Endothelial cells release soluble factors that support the long-term survival of filarial worms in vitro

Abstract

The inability to maintain filarial nematodes in long-term in vitro culture greatly limits research into the basic biology of these parasites and hinders in vitro screening of novel anti-filarial agents. In this study, we sought to characterize nutrients that promote the long-term survival of filarial worms in vitro. Using microfilariae (MF) obtained from gerbils infected with Litomosoides sigmodontis, a filarial parasite of rodents, we found that Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) resulted in MF survival of only 5 days. However, co-culturing MF with a mouse endothelial cell line (EOMA) enabled survival for 40 days. Culturing EOMA cells in transwell plates extended MF survival to the same degree as direct co-culture, suggesting that the factors microfilariae require are soluble in nature. Heat inactivation of EOMA conditioned media at 56 °C reduced MF survival by approximately 50%, and heat inactivation at 100 °C reduced survival to 3 days, demonstrating that both heat labile and heat stable factors are involved. EOMA cells require FBS to produce these factors, as conditioned media collected from EOMA cells grown in the absence of FBS failed to prolong survival. The removal of lipids also abrogated survival, indicating MF are likely utilizing lipid factors released by EOMA cells. Dialysis experiments demonstrate that at least some of the required factors are between 0.1 and 1 kDa in size. Importantly, L. sigmodontis adult worms also show significantly extended survival when cultured in EOMA conditioned media. Together, these results suggest that EOMA-produced factors include lipid-containing molecules, heat labile molecules (likely a protein), and micronutrients between 0.1 and 1 kDa in size. These studies have established a cell-free approach to maintaining MF and adult stage filarial worms in long-term in vitro culture and have taken important steps towards biochemically characterizing host-derived nutrients required for parasite survival.

Keywords: Endothelial cells; Filariae; In vitro cell culture; Life cycle.

Fig. 1

Co-culture with mouse endothelial cell and mosquito lines enhances in vitro survival of MF. (A) MF survival in DMEM supplemented with various concentrations of FBS. DMEM = DMEM with 10% FBS. DMEM – FBS = DMEM with 0% FBS. DMEM 20% FBS = DMEM with 20% FBS. All groups were significantly different from one another. Median survival was calculated as 2.38 days for DMEM – FBS, 3.17 days for DMEM, and 3.61 days for DMEM + 20% FBS. (B) Co-culture of MF with a murine endothelial cell line in DMEM (EOMA co-culture) or with DMEM alone. (C) Maximum duration of MF survival when co-cultured with mosquito (C6/36), murine myeloma (MYE), and EOMA cell lines. As a control, MF were cultured in EMEM and DMEM supplemented with 10% FBS.

Fig. 2

Factors that prolong survival are soluble and constitutively produced when EOMA cells are grown in the presence of FBS. (A) Maximum duration of MF survival when cultured with EOMA cells in 0.22 μm transwell plates. (B) MF survival in CM from various cell lines. All groups were significantly different from one another, except for DMEM vs. RBL-2H3 CM. Median survival was calculated as 2.46 days for DMEM, 2.97 days for RBL CM, 3.60 for IMDM, 5.76 days for MYE CM, and 19.01 days for EOMA CM. (C) MF survival in CM derived from EOMA cells grown for 3 days in the absence of FBS. DMEM = DMEM with 10% FBS. EOMA CM = CM from EOMA cells grown in DMEM with 10% FBS. EOMA CM − FBS = CM from EOMA cells grown in DMEM without FBS. EOMA CM – FBS/+FBS = CM of EOMA cells grown in DMEM without FBS, followed by subsequent addition of 10% FBS. All groups were significantly different from one another. Median survival was calculated as 3.31 days for DMEM, 4.06 days for EOMA CM − FBS, 5.41 days for EOMA CM − FBS/+FBS, and 19.84 days for EOMA CM.

