Pyriproxyfen is a juvenile hormone mimic of vital importance for insect development with little risk to humans. This study was performed to investigate whether large doses of pyriproxyfen affect the immune response in mammals. Mice were immunized thrice with ovalbumin in 5% ethanol, with or without pyriproxyfen or alum. Large doses of pyriproxyfen (9 or 15 mM) significantly enhanced specific total IgG immune response. This enhancement was no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, pyriproxyfen induced higher titers of IgG2a and enhanced tumor necrosis factor-alpha and gamma-interferon responses whereas alum induced IgG1 with enhanced interleukin-4 and -10. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that of alum.
cluster of differentiation 4
juvenile hormone analog
myeloid differentiation factor 88
phosphate-buffered saline with Tween-20
standard error of the mean
T-helper type 1
T-helper type 2
tumor necrosis factor-alpha
Juvenile hormones are a group of sesquiterpenes that regulate diverse aspects of insect physiology and ensure the growth and development of larvae while preventing metamorphosis . Several JHAs that are stable in the environment and mimic the biological action of JH as insect growth regulators, including methoprene, fenoxycarb and pyriproxyfen, have been synthesized . Although the mechanisms of action of JHs remain unclear, the lipophilic nature of sesquiterpene JHs suggests an intracellular receptor-mediated action. Recently, Met protein, which possesses a basic helix–loop–helix motif followed by two PAS domains, was reported to bind to pyriproxyfen and function as a JH-dependent transcription receptor . Therefore, pyriproxyfen is a potent ligand for Met, mimicking the function of JH and thus preventing adult transition. Previous studies in a mouse model have indicated that pyriproxyfen is stable and safe up to 5 g/kg when administered orally and is rapidly biodegraded after administration . However, the effects of large doses of pyriproxyfen on mammalian immune response are still unknown. Therefore, we explored whether large doses of pyriproxyfen affect the immune response. We aimed to determine the IgG immune response to pyriproxyfen and the widely used model antigen OVA. We also monitored other aspects of the immune profile in response to pyriproxyfen, including IgG subtypes such as IgG1 or IgG2a, IgE production and cytokines.
The four-week-old female BALB/c mice used in this study were purchased from Kyudo (Saga, Japan) and housed in a controlled specific pathogen-free environment with a 12 hr light/dark cycle (lights on from 07:00 to 19:00) and temperature and humidity controlled to 23 ± 2°C and 55 ± 5%, respectively. Feed (CE-2; Clea Japan, Tokyo, Japan) and water were provided ad libitum. All procedures related to the animals and their care were approved (Certificate No. 1104474) by the Laboratory Animal Care and Use Committee of Fukuoka University.
For immunization, OVA (Sigma–Aldrich, St. Louis, MO, USA) was dissolved in PBS at a concentration of 5 μg/mL. Initially, 1.9, 5.8 and 9.7 mg of pyriproxyfen (Fig. 1) (Wako Pure Chemical Industries, Osaka, Japan) were dissolved in 100 μL of 99% ethanol and made up to 1 mL with PBS. Subsequently, 100 μL of each pyriproxyfen solution was diluted with an equivalent volume of OVA solution to provide the desired concentrations of 3, 9 and 15 mM, respectively. The control sample was made by using PBS to create 10% ethanol and then diluting this down to 5% ethanol with OVA solution to obtain the desired concentration. Imject Alum (alum; Thermo Scientific, Rockford, IL, USA) solution was prepared by mixing 1 μL of alum (40 μg/μL) in 100 μL of OVA solution according to the manufacturer's protocol and finally diluting to 200 μL with PBS to obtain the desired concentration of 200 μg/mL. All immunizations were performed by intraperitoneal injection in a volume of 200 μL.
