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

Eosinophil-Mediated Immune Control of Adult Filarial Nematode Infection Can Proceed in the Absence of IL-4 Receptor Signaling

Helminth infections are accompanied by eosinophilia in parasitized tissues. Eosinophils are effectors of immunity to tissue helminths. We previously reported that in the context of experimental filarial nematode infection, optimum tissue eosinophil recruitment was coordinated by local macrophage populations following IL-4R–dependent in situ proliferation and alternative activation. However, in the current study, we identify that control of chronic adult filarial worm infection is evident in IL-4Ra–deficient (IL-4Ra2/2) mice, whereby the majority of infections do not achieve patency. An associated residual eosinophilia was apparent in infected IL-4Ra2/2 mice. By treating IL-4Ra2/2 mice serially with anti-CCR3 Ab or introducing a compound deficiency in CCR3 within IL-4Ra2/2 mice, residual eosinophilia was ablated, and susceptibility to chronic adult Brugia malayi infection was established, promoting a functional role for CCR3-dependent eosinophil influx in immune control in the absence of IL-4/IL13–dependent immune mechanisms. We investigated additional cytokine signals involved in residual eosinophilia in the absence IL-4Ra signaling and defined that IL-4Ra2/2/IL-52/2 double-knockout mice displayed significant eosinophil deficiency compared with IL-4Ra2/2 mice and were susceptible to chronic fecund adult filarial infections. Contrastingly, there was no evidence that either IL-4R–dependent or IL-4R–independent/CCR3/IL-5–dependent immunity influenced B. malayi microfilarial loads in the blood. Our data demonstrate multiplicity of Th2-cytokine control of eosinophil tissue recruitment during chronic filarial infection and that IL-4R–independent/IL-5– and CCR3-dependent pathways are sufficient to control filarial adult infection via an eosinophil-dependent effector response prior to patency

Interleukin-4 activated macrophages mediate immunity to filarial helminth infection by sustaining CCR3-dependent eosinophilia

Eosinophils are effectors in immunity to tissue helminths but also induce allergic immunopathology. Mechanisms of eosinophilia in non-mucosal tissues during infection remain unresolved. Here we identify a pivotal function of tissue macrophages (Mϕ) in eosinophil anti-helminth immunity using a BALB/c mouse intra-peritoneal Brugia malayi filarial infection model. Eosinophilia, via C-C motif chemokine receptor (CCR)3, was necessary for immunity as CCR3 and eosinophil impairments rendered mice susceptible to chronic filarial infection. Post-infection, peritoneal Mϕ populations proliferated and became alternatively-activated (AAMϕ). Filarial AAMϕ development required adaptive immunity and interleukin-4 receptor-alpha. Depletion of Mϕ prior to infection suppressed eosinophilia and facilitated worm survival. Add back of filarial AAMϕ in Mϕ-depleted mice recapitulated a vigorous eosinophilia. Transfer of filarial AAMϕ into Severe-Combined Immune Deficient mice mediated immunological resistance in an eosinophil-dependent manner. Exogenous IL-4 delivery recapitulated tissue AAMϕ expansions, sustained eosinophilia and mediated immunological resistance in Mϕ-intact SCID mice. Co-culturing Brugia with filarial AAMϕ and/or filarial-recruited eosinophils confirmed eosinophils as the larvicidal cell type. Our data demonstrates that IL-4/IL-4Rα activated AAMϕ orchestrate eosinophil immunity to filarial tissue helminth infection

A mouse infection model and long-term lymphatic endothelium co-culture system to evaluate drugs against adult Brugia malayi

