Skip to main content
Download PDF

Ancylostoma ceylanicum infective third-stage larvae are activated by co-culture with HT-29-MTX intestinal epithelial cells

Parasites & Vectors volume 10, Article number: 606 (2017) Cite this article

Abstract

Background

Human hookworm larvae arrest development until they enter an appropriate host. This makes it difficult to access the larvae for studying larval development or host-parasite interactions. While there are in vivo and in vitro animal models of human hookworm infection, there is currently no human, in vitro model. While animal models have provided much insight into hookworm biology, there are limitations to how closely this can replicate human infection. Therefore, we have developed a human, in vitro model of the initial phase of hookworm infection using intestinal epithelial cell culture.

Results

Co-culture of the human hookworm Ancylostoma ceylanicum with the mucus-secreting, human intestinal epithelial cell line HT-29-MTX resulted in activation of infective third-stage larvae, as measured by resumption of feeding. Larvae were maximally activated by direct contact with fully differentiated HT-29-MTX intestinal epithelial cells. HT-29-MTX cells treated with A. ceylanicum larvae showed differential gene expression of several immunity-related genes.

Conclusions

Co-culture with HT-29-MTX can be used to activate A. ceylanicum larvae. This provides an opportunity to study the interaction of activated larvae with the human intestinal epithelium.

Background

Hookworm infection continues to be a problem in developing regions of Asia, Africa and Latin America [1]. Infection with hookworm contributes to the cycle of poverty by causing poor growth, anemia, and impaired school performance in children [1, 2]. Hookworm infection also causes illness in adults, especially in those who are immunocompromised or afflicted by other diseases such as malaria and tuberculosis [3].

There are two major hookworm species of humans: Ancylostoma duodenale and Necator americanus [4]. Ancylostoma ceylanicum is an emerging hookworm of humans in southern Asia [5,6,7]. Unlike the major hookworm species, A. ceylanicum can infect dogs and hamsters. The ability of A. ceylanicum to parasitize laboratory animals has made it a useful model for the study of human hookworm infection [8].

Hookworm require their host in order to complete development. Eggs in feces from an infected host hatch in the soil and develop to infective third-stage larvae (iL3). At this point, the larvae arrest and will not resume development until they enter their host. Upon entry to the host, the larvae activate, which entails resumption of feeding, secretion of infection-related proteins, and transcriptional changes [9,10,11,12,13]. Activated larvae migrate to the intestine where they continue development through the fourth larval stage (L4), attach to the intestinal wall, and mature to adulthood. There are two routes by which iL3 larvae can migrate to the intestine [4]. Ancylostoma sp., if ingested, can travel through the stomach and directly to the intestine. Alternatively, both Ancylostoma and Necator sp. can enter through the skin upon contact with contaminated soil and travel through the blood stream to the lungs. From the lungs, they are coughed up, swallowed, and travel through the stomach to the intestine.

Since hookworm only continue development beyond the iL3 stage inside their host, the initiation of parasitism is difficult to study. The signaling events allowing the larvae to transition from free-living iL3 to parasitic L4 are still unknown. Elucidation of these events could allow for the development of novel parasite control interventions and identification of immunomodulatory natural products, as well as further our understanding of hookworm biology [12, 14, 15].

In light of this problem, an in vitro method for the study of activation of A. ceylanicum iL3 larvae outside of the host has been developed [12, 16, 17]. In this method, larvae are incubated with canine serum and reduced glutathione, resulting in activation, the earliest stage of larval response to the host environment. Although these worms do activate, they do not continue development to L4. Additionally, recent work has shown that larvae activated by this method do not have the same transcriptional profile as larvae activated in vivo [13, 18].

In order to improve upon the existing in vitro model, we have begun development of an alternative, human model for the activation of iL3. In this model, A. ceylanicum iL3 are incubated with human intestinal epithelial cells. Using this model, we have successfully activated iL3 worms, causing them to resume feeding to a level exceeding what is observed after percutaneous infection in vivo [19].

Methods

Parasites

An Indian strain of A. ceylanicum (USNPC No. 102954) was raised in Syrian hamsters as described previously [20]. Hamsters were maintained at the George Washington University in accordance with institutional animal care and use committee guidelines. Infective A. ceylanicum L3 s were recovered from coprocultures after approximately 1 week at 27 °C by modified Baermann technique and stored up to 4 weeks in BU buffer (50 mM Na2HPO4/22 mM KH2PO4/70 mM NaCl, pH 6.8) [21] at room temperature.