To evaluate OVA-specific total IgG immune responses induced by pyriproxyfen, groups of 17 mice were immunized on Weeks 0, 3 and 6 with OVA in 5% ethanol (negative control), OVA containing alum (positive control) or pyriproxyfen (15 mM). Blood samples were collected from each mouse via the tail vein at 3, 5, 7 and 8 weeks. After collection, blood samples were centrifuged at 12,000 rpm for 15 min to obtain sera. The sera were heat-inactivated at 50°C for 30 min and kept at −20°C until use. Below is a brief description of detection by ELISA of OVA-specific total IgG immune responses in sera. First, 96-well ELISA plates were coated overnight at 4°C with 10 μg/mL OVA in 0.05 M bicarbonate buffer (pH 9.6), and then washed and blocked with 1% BSA (Biochemical Reagents, Kyoto, Japan). Washes were performed in between steps with PBST and PBS. Serum samples were diluted 1:200 with PBS and applied to the plates in duplicate and in twofold serial dilutions to 1:1,638,400 for 2 hrs at 37°C. After washing, secondary antibody–alkaline phosphatase-conjugated anti-mouse IgG (Cell Signaling Technology, Danvers, MA, USA; 1:4,000) was added to the corresponding plates, which were again incubated at 37°C for 2 hrs. Finally, after extensive washing, 0.1 mL of p-nitrophenyl phosphate solution (Sigma–Aldrich) was added to each well and the OD read at 405 nm with a microplate reader (ImmunoMini Nj-2300; Nunc, Rochester, NY, USA). Values of end-point total IgG titers above the background cutoff level (in which the optical density was at least twofold greater in the OVA-coated wells than non-coated wells) were considered positive. Titers are shown as end-point dilutions. The end-point titers were expressed as means ± SEM and compared by nonparametric Mann–Whitney's U-test. In all analyses, P < 0.05 was taken to indicate statistical significance.
To characterize the ability of pyriproxyfen to enhance the immune response, we first examined the total IgG immune response to pyriproxyfen with OVA-immunized mice at different time points. Figure 2 shows the end-point titers of total IgG. As shown in Figure 2a, b, at Weeks 3 and 5 there were no significant differences in OVA-specific total IgG titers between pyriproxyfen with OVA-immunized mice and controls. However, significant increases in OVA-specific total IgG titers were observed by Week 7, which increased by Week 8 (three- and fourfold greater, respectively) compared to controls (P = 0.04 and P = 0.02, respectively; Fig. 2c, d). OVA administered with alum induced a rapid significant increase in OVA-specific total IgG titer by Week 3 (1.5-fold greater than control; P = 0.02, Fig. 2a) and finally increased by threefold at 7 and 8 weeks (P = 0.02 and P = 0.02, respectively; Fig. 2c, d). However, there were no significant differences in OVA-specific total IgG titers between mice immunized with pyriproxyfen and alum at Weeks 7 or 8. The observation that OVA with alum-immunized mice, the positive controls, showed significant enhancement of the total IgG immune response (Fig. 2c, d) confirms the accuracy of these experiments. Therefore, these observations suggest that pyriproxyfen enhances the total IgG immune response.
A dose–response assay was performed to further characterize enhancement of the total IgG immune response by pyriproxyfen. Groups of six mice were immunized on Weeks 0, 3 and 6 with OVA in 5% ethanol, with or without alum, or increasing concentrations of pyriproxyfen (3, 9 and 15 mM), and blood samples were collected on Week 8 and subjected to ELISA to detect OVA-specific total IgG immune responses in sera. Interestingly, as shown in Figure 3, a small dose of pyriproxyfen (3 mM) failed to enhance OVA-specific total IgG immune responses at 8 weeks compared with controls. However, pyriproxyfen at doses of 9 and 15 mM resulted in higher titers of OVA-specific total IgG than in controls (two- and fivefold greater; P = 0.01 and P = 0.002, respectively). There were no significant differences in the titers of total IgG immune response between groups treated with 9 and 15 mM pyriproxyfen. These results indicate that OVA-specific total IgG titers increased significantly in a dose-dependent manner.