The development of new drugs targeting adult-stage lymphatic filarial nematodes is hindered by the lack of a robust long-term in vitro culture model. Testing potential direct-acting and anti-Wolbachia therapeutic candidates against adult lymphatic filariae in vitro requires their propagation via chronic infection of gerbils. We evaluated Brugia malayi parasite burden data from male Mongolian gerbils compared with two immune-deficient mouse strains highly susceptible to B. malayi: CB.17 Severe-Combined Immmuno-Deficient (SCID) and interleukin-4 receptor alpha, interleukin-5 double knockout (IL-4Rα-/-IL-5-/-) mice. Adult worms generated in IL-4Rα-/-IL-5-/- mice were tested with different feeder cells (human embryonic kidney cells, human adult dermal lymphatic endothelial cells and human THP-1 monocyte differentiated macrophages) and comparative cell-free conditions to optimise and validate a long-term in vitro culture system. Cultured parasites were compared against those isolated from mice using motility scoring, metabolic viability assay (MTT), ex vivo microfilariae release assay and Wolbachia content by qPCR. A selected culture system was validated as a drug screen using reference anti-Wolbachia (doxycycline, ABBV-4083 / flubentylosin) or direct-acting compounds (flubendazole, suramin). BALB/c IL-4Rα-/-IL-5-/- or CB.17 SCID mice were superior to Mongolian gerbils in generating adult worms and supporting in vivo persistence for periods of up to 52 weeks. Adult females retrieved from BALB/c IL-4Rα-/-IL-5-/- mice could be cultured for up to 21 days in the presence of a lymphatic endothelial cell co-culture system with comparable motility, metabolic activity and Wolbachia titres to those maintained in vivo. Drug studies confirmed significant Wolbachia depletions or direct macrofilaricidal activities could be discerned when female B. malayi were cultured for 14 days. We therefore demonstrate a novel methodology to generate adult B. malayi in vivo and accurately evaluate drug efficacy ex vivo which may be adopted for drug screening with the dual benefit of reducing overall animal use and improving anti-filarial drug development

NKp46+ natural killer cells develop an activated/memory-like phenotype and contribute to innate immunity against experimental filarial infection

Lymphatic filariasis and onchocerciasis are major neglected tropical diseases affecting over 90 million people worldwide with painful and profoundly disfiguring pathologies (such as lymphoedema or blindness). Type 2 inflammation is a hallmark of filarial nematode tissue infection and is implicated both in eosinophil dependent immunity and lymphatic or ocular immunopathologies. Type-2 innate lymphoid cells (ILC2) are known to play an important role in the initiation of type 2 inflammation in helminth infection. We therefore tracked comparative IL-12Rβ2+ ILC1, ST2+ ILC2 and NKp46+ natural killer (NK) innate lymphoid cell population expansions during Brugia malayi experimental peritoneal filarial infections using either immunocompetent or immunodeficient mice. In immunocompetent BALB/c animals, NKp46+ NK cells rapidly expanded representing over 90% of the ILC population in the first week of infection, whereas, surprisingly, ST2+ ILC2 failed to expand. NKp46+ NK cell expansions were confirmed in RAG2 deficient mice lacking adaptive immunity. Ablation of the NKp46+ NK cell compartment in RAG2 common gamma chain (gc) mice led to increased susceptibility to chronic adult B. malayi infection. This data was recapitulated using an Onchocerca ochengi male worm peritoneal implant model. When NKp46+ NK cells were depleted in RAG2 deficient mice using anti-NKp46 or asialo GM1 antibody injections over the first five weeks of B. malayi infection, susceptibility to adult B. malayi infection was significantly increased by 2-3 fold with concomitant impairment in eosinophil or neutrophil recruitments. Finally, we demonstrate that in RAG2 deficient mice, drug clearance of a primary adult B. malayi infection followed by challenge infection leads to resistance against early larval B. malayi establishment. This innate resistance is associated with bolstered NK and eosinophils whereby NKp46+ NK cells express markers of memory-like/enhanced activation (increased expression of interferon gamma and Ly6C). Our data promotes a novel functional role for NKp46+ NK cells in immunoprotection against experimental primary and secondary filarial infection which can proceed in the absence of adaptive immune regulation

Tetracyclines improve experimental lymphatic filariasis pathology by disrupting interleukin-4 receptor-mediated lymphangiogenesis

Lymphatic filariasis is the major global cause of nonhereditary lymphedema. We demonstrate that the filarial nematode Brugia malayi induced lymphatic remodeling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type 2 adaptive immunity, the interleukin-4 receptor, and recruitment of C-C chemokine receptor-2–positive monocytes and alternatively activated macrophages with a prolymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type 2 prolymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarization of alternatively activated macrophages, and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism of action for the antimorbidity effects of doxycycline in filariasis and support clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphedemas of chronic inflammatory origin

Fungal-mediated lung allergic airway disease: The critical role of macrophages and dendritic cells.

