Filarial Excretory-Secretory Products Induce Human Monocytes to Produce Lymphangiogenic Mediators

<div><p>The nematodes <i>Wuchereria bancrofti</i> and <i>Brugia</i> spp. infect over 120 million people worldwide, causing lymphedema, elephantiasis and hydrocele, collectively known as lymphatic filariasis. Most infected individuals appear to be asymptomatic, but many exhibit sub-clinical manifestations including the lymphangiectasia that likely contributes to the development of lymphedema and elephantiasis. As adult worm excretory-secretory products (ES) do not directly activate lymphatic endothelial cells (LEC), we investigated the role of monocyte/macrophage-derived soluble factors in the development of filarial lymphatic pathology. We analyzed the production of IL-8, IL-6 and VEGF-A by peripheral blood mononuclear cells (PBMC) from naïve donors following stimulation with filarial ES products. ES-stimulated PBMCs produced significantly more IL-8, IL-6 and VEGF-A compared to cells cultured in medium alone; CD14⁺ monocytes appear to be the primary producers of IL-8 and VEGF-A, but not IL-6. Furthermore, IL-8, IL-6 and VEGF-A induced <i>in vitro</i> tubule formation in LEC Matrigel cultures. Matrigel plugs supplemented with IL-8, IL-6, VEGF-A, or with supernatants from ES-stimulated PBMCs and implanted <i>in vivo</i> stimulated lymphangiogenesis. Collectively, these data support the hypothesis that monocytes/macrophages exposed to filarial ES products may modulate lymphatic function through the secretion of soluble factors that stimulate the vessel growth associated with the pathogenesis of filarial disease.</p></div

Quantitative assessment of the presence of podoplanin positive areas (lymphatic endothelial elements) and vWF positive areas (blood endothelial elements) in treated Matrigel plugs recovered from rats 9 days after sub-cutaneous implantation.

<p>A total of 9 areas were examined in each sample (54 areas per treatment) and assessed using a Chalkley Point Array count (CPA).</p><p>* Significantly different (p<0.005) from anti-podoplanin Groups 1 and 2.</p><p>** Significantly different (p<0.05) from anti-podoplanin Groups 1 and 2.</p><p>*** Significantly different (p<0.005) from anti-vWF Groups 1 and 2.</p><p>**** Significantly different (p<0.05 from anti-vWF Groups 1 and 2.</p

Filarial ES-induced lymphangiogenic mediators induce LEC tubule formation <i>in vitro</i>.

<p>LECs were grown on Matrigel in the presence or absence of IL-8, IL-6 or VEGF-A and lymphatic networks were photographed (A). (B) The number of tubules was quantified using image analysis software. The data represented here are the means +SEM of one experiment representative of 4 independent experiments performed in triplicate.</p

Cellular responses in the central assessment area of Matrigel plugs.

<p>Matrigel was injected in the presence or absence of 10/mL IL-8, IL-6 or VEGF-A in 0.5 mL. Matrigel alone was injected as a control. Matrigel was supplemented with supernatants collected from ES-stimulated PBMCs or PBMCs cultured in media as a control and injected into rats. After 9 days, Matrigel plugs were excised, sectioned and analyzed. Representative observations are presented from a single experiment using 6 rats per group. (A) Matrigel alone (control) - H&E stain. (B) Vascular response in VEGF-A plug - H&E stain. (C) Cellular response in PBMC+ES Matrigel plug – H&E stain. (D) Lymphatic vessels (green arrow) together with blood vessels (black arrow) in an IL-6-treated Matrigel plug. (E) Example of anti-vWF staining of blood vessels in PBMC+ES Matrigel plug. (F) High power of the anti-podoplanin staining in an IL-6-containing Matrigel plug at 9 days. The scale bars represent 50 microns in A, B, D and E; 100 microns in 5C; and 10 microns in 5F.</p

<i>Brugia</i> ES products induce the production of IL-8 and VEGF-A by human CD14⁺ monocytes.

<p>Human CD14⁺ monocytes were isolated and compared to CD14-depleted cells for IL-8, IL-6 and VEGF-A production in response to worm ES products or LPS. Cell supernatants were assessed for the presence of (A) IL-8 (n = 12), (B) IL-6 (n = 7) and (C) VEGF-A (n = 7) after 72 h of stimulation. Data presented represents the mean +SEM of at least 7 people per factor and comparisons were made using the Signed Rank test. LPS was used as a positive control and stimulated the production of IL-8 (p<0.003) and IL-6 (p<0.02) compared to cells cultured in media alone.</p

<i>Brugia</i> ES products induce the production of lymphangiogenic molecules by human PBMCs.