A time-dependent assay was performed to evaluate how long pyriproxyfen remains capable of enhancing the IgG immune response. Groups of 12 mice were immunized with OVA in 5% ethanol or OVA containing alum, according to the above schedule, and pyriproxyfen (15 mM) injected followed by injection of OVA (0.5 μg) at 0, 3 and 24 hrs. Blood samples were collected on Week 8 and subjected to ELISA to detect OVA-specific total IgG immune responses in sera. As shown in Figure 4, when OVA was injected at 0 and 3 hrs after injecting pyriproxyfen, the OVA-specific total IgG titers were significantly higher (threefold) than those of controls (P = 0.008 and P = 0.006, respectively). Immunization with OVA in alum also resulted in a significantly increased OVA-specific total IgG titer (P = 0.01). As expected, there were no significant differences between the alum, 0 and 3 hr groups. In addition, the differences in total IgG titer between these groups and the control remained insignificant in the 24 hr group. In the present study, large doses of pyriproxyfen (9 or 15 mM) greatly increased total IgG antibody titers, whereas a small dose (3 mM) did not induce a significant increase in this titer (Fig. 3). These results indicate that administration of a small dose of pyriproxyfen has no immune-enhancing effect. The World Health Organization accepts a titer of pyriproxyfen of up to ca. 1 μM (0.3 mg/L) in human drinking water . In the present study, we observed no adverse effects on mice at the largest dose of pyriproxyfen tested, suggesting that pyriproxyfen is safe for mammals. However, administration of a large dose of pyriproxyfen specifically enhanced the total IgG immune response with high antibody titers. Interestingly, this enhancement of total IgG immune response by pyriproxyfen was time-restricted (Fig. 4). [14C]Pyriproxyfen orally administered to rats is rapidly eliminated from the body within 48 hrs, predominantly in the feces (90%) with 4–11% in the urine . This rapid elimination of pyriproxyfen from the body may explain the time-restricted nature of the enhancement of total IgG immune response by administration of large doses of pyriproxyfen, which may in turn decrease any negative effect of pyriproxyfen on mammalian immune responses. These two characteristics suggest that pyriproxyfen is a safe chemical for enhancing the total IgG immune response in vivo.
To determine the effect of pyriproxyfen on IgG subtypes, mice were immunized with OVA (in 5% ethanol) alone or with pyriproxyfen (15 mM) or alum, and the titers of IgG1/IgG2a subtypes measured. Groups of six mice were immunized at 3-week intervals (on Weeks 0, 3 and 6) and blood samples collected on Weeks 5 and 8. ELISAs to measure the titers of OVA-specific IgG subtypes were performed similarly, with minor modifications. In this instance, the initial dilutions of serum samples were 1:3000 and 1:100 for the IgG1 and IgG2a antibody-binding assays, respectively, and in the next step, the secondary antibodies (goat anti-mouse IgG1 and IgG2a [Southern Biotech, Birmingham, AL, USA]; 1:4000) were assessed. Figure 5a shows that at Week 5 there were no significant differences in OVA-specific IgG1 or IgG2a titers compared with controls among mice immunized with pyriproxyfen and alum. Figure 5b shows that pyriproxyfen significantly enhanced OVA-specific IgG2a titers compared to controls at 8 weeks (eightfold greater; P = 0.002), whereas the difference in the OVA-specific IgG1 immune response compared to the control remained insignificant. As expected, immunization with OVA containing alum resulted in a significantly greater OVA-specific IgG1 titer (fourfold greater, P = 0.01) than in the control at 8 weeks (Fig. 5b). These observations suggest that the IgG subtypes assessed, IgG2a and IgG1, reached significantly increased titers after immunization three times with pyriproxyfen or alum in OVA. The titers of IgE were also measured to determine the effect of pyriproxyfen on IgE production. For this, mice were immunized three times with OVA (in 5% ethanol) alone or with pyriproxyfen (15 mM) or alum and the titers of IgE measured. Groups of six mice were immunized at 3 week intervals (Weeks 0, 3 and 6) and blood samples collected on Weeks 8. ELISA for measuring the IgE titer was performed according to a method similar to that described above except the initial dilution of serum samples was 1:10 for the IgE antibody binding assay and the secondary antibody used was goat anti-mouse IgE (Southern Biotech) (1:4000). As shown in Figure 5c, there were no significant differences in OVA-specific IgE titer between mice immunized with OVA plus pyriproxyfen and controls. Compared to the controls, at 8 weeks OVA-specific IgE titers were increased only in mice immunized with OVA containing alum (P = 0.01).
