Defining the target and the effect of imatinib on the filarial c-Abl homologue

Abstract

Background: Previously we demonstrated the micro- and macrofilaricidal properties of imatinib in vitro. Here we use electron and multiphoton microscopy to define the target of imatinib in the adult and microfilarial stages of Brugia malayi and assess the effects of pharmacologically relevant levels of imatinib on the adult parasites.

Methods: After fixation of adult B. malayi males and females, sections were stained with polyclonal rabbit anti-c-Abl antibody (or isotype control) and imaged with multiphoton fluorescent microscopy. Microfilariae were fixed and labeled with rabbit anti-c-Abl IgG primary antibody followed by anti-rabbit gold conjugated secondary antibody and imaged using transmission electron microscopy (TEM; immunoEM). In addition, adult B. malayi males and females were exposed to 0 or 10μM of imatinib for 7 days following which they were prepared for transmission electron microscopy (TEM) to assess the drug's effect on filarial ultrastructure.

Results: Fluorescent localization of anti-c-Abl antibody demonstrated widespread uptake in the adult filariae, but the most intense signal was seen in the reproductive organs, muscle, and intestine of both male and female worms. Fluorescence was significantly more intense in the early microfilarial stage (i.e. early morula) compared with later development stages (i.e. pretzel). Anti-c-Abl antibody in the microfilariae localized to the nuclei. Based on TEM assessment following imatinib exposure, imatinib appeared to be detrimental to embryogenesis in the adult female B. malayi.

Conclusions: At pharmacologically achievable concentrations of imatinib, embryogenesis is impaired and possibly halted in adult filariae. Imatinib is likely a slow microfilaricide due to interference in intra-nuclear processes, which are slowly detrimental to the parasite and not immediately lethal, and thus may be used to lower the levels of L. loa microfilariae before they are treated within the context of conventional mass drug administration.

Fig 1. Expression of c-Abl homologue in B. malayi adults.

(A-C) Adult females and (E) adult males were exposed to polyclonal anti-c-Abl rabbit antibody or (D, F) isotype control rabbit IgG and counterstained with Alexa Fluor 594 labeled goat anti-rabbit IgG. High levels of expression are seen throughout the hypodermis (including hypodermal cords), gastrointestinal tract, uteri (A, female), and vas deferens (E, male). Scale bars are 20μm. O, fertilized ova; U, uterus; M, muscle; L, lateral chord; EM, early morula stage; P, early pretzel stage; ES, excretory secretory canal; S, spermatids; VD, Vas Deferens; D, dorsal cord; V, ventral cord; HN, hypodermal nuclei; I, intestine. Images are representative of at least 6 immunofluorescent microscopy experiments.

Fig 2. C-Abl expression in developing microfilariae decreases over the course of maturation.

C-Abl expression was measured as mean fluorescence intensity in the internal structures of adult worms. (A, C) The intensity of fluorescence between the reproductive apparatus (A, uterus, ovaries, early morula, pretzel; C, vas deferens, spermatids), muscle, glycogen, and intestine is significantly more in the structures treated with anti-c-Abl antibody compared with isotype control. (A) Adult females show over the course of embryonic development a decrease in c-Abl expression from the early morula to the pretzel stage (p = 0.0035). Higher c-Abl expression is also seen in muscle compared with glycogen in females (A, p = 0.0187) as well as males (C, p = 0.0155). (B, D) Fluorescence intensity generated by the nuclear stain DAPI, is essentially identical between the c-Abl and isotype control conditions. Three sections per condition were analyzed, and the experiment was repeated 3 times; means ± SEM.

Fig 3. Localization of c-Abl in B. malayi microfilariae by immune-transmission electron microscopy.

A)Staining with isotype control antibody (18,500X). B)Localization of c-Abl-like protein in the nuclei of MF (black dots, 13,000X). Magnification: bar 500 nm. n—nucleus, mu–muscle, cu—cuticle.

Fig 4. Changes in adult female B. malayi treated for 7 days with 10 μM imatinib compared with untreated cultured control worms.

A) Low power (2,900X) demonstrating overall significant necrosis of developing microfilariae (C) compared with the untreated females (A). B) Higher magnification of females (6,800X) shows treated females (D-F) have numerous electron dense vacuoles (EDV), distended cisternae of the endoplasmic reticulum (CER), distorted microfilariae morphology and a spectrum of microfilarial necrosis compared with untreated (B) worms. Inset of cilium is 68,000X. m–Developing microfilariae, ue–Uterine epithelium, uw–Uterine Wall, h–Hypodermis of the lateral cord, cu–Cuticle, nm–Necrotic Microfilaria, n–Nucleus, s–Spermatocyte, mi–Mitochondria, c–Cilium, bi–Basal infoldings, b–Basement membrane, g–Glycogen, nc–Nerve Cord, w–Wolbachia, edv–Electron Dense Vacuoles, cer-cisternae of endoplasmic reticulum.

Fig 5. Hypodermis unchanged in adult female B. malayi treated for 7 days with 10 μM imatinib compared with untreated cultured control worms.

A) Untreated hypodermis Wolbachia (11,000X) appear similar to B) Wolbachia (W, arrows) from treated females. C) Untreated female cuticle, muscle, and hypodermis appear similar to D) treated the treated females (11,000X). h–Hypodermis of the lateral cord, cu–Cuticle, n–Nucleus, bi–Basal infoldings, mu–Muscle, b–Basement membrane, g–Glycogen, nc–Nerve Cord, w–Wolbachia.

Fig 6. Nerve and intestine unchanged in adult female B. malayi treated for 7 days with 10 μM imatinib compared with untreated cultured control worms.

B) Nerve chord of treated adult female (9,300X) without a significant difference as compared to controls (A). D) No change in the intestine morphology (6,800X) compared with untreated controls (C). n–Nucleus, bi–Basal infoldings, mu–Muscle, b–Basement membrane, g–Glycogen, nc–Nerve Cord, mi–Mitochondria, mv–Microvilli, tj–Tight Junction, lv–Lipid Vacuole, iw–Intestinal Wall.

Fig 7. Imatinib-treated adult male B. malayi do not show significant alterations at pharmacologically achievable dosing.

Adult males (4,800X) exposed to imatinib at 10 μM demonstrate minimal disorganization of glycogen and muscle compared with controls. uw–uterine wall, n–hypodermal nuclei, g–glycogen, mu–muscle, cu–cuticle, i–intestine.