Authors

Discussion

Lymphatic filariasis (elephantiasis) is the third major cause of global disability and the number one causative agent of non-hereditary lymphoedema (LE). An anti-morbidity effect of the second-generation tetracycline, doxycycline (DOX), has been promoted in filarial lymphoedema which is disassociated from active filarial infection, anti-Wolbachia activity or more broad-spectrum antibiotic effects. Here, we utilise in vitro and in vivo preclinical models of human filarial infection and vascular remodelling to interrogate the direct anti-lymphangiogenic effects of treatment with DOX or the related second-generation tetracycline, minocycline (MIN).

Two weeks following subcutaneous hind-limb infections of B. malayi infectious stage (L3) larvae, extensive lymphatic remodelling in limb superficial dermal lymphatic system could be visualised using intra-vital Near Infra-Red optical bioimaging. Further, impaired lymphatic drainage/back-flow from the site of infection was evident by Evans Blue dermal retention. Significant increases in vascular endothelial growth factor (VEGF)C was detectable in circulation following infection. In mice receiving human dose bioequivalent DOX (40mg/kg bid) for two weeks co-incident with infection, the extent of aberrant lymphatic remodelling and impaired lymphatic function were reduced. This ameliorating effect on lymphatic pathology was not due to anti-Wolbachia efficacy of DOX because larval recovery rates were similar between DOX and vehicle treated animals at 2 weeks and TLR6-/- mice deficient in the Wolbachia innate inflammatory pathway exhibited a similar degree of remodelling and lymphatic insufficiency. Further, evidence against an ‘off-target’, broad spectrum anti-microbial effect of DOX in modifying pathology was evident by observing a similar degree of lymphatic pathology in vehicle and oral amoxicillin (dose) treated mice.

We explored a direct mechanism of tetracyclines attenuating lymphangiogenesis using longitudinal in vitro human endothelial cell imaging. We determined that DOX and MIN are effective inhibitors of VEGFA or C-induced endothelial cell proliferation. This effect was more selective for lymphatic endothelial cells versus blood endothelial cells and was dose dependent with effects manifest after concentration exposures ≥6 micromolar. Preliminary data indicates at these dosages, DOX interferes with intracellular calcium levels, suggesting a calcium-binding anti-proliferative mode-of-action.