Cytokine profiles were also checked to confirm the basis for immune responses after the addition of pyriproxyfen. Two groups of five mice were immunized on Weeks 0, 3 and 6 and injected with OVA (in 5% ethanol) with or without pyriproxyfen (15 mM) and alum, prior to spleen collection on Weeks 3 and 8 and measurement of cytokine concentrations by sandwich ELISA. The spleens were dissected out from the mice under aseptic conditions. Single-cell suspensions were prepared by homogenizing each spleen in 3 mL of RPMI 1640 medium (Sigma–Aldrich) followed by centrifugation for 5 mins at 1200 rpm at 4°C. The precipitates were then mixed with an additional 3 mL of RPMI 1640 medium to remove the fat bodies and centrifuged under the same conditions for 8 mins. To lyse contaminating erythrocytes, 1 mL of 0.83% NH4Cl:Tris aminomethane 20.59 g/L, 9:1 (pH 7.2) was mixed with the precipitate and centrifuged at 1500 rpm for 5 mins at 4°C. Finally, the pelleted cells were resuspended in RPMI 1640 medium with 10% heat inactivated FBS (Biowest, Nuaile, France). Viable cell numbers were counted with a hemocytometer by the trypan blue dye exclusion technique. Splenocytes were seeded in 12-well plates at a concentration of 2 × 107 cells/mL and restimulated with 0.5 mg/mL OVA. The plates were incubated at 37°C in a humidified 5% CO2 environment. The culture supernatants were collected after 24 and 72 hrs for measurement of cytokines. The concentrations of cytokines in the supernatants were assessed by sandwich ELISA according to the manufacturer's instructions (Duosets; R & D Systems, Minneapolis, MN, USA) and calculated by interpolation of cytokine standard curves. Student's t-test was used for statistical analysis of the cytokine profiles. IL-10, IL-13 and TNF-α were detected in the culture supernatants collected after 24 hrs, whereas IFN-γ and IL-4 were detected in those collected after 72 hrs. As shown in Figure 6, as evidenced by cytokine concentrations in the supernatants of the splenocytes, there were no significant differences in IL-4, IL-10 or IL-13 production in OVA with pyriproxyfen-immunized mice compared to controls at Weeks 3 or 8. However, mice immunized with OVA with pyriproxyfen showed significantly greater concentrations of TNF-α on both Weeks 3 and 8 (907.9 ± 57.9 and 363.0 ± 72.8 pg/mL, respectively) than did controls (479.6 ± 59.7 and 149.1 ± 34.7 pg/mL; P = 0.04 and P = 0.03, respectively). In addition, as shown in Figure 6, the concentration of TNF-α on Week 3 was significantly higher than that on Week 8 (P = 0.02). The concentrations of IFN-γ were significantly higher at both time points (370.6 ± 45.34 and 273.0 ± 66.2 pg/mL, respectively) compared to controls (83.5 ± 29.2 and 68.9 ± 32.9 pg/mL; P = 0.001 and P = 0.01, respectively). In alum containing OVA immunized mice, the concentrations of IL-4 were significantly higher than those of controls (290.9 ± 22.1 vs. 113.3 ± 5.6 pg/mL; P = 0.001) on Week 8 only. The concentrations of IL-10 were significantly higher (700.2 ± 85.0 and 555.1 ± 32.1 pg/mL, respectively) than those of the controls at both time points (395.1 ± 92.8 and 420.9 ± 20.9 pg/mL, P = 0.04 and P = 0.01, respectively). However, there were no significant differences in production of IL-13 in OVA between alum-immunized mice and controls on Weeks 3 or 8.