Cell culture

HT-29-MTX (from Dr Thécla Lesuffleur, INSERM U560, Lille, France) were maintained in DMEM with 10% fetal bovine serum (FBS) and 1X Antibiotic-Antimycotic (Gibco, Gaithersburg, MD, USA; final concentration of 100 units/ml penicillin, 100 μg/ml streptomycin, and 0.25 μg/ml Amphotericin B) at 37 °C with 5% CO2. Prior to seeding trays for A. ceylanicum activation assays, cells were treated with 0.25% (v/v) trypsin and 0.02% EDTA in phosphate-buffered saline, pH 7.4 (PBS).

Ancylostoma ceylanicum activation assays

HT-29-MTX were seeded into 12-well culture trays (VWR, 10,062-894), with 2 × 104 cells per well. For the first 2 days of culture, the cells were incubated in DMEM with 10% FBS and 1× Antibiotic-Antimycotic (Gibco). On day 3 of culture, serum-free media (DMEM with 1X Antibiotic-Antimycotic, 1× Insulin-Transferrin-Selenium, 1× GlutaMax, and 1× MEM NEAA) was substituted for the previous media. All serum-free media (SFM) supplements were products of Gibco. Cells were maintained in SFM, with media changes every other day. On day 6, day 13, or day 20, A. ceylanicum iL3 were prepared and approximately 100 were added to each cell culture well with 2 μl of Fungizone (Gibco). To prepare A. ceylanicum iL3 for incubation in cell culture, they were pre-treated with 0.05% sodium hypochlorite in 1× PBS for 3 min and then washed twice in 1× PBS. Where indicated, they were incubated in 1% HCl for 30 min and/or incubated in 5% porcine bile in 1× PBS for 2 h. After each treatment they were washed twice in 1× PBS. The worms were then added to cell culture, buffer, or media and incubated for 36 h at 37 °C and 5% CO2. Larvae were incubated for 36 h because previous work has shown that by 36 h the maximum number of larvae are activated [17]. After 36 h, the worms were assayed for resumption of feeding, according to the method described in Hawdon & Schad [16]. Briefly, they were incubated in 2.5 mg/ml FITC-labeled BSA (Invitrogen, Carlsbad, CA, USA) for 2 h at 37 °C and 5% CO2, washed in 1× PBS, and observed by fluorescent microscopy. Individuals with a fluorescent intestinal lumen were counted as activated.

For A. ceylanicum activation assays in the absence of direct contact with the cells, HT-29-MTX were seeded on the bottom of 6-well transwell insert trays (Costar, 3450), with 2 × 104 cells per well. After day 2, they were maintained in SFM, as described above, for 20 days. On day 6, 13, or 20, pre-treated A. ceylanicum iL3 larvae were added to the upper portion of the transwell insert and incubated as described above.

A minimum of three biological replicates were conducted for each data point. Where noted, the percentage of larvae feeding was normalized to the percentage of larvae feeding in the SFM or PBS control, as appropriate for the experiment, with the percentage of larvae feeding in the control group being set as zero prior to averaging the results for each biological replicate. This normalization accommodates for batch-to-batch variability in the activation capacity of the larvae. Two-way analysis of variance was performed to look for interactions between variables. Where no interactions were observed, pairwise comparisons using t-tests with pooled standard deviations were performed.

Treatment of HT-29-MTX with A. ceylanicum larvae

HT-29-MTX were seeded and maintained as for the 20-day A. ceylanicum activation assays. For treatment of HT-29-MTX cells, A. ceylanicum larvae were prepared as above with 0.05% sodium hypochlorite treatment followed by 1% HCl treatment. Buffer washes were performed as described above. On day 20, HT-29-MTX cells were treated with approximately 100 A. ceylanicum larvae in 20 μl of buffer or with buffer alone and incubated at 37 °C and 5% CO2 for 24 h. After 24 h the cells were harvested for RNA isolation.