Fig. 3

EOMA CM contains factors that are sensitive to heat treatment and lipid depletion. (A) MF survival in DMEM and EOMA CM heat treated at 56 °C for 1 or 4 h. All groups were significantly different from one another, except for DMEM vs. DMEM HT 56 °C. Median survival was calculated as 2.88 days for DMEM HT 56 °C, 3.6 days for DMEM, 7.69 days for EOMA CM HT 56 °C 4 h, 8.82 days for EOMA CM HT 56 °C, and 16.57 days for EOMA CM. (B) MF survival in DMEM and EOMA CM heat treated at 100 °C for 1 h. DMEM HT 100 °C + FBS = DMEM heat treated at 100 °C for 1 h followed by subsequent addition of 10% FBS. EOMA CM HT 100 °C + FBS = EOMA CM heat treated at 100 °C for 1 h followed by subsequent addition of 10% FBS. Groups were statistically different from one another except for: DMEM vs. EOMA CM HT 100 °C + FBS, DMEM HT 100 °C vs. DMEM HT 100 °C + FBS, DMEM HT 100 °C vs. EOMA CM HT 100 °C, and DMEM HT 100 °C + FBS vs. EOMA CM HT 100 °C. Median survival was calculated as 1.34 days for EOMA CM HT 100 °C, 1.53 days for DMEM HT 100 °C, 1.54 days for DMEM HT 100 °C + FBS, 2.49 days for DMEM, 2.55 days for EOMA CM 100 °C, and 16.31 days for EOMA CM. (C) MF survival in DMEM and EOMA CM depleted of lipids and lipoproteins with LRA. DMEM LRA = DMEM treated with LRA. DMEM LRA + 10% FBS = DMEM treated with LRA, followed by subsequent addition of 10% FBS. EOMA LRA = EOMA CM treated with LRA. EOMA LRA + 10% FBS = EOMA CM treated with LRA, followed by subsequent addition of 10% FBS. EOMA CM was statistically different from all other groups. Additionally, DMEM vs. DMEM LRA, DMEM LRA vs. EOMA CM LRA, and DMEM LRA vs. EOMA CM LRA + FBS were statistically different from one another. Median survival was calculated as 1.90 days for DMEM LRA, 1.99 days for DMEM LRA + FBS, 2.36 days for EOMA CM LRA + FBS, 2.62 days for EOMA CM LRA, 2.88 days for DMEM, and 16.03 days for EOMA CM.

Fig. 4

MF require factors between 0.1–1 kDa in size. MF were cultured in EOMA CM dialyzed into DMEM using 1000, 20, 1, and 0.1 kDa molecular weight cutoffs. DMEM = DMEM supplemented with 10% FBS. EOMA CM > 1000 kDa = EOMA CM including all factors greater than 1000 kDA. EOMA CM > 20 kDa = EOMA CM including all factors greater than 20 kDa. EOMA CM > 1 kDa = EOMA CM including all factors greater than 1 kDA. EOMA CM > 0.1 kDA = EOMA CM including all factors greater than 0.1 kDa. All groups were statistically different from one another except for: DMEM vs. EOMA CM > 1000 kDa, and EOMA CM > 1000 kDa vs. EOMA CM > 1 kDa. Median survival was calculated as 3.12 days for DMEM, 3.25 days for EOMA CM > 1000 kDa, 3.32 days for EOMA CM > 1 kDa, 3.48 days for EOMA CM > 20 kDa, 9.10 days for EOMA CM > 0.1 kDa, and 17.83 days for EOMA CM.

Fig. 5

C3 is upregulated in EOMA CM and modestly prolongs MF survival. (A) Comparative 2D DIGE gel of EOMA CM (green) and RBL-2H3 CM (red). 32 spots that exhibited >1.5 fold upregulation in EOMA CM were analyzed by mass spectrometry (Table 1). A cluster analysis was performed to determine which functional groups were enhanced in EOMA CM (Table 2). (B) Supplementation of DMEM with proteins from Table 2 that were categorized as secreted/extracellular. A single dose of 20 μg of recombinant mouse C3, recombinant apolipoprotein E, or native albumin was added to DMEM with 10% FBS. DMEM + Recombinant C3 was statistically different from all other groups. Median survival was calculated as 2.15 days for DMEM + Recombinant ApoE, 2.41 days for DMEM + Native Albumin, 2.88 days for DMEM, and 3.89 days for DMEM + Recombinant C3.

Fig. 6

The use of serum replacements, added cholesterol, or nucleosides did not enhance MF survival. (A) 10% Cell-Ess or 1X Fatty Acid Supplement was added to unsupplemented DMEM as serum replacements. Additionally, 10 μg/ml of cholesterol was added to DMEM with 10% FBS. All groups were statistically different from one another except for DMEM vs. DMEM + Cholesterol. Median survival was calculated as 0.05 days for DMEM 10% Cell-Ess, 1.50 days for DMEM + Fatty Acid Supplement, 2.54 days for DMEM, and 2.57 days for DMEM + Cholesterol. (B) A nucleoside supplement (1X) was added to DMEM with 10% FBS. There was no difference between groups. Median survival was calculated as 2.49 days for DMEM + Nucleosides, and 2.53 days for DMEM.

Fig. 7

EOMA CM prolongs survival of adult filarial worms. (A) Culture of L. sigmodontis adult female worms in 0.22 μm transwell plates with EOMA cells with no media exchanged. Graph depicts a representative experiment. (B) L. sigmodontis adult female worms cultured in CM. One adult female worm was cultured per 1 ml of CM, and CM was exchanged every other day. EOMA CM significantly enhanced survival compared to DMEM (p = 0.012).