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. 2012 Jun;80(6):2141-9.
doi: 10.1128/IAI.00024-12. Epub 2012 Apr 2.

Ingested human insulin inhibits the mosquito NF-κB-dependent immune response to Plasmodium falciparum

Nazzy Pakpour  1 , Vanessa Corby-Harris, Gabriel P Green, Hannah M Smithers, Kong W Cheung, Michael A Riehle, Shirley Luckhart
Affiliations

Ingested human insulin inhibits the mosquito NF-κB-dependent immune response to Plasmodium falciparum

Nazzy Pakpour et al. Infect Immun. 2012 Jun.

Abstract

We showed previously that ingested human insulin activates the insulin/IGF-1 signaling pathway in Anopheles stephensi and increases the susceptibility of these mosquitoes to Plasmodium falciparum. In other organisms, insulin can alter immune responsiveness through regulation of NF-κB transcription factors, critical elements for innate immunity that are also central to mosquito immunity. We show here that insulin signaling decreased expression of NF-κB-regulated immune genes in mosquito cells stimulated with either bacterial or malarial soluble products. Further, human insulin suppressed mosquito immunity through sustained phosphatidylinositol 3-kinase activation, since inhibition of this pathway led to decreased parasite development in the mosquito. Together, these data demonstrate that activation of the insulin/IGF-1 signaling pathway by ingested human insulin can alter NF-κB-dependent immunity, and ultimately the susceptibility, of mosquitoes to P. falciparum.

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Figures

Fig 1
Fig 1
Human insulin decreases LPS-induced antimicrobial promoter activity in immortalized A. stephensi cells. ASE cells were transfected with Defensin, Cecropin, or Gambicin luciferase promoter-reporter plasmid constructs and then stimulated 24 h later with 1.7 μM human insulin, 100 μg of LPS/ml, or LPS and decreasing concentrations of human insulin. Graphs represent means ± the standard errors of the mean (SEM) of luciferase activity (relative light units [RLU]) normalized to untreated controls (n = 3). Pairwise comparisons of treatments were analyzed by using the Student t test (*, P < 0.05).
Fig 2
Fig 2
Inhibition of PI3K, but not MEK, reverses the negative effects of human insulin on LPS-induced antimicrobial promoter activity in immortalized A. stephensi cells. ASE cells were transfected with the Defensin luciferase promoter-reporter plasmid (pDef-luc) and treated with specific small molecule inhibitors of PI3K (20 μM LY294002 [LY] or 1 μM wortmannin [W]) (A) or MEK (10 μM PD98059 [PD] or 10 μM U0126 [UO]) (C) for 1 h prior to stimulation with 1.7 μM human insulin, 100 μg of LPS/ml, or LPS+insulin. Luciferase activities were determined 24 h after stimulation. The graphs represent the means ± the SEM of luciferase activity (relative light units [RLU]) normalized to untreated controls (not shown) (n = 5). Pairwise comparisons of treatments were analyzed by using the Student t test (*, P < 0.05). Representative, replicated Western blots of phosphorylated ERK (p-ERK) and FOXO (p-FOXO) from cell lysates treated as described above for 5 min are shown to confirm the inhibition of the IIS pathway by PI3K (B) or MEK (D) inhibitors. GAPDH provided an assessment of protein loading (n = 2).
Fig 3
Fig 3
Stimulation of immortalized A. stephensi cells with human insulin results in sustained FOXO, but not ERK, phosphorylation. Cell lysates from ASE cells treated with 100 μg of LPS/ml, 1.7 μM human insulin, or LPS+insulin were collected at the indicated time points after treatment, and the levels of phosphorylated ERK (pERK) and FOXO (pFOXO) were determined by Western blotting. The graphs represent the average fold change ± the SEM of phospho-specific protein levels normalized to untreated controls (n = 4). The dashed line represents phospho-specific protein levels in untreated controls. GAPDH provided an assessment of protein loading. Pairwise comparisons of treatments versus controls at each time point were analyzed by using the Student t test (*, P < 0.05; **, P < 0.10).
Fig 4
Fig 4
Provision of human insulin increases survival of female A. stephensi fed heat-killed bacteria (HK-bacteria). Approximately 50 3- to 5-day-old female A. stephensi were fed a single blood meal containing PBS as a control, 170 pM human insulin, HK-bacteria, or HK-bacteria+human insulin. All mosquitoes were provided 10% sugar ad libitum after blood feeding. Daily mortality for each treatment was recorded, and dead mosquitoes were removed and counted daily for 18 days. (A) A representative survivorship curve is shown. Life span experiments were replicated four times with separate cohorts of mosquitoes. (B) Summary of sample sizes, medians, means, and statistical significance comparing mosquitoes fed HK-bacteria versus HK-bacteria+insulin.
Fig 5
Fig 5
Antimicrobial promoter activity is increased by stimulation with P. falciparum soluble products, and this activity is decreased by human insulin in immortalized mosquito cells. ASE cells were transfected with Defensin, Cecropin, or Gambicin luciferase promoter-reporter plasmid constructs and stimulated 24 h later with 150 × 106 parasite equivalents of P. falciparum soluble products (PfsPs), 1.7 μM human insulin, or PfsPs plus 1.7 or 0.17 μM insulin. The luciferase activity (relative light units [RLU]) was normalized to untreated controls. The graphs show the fold change relative to untreated controls (A) or PfsPs-treated cells (B). Horizontal lines represent means from three to six independent experiments. Pairwise comparisons of treatments were analyzed by using the Student t test; the P values are indicated.
Fig 6
Fig 6
Inhibition of PI3K signaling in insulin-fed A. stephensi results in decreased P. falciparum development. (A) Mosquitoes were fed a meal of saline or washed RBCs supplemented with PBS as a control (buffer), 20 μM LY294002 (LY), 170 pM human insulin (Ins), or insulin+LY294002 (ins+LY). The graphs represent the fold change of phosphorylated FOXO (p-FOXO) normalized to untreated controls (n = 4). GAPDH provided an assessment of protein loading. The data were not normally distributed; therefore, pairwise comparisons of treatments were analyzed by Wilcoxon test (P values are shown). A representative Western blot of p-FOXO and GAPDH is shown. (B) Mosquitoes were fed with P. falciparum-infected RBCs supplemented with PBS (buffer) as a control, 20 μM LY294002 (LY), 170 pM human insulin (Ins), or insulin+LY294002 (Ins+LY). The data from three independent experiments with separate cohorts of mosquitoes were analyzed for the main effects of experiment and treatment. Horizontal lines indicate the means for three combined sets of 50 mosquitoes (150 mosquitoes total) per treatment group. The data were not normally distributed and, therefore, analyzed using the Kruskal-Wallis test and Dunn's post test. Significant differences between treatment groups are indicated. (C) The prevalence of infection (mosquitoes with at least one P. falciparum oocyst) is shown as a percentage of the dissected mosquitoes (n = 3). No significant differences were noted. (D) P. falciparum cultures were incubated with 2, 20, or 200 μM LY294002. The graph represents the average relative growth compared to DMSO controls at 48 h (□) or 96 h (■) (n = 2).
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