Investigating the cellular, molecular and immunological mechanisms of pathological filarial associated lymphatic remodelling

Pathological manifestations of Lymphatic filariasis (LF), namely: hydrocele and lymphoedema (LE), results in severe chronic morbidity, remaining a leading cause of global disability. Treatment for the ~40 million symptomatic LF patients worldwide is currently limited to morbidity management to slow progression of disease. There is an urgent need to identify and translate novel therapeutic strategies to improve on standard care and effectively reverse LF pathology. A novel in vivo murine limb pathology model, utilising Brugia malayi infective L3 larvae (BmL3) was developed and an in vivo intravital optical imaging system utilised to longitudinally track lymphatic alterations and developing pathology following infection. Lymphatic fluorescent reporter mice (Prox1-GFP) were also used to image changes in lymphatic architecture. Multiplex (Luminex) protein assays on harvested plasma were undertaken to investigate associations between changes in circulating lymphangiogenic factors and lymphatic remodelling following filarial infection. Immune-deficient murine knockout strains, targeted immune cell ablations, and manipulation of specific lymphangiogenic molecules were utilised in the model to investigate cellular, molecular and immunological mechanisms of lymphatic pathology. Significant lymphatic remodelling and lymphatic insufficiency were consistently observed as rapidly as 6 days following BmL3 infection in lymphatic tissues where active parasitism could be determined by fluorescent BmL3 labelling experiments. Severity of BmL3 induced lymphatic remodelling and pathology was mouse strain-dependent and associated with significantly altered plasma concentrations of a milieu of lymphangiogenic factors including: Vascular Endothelial Growth Factors (VEGFA, C), Transforming Growth Factor-β superfamily members (BMP-10, endoglin, follistatin, sALK-1, TGF-β) and prolactin. Both elevated prolymphangiogenic secretions and pathology persisted for 12 weeks post-infection at a point where active parasitism was not evident (no adult infections or blood microfilaraemias). Monocytes and macrophages isolated from pathological lymphatic tissues and adjacent draining lymph nodes, secreted significant levels of prolymphangiogenic factors including sALK-1 and VEGF-C. Macrophages isolated from pathological lymphatic / lymphoid 17 tissues expressed markers consistent with blood monocyte recruitment and alternative activation. Clodronate liposome mediated ablations of phagocytic cells, including monocytes and macrophages or specific anti-CCR2 antibody ablation of CCR2+ monocytes in wild type (WT) mice also ameliorated filarial lymphatic insufficiency and dilation. BmL3 infected Severe-Combined or Th2 adaptive immune deficient (SCID, IL-4Rα-/-) mice displayed significantly abrogated lymphatic remodelling and pathology. Human lymphatic endothelial cells proliferated in response to monocyte-differentiated macrophage secretions after stimulation with live BmL3, L3 extracts or recombinant IL-4+IL-13. Together, the data demonstrates that lymphatic remodelling and insufficiency is rapidly induced following BmL3 infection. Further, the data highlights a novel Th2/monocyte/macrophage signalling axis as a key driver of filarial lymphatic remodelling and pathology. Inhibiting, or reversing, filarial remodelling, through targeting the identified adaptive Th2 signalling mechanisms may represent novel therapeutic strategies to ameliorate LF-related pathology.