In the present study, particularly high IgG2a titers and upregulation of TNF-α and IFN-γ were observed in mice immunized with pyriproxyfen along with OVA, but not in those immunized with OVA in alum (Figs. 5 and 6). Therefore, it is reasonable to suggest that the mechanism of immune enhancement by pyriproxyfen differs from that of alum. It has been reported that IFN-γ enhances secretion of IgG2a and suppresses production of IgG1 and IgE by murine splenic B cells stimulated with bacterial LPS in vitro [5, 6], whereas IL-4 distinctly promotes secretion of IgG1 and IgE from B cells stimulated with LPS [6, 7]. Furthermore, Constant et al. reported that IFN-γ and TNF-α are secreted from the Th1 subset of CD4+ T cells, which induces B-cells to produce IgG2a leading to Th1 immune response, and that IL-4 is secreted from Th2 subsets of CD4+ T cells and is associated with the Th2 immune response . In addition, IL-10 has been reported to inhibit the Th1 immune response by inhibition of TNF-α and IFN-γ production . Taken together, these reports suggest that production of IgG2a with increased TNF-α and IFN-γ concentrations are characteristic of Th1 CD4+ T cell responses, whereas IgG1 along with increased IL-4 and IL-10 concentrations are characteristic of Th2 CD4+ T cell responses. However, we did not use CD4+ T cells specifically, but rather used erythrocyte-depleted total spleen cells, which may have included T and B lymphocytes, dendritic cells and macrophages. Therefore, our study does not clearly provide evidence for shifting of Th1 or Th2 cell responses with pyriproxyfen. A flow cytometry  or magnetic cell sorting assay  would be necessary for further assessment of Th1/Th2 CD4+ T cell responses.
Although the present study has demonstrated IgG immune responses to pyriproxyfen, the mechanism(s) for these actions of this lipophilic hormone remain unknown. Being a member of the terpene family, pyriproxyfen may have a mechanism of action similar to those of other terpene-based immune enhancers such as MF59 adjuvant, which includes squalene, a 30-carbon molecule. However, unlike pyriproxyfen, MF59 induces a Th2-type immune response with increased concentrations of IL-4, IL-5, other cytokines and IgG1 , this being mediated via a TLR-independent MyD88-dependent signaling pathway . On the other hand, pyriproxyfen, a JHA, has 20 carbon atoms, which is close to the number in JH C15 . Interestingly, the hydroxy fatty acyl chains of lipid A, the bioactive component of LPS from gram-negative bacteria, consist of 12–16 carbon atoms . In this respect, therefore, pyriproxyfen is more similar to lipid A than to squalene (MF59). Furthermore, lipid A reportedly induces a strong Th1 immune response and a TLR-4-dependent MyD88 signaling pathway regulates its mechanism of action [15, 16]. Based on these observations, it is reasonable to infer that pyriproxyfen in the presence of antigen may have a mechanism of action involving the TLR-4-dependent MyD88 signaling pathway, similar to that of lipid A rather than MF59.
In conclusion, the results of the present study suggest that pyriproxyfen is capable of enhancing total IgG immune response. Importantly, large doses of pyriproxyfen significantly enhance the total IgG immune response. However, the enhancement is no longer present 24 hr after treatment with pyriproxyfen. These results suggest that pyriproxyfen is a safe chemical. Moreover, unlike alum, pyriproxyfen induces an increase in titers of IgG2a and enhanced TNF-α and IFN-γ. These observations indicate that the mechanism of immune enhancement by pyriproxyfen may differ from that which has been well established for alum.
The authors are grateful to the students of the Department of Microbiology, Faculty of Pharmaceutical Sciences, Fukuoka University, for their cooperation during these experiments. The first author was supported by a scholarship from the Ministry of Science and Education, Japan.
None of the authors has any conflict of interest associated with this study.