<p>PBMCs were isolated from a minimum of 10 healthy human volunteers and 1×10⁶ cells were stimulated with or without ES for 72 h. Cell supernatants were assessed for the presence of IL-8, IL-6 and VEGF-A by luminex bead analysis. <i>Brugia</i> ES products induced the production of (A) IL-8 (n = 15), (B) IL-6 (n = 10) and (C) VEGF-A (n = 15) by PBMCs compared to cells in media alone as assessed by the Signed Rank test. Medians are presented as bars.</p

Cytokine and growth factor levels (pg/mL) in PBMC supernatants^a.

a<p>1×10⁶ PBMCs were stimulated with worm ES or cultured in media alone for 72 h.</p><p>Supernatants from 5 different individuals were pooled and cytokines and growth factors were analyzed by luminex bead technology. Matrigel plugs were supplemented with 80 µL of the pooled supernatants and used for rat <i>in vivo</i> vessel formation experiments.</p

Lymphatic Filariasis Elimination in American Samoa: Evaluation of Molecular Xenomonitoring as a Surveillance Tool in the Endgame

<div><p>The Global Programme to Eliminate Lymphatic Filariasis has made significant progress toward interrupting transmission of lymphatic filariasis (LF) through mass drug administration (MDA). Operational challenges in defining endpoints of elimination programs include the need to determine appropriate post-MDA surveillance strategies. As humans are the only reservoirs of LF parasites, one such strategy is molecular xenomonitoring (MX), the detection of filarial DNA in mosquitoes using molecular methods (PCR), to provide an indirect indicator of infected persons nearby. MX could potentially be used to evaluate program success, provide support for decisions to stop MDA, and conduct post-MDA surveillance. American Samoa has successfully completed MDA and passed WHO recommended Transmission Assessment Surveys in 2011 and 2015, but recent studies using spatial analysis of antigen (Ag) and antibody (Ab) prevalence in adults (aged ≥18 years) and entomological surveys showed evidence of possible ongoing transmission. This study evaluated MX as a surveillance tool in American Samoa by linking village-level results of published human and mosquito studies. Of 32 villages, seropositive persons for Og4C3 Ag were identified in 11 (34.4%), for Wb123 Ab in 18 (56.3%) and for Bm14 Ab in 27 (84.4%) of villages. Village-level seroprevalence ranged from 0–33%, 0–67% and 0–100% for Og4C3 Ag, Wb123 Ab and Bm14 Ab respectively. PCR-positive <i>Aedes polynesiensis</i> mosquitoes were found in 15 (47%) villages, and their presence was significantly associated with seropositive persons for Og4C3 Ag (67% vs 6%, <i>p</i><0.001) and Wb123 Ab (87% vs 29%, <i>p</i> = 0.001), but not Bm14 Ab. In villages with persons seropositive for Og4C3 Ag and Wb123 Ab, PCR-positive <i>Ae</i>. <i>polynesiensis</i> were found in 90.9% and 72.2% respectively. In villages without seropositive persons for Og4C3 Ag or Wb123 Ab, PCR-positive <i>Ae</i>. <i>polynesiensis</i> were also absent in 94.1% and 70.6% of villages respectively. Our study provides promising evidence to support the potential usefulness of MX in post-MDA surveillance in an <i>Aedes</i> transmission area in the Pacific Islands setting.</p></div

Association between PCR-positive pools of mosquitoes and seropositive villages for Og4C3 Ag, Wb123 Ab, and Bm14 Ab.

<p>Association between PCR-positive pools of mosquitoes and seropositive villages for Og4C3 Ag, Wb123 Ab, and Bm14 Ab.</p

Probabilities of identifying seropositive villages for Og4C3 Ag, Wb123 Ab and Bm14 Ab based on the presence of PCR-positive pools of a) Ae. polynesiensis, b) any mosquito species, and c) other mosquito species.

<p>Probabilities of identifying seropositive villages for Og4C3 Ag, Wb123 Ab and Bm14 Ab based on the presence of PCR-positive pools of a) Ae. polynesiensis, b) any mosquito species, and c) other mosquito species.</p