Fungi are abundant in the environment, causing our lungs to be constantly exposed to a diverse range of species. While the majority of these are cleared effectively in healthy individuals, constant exposure to spores (especially Aspergillus spp.) can lead to the development of allergic inflammation that underpins and worsen diseases such as asthma. Despite this, the precise mechanisms that underpin the development of fungal allergic disease are poorly understood. Innate immune cells, such as macrophages (MΦs) and dendritic cells (DCs), have been shown to be critical for mediating allergic inflammation to a range of different allergens. This review will focus on the crucial role of MΦ and DCs in mediating antifungal immunity, evaluating how these immune cells mediate allergic inflammation within the context of the lung environment. Ultimately, we aim to highlight important future research questions that will lead to novel therapeutic strategies for fungal allergic diseases

Recursos calcários associados aos depósitos biodetríticos da região sul do Rio grande do Sul para uso industrial

O objetivo do trabalho é o de caracterizar quimicamente o calcário biodetrítico da plataforma continental e zona costeira da região sul do Rio Grande do Sul, visando sua utilização em ração animal. Foram analisadas quatorze amostras representativas da região, nas quais foram determinados os teores de fósforo, flúor, cálcio, magnésio, resíduo insolúvel e matéria orgânica. A separação granulométrica das frações mostrou que somente os intervalos 4mm e 2mm apresentam viabilidade econômica industrial. O material separado apresentou alto teor de cálcio (33,13%) e baixos teores de magnésio (0,63), flúor (0,139ppm), fósforo (0,398%) e matéria orgânica (0,0984%), sendo estes valores correspondentes à média das dez amostras analisadas. Palavras-Chave: calcário de conchas, composição de calcário, calcário para raçã

Recursos calcários associados aos depósitos biodetríticos da região sul do Rio grande do Sul para uso industrial

O objetivo do trabalho é o de caracterizar quimicamente o calcário biodetrítico da plataforma continental e zona costeira da região sul do Rio Grande do Sul, visando sua utilização em ração animal. Foram analisadas quatorze amostras representativas da região, nas quais foram determinados os teores de fósforo, flúor, cálcio, magnésio, resíduo insolúvel e matéria orgânica. A separação granulométrica das frações mostrou que somente os intervalos 4mm e 2mm apresentam viabilidade econômica industrial. O material separado apresentou alto teor de cálcio (33,13%) e baixos teores de magnésio (0,63), flúor (0,139ppm), fósforo (0,398%) e matéria orgânica (0,0984%), sendo estes valores correspondentes à média das dez amostras analisadas. Palavras-Chave: calcário de conchas, composição de calcário, calcário para raçã

Tetracyclines improve experimental lymphatic filariasis pathology by disrupting interleukin-4 receptor–mediated lymphangiogenesis

Lymphatic filariasis is the major global cause of nonhereditary lymphedema. We demonstrate that the filarial nematode Brugia malayi induced lymphatic remodeling and impaired lymphatic drainage following parasitism of limb lymphatics in a mouse model. Lymphatic insufficiency was associated with elevated circulating lymphangiogenic mediators, including vascular endothelial growth factor C. Lymphatic insufficiency was dependent on type 2 adaptive immunity, the interleukin-4 receptor, and recruitment of C-C chemokine receptor-2-positive monocytes and alternatively activated macrophages with a prolymphangiogenic phenotype. Oral treatments with second-generation tetracyclines improved lymphatic function, while other classes of antibiotic had no significant effect. Second-generation tetracyclines directly targeted lymphatic endothelial cell proliferation and modified type 2 prolymphangiogenic macrophage development. Doxycycline treatment impeded monocyte recruitment, inhibited polarization of alternatively activated macrophages, and suppressed T cell adaptive immune responses following infection. Our results determine a mechanism of action for the antimorbidity effects of doxycycline in filariasis and support clinical evaluation of second-generation tetracyclines as affordable, safe therapeutics for lymphedemas of chronic inflammatory origin