29.