RNA isolation

RNA was isolated from and RNA-Seq performed on a total of four samples. Two samples were HT-29-MTX cells treated with A. ceylanicum larvae and two control samples were HT-29-MTX cells treated with buffer alone. Total RNA from the samples was purified using Trizol (Thermo Fisher, Waltham, MA, USA). The purification procedure deviated from the commercial Trizol protocol in the following regards: (i) after the first phase separation, an additional chloroform extraction step of the aqueous layer was performed using Phase-lock Gel tubes (5 prime), (ii) 1 μl of GlycoBlue (Thermo Fisher) was added immediately prior to the isopropanol precipitation, and (iii) the RNA was washed twice with 75% ethanol. RNA sample quality was confirmed by spectrophotometry (NanoDrop) and with a fragment analyzer (AATI Fragment Analyzer). For each sample, 1 μg of total RNA was used for further analysis. PolyA+ RNA was isolated with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB).

RNA sequencing

RNA-Seq was performed using the RNA Sequencing Core at Cornell University. TrueSeq-barcoded RNAseq libraries were generated with the NEBNext Ultra Directional RNA Library Prep Kit (NEB). Each library was quantified with a Qubit 2.0 (dsDNA HS kit; Thermo Fisher) and the size distribution was determined using a fragment analyzer (Advanced Analytical) prior to pooling. Libraries were sequenced on the NEXTseq 500. At least 20 M single-end 75 bp reads were generated per library. Reads were trimmed for low quality and adaptor sequences with Cutadapt v1.8. Reads were mapped to the reference genome UCSC hg19 using Tophat v2.0. Cufflinks v2.2 was used to generate FPKM values and to complete statistical analysis on differential gene expression.

Results

In designing an in vitro method for the activation of A. ceylanicum iL3 larvae, we aimed to mimic the oral route of infection, where iL3 larvae are consumed and pass through the stomach into the small intestine [19]. Therefore, after sterilization in 0.05% sodium hypochlorite, A. ceylanicum were treated with 1% HCl to mimic passage through the stomach. After HCl treatment, larvae were treated with 5% porcine bile, in a manner similar to that used by Li et al. [22] in Trichinella activation. Larvae would encounter bile acids as they pass through the duodenum.

After these pre-treatments, larvae were washed and incubated with HT-29-MTX intestinal epithelial cells (IECs) for 36 h at 37 °C and 5% CO2. HT-29 is a cell line derived from a human colon carcinoma [23]. Colon cancer cell lines, when differentiated, are structurally and functionally similar to the epithelium of the small intestine [24]. Methotrexate-adapted HT-29 cells (HT-29-MTX), when grown to post-confluency, are fully differentiated and composed of mucus-secreting goblet cells and polarized, absorptive enterocytes with brush borders [23,24,25]. HT-29-MTX cells were cultured in SFM, as previous work has shown that incubation of A. ceylanicum iL3 with serum alone is sufficient to induce activation of the larvae [12, 16, 17].

As a measure of activation, we calculated the percentage of the larvae that were feeding after incubation with the IECs. Larvae were counted as feeding if they were observed to have fluorescent protein in their intestines after two-hours of incubation with FITC-BSA.

First we determined the effect that each step of the treatment protocol had on A. ceylanicum activation. Incubation in SFM alone increases the percent of activated A. ceylanicum iL3 by 3 %, compared to larvae incubated in PBS. Pre-treatment with HCl did not increase the percentage of feeding larvae over incubation in SFM without pre-treatment (Fig. 1). Although treatment with HCl did not increase the percentage of feeding larvae, we continued to include this step in our larval preparation as an additional sterilization step.

Fig. 1
figure 1

Treatment with 5% bile increases the activation of Ancylostoma ceylanicum iL3 larvae. Treatment of A. ceylanicum iL3 with 5% bile increases the percent of activated larvae (P = 0.0017). Each bar represents the mean of three trials normalized to the percent feeding in PBS alone. Error bars represent the standard error of the mean. Each trial had n > 50. Data from each trial was normalized to the percent activation observed in observed in cell-free controls. Abbreviation: SFM, serum-free media

Full size image

We hypothesized that bile acid pre-treatment may play a role in activation of A. ceylanicum iL3. Treatment with bile did significantly increase feeding by approximately 10% (F (1, 9) = 16.5379, P = 0.0017) (Fig. 1). While bile treatment alone was not sufficient to induce a high level of feeding in the larvae, it may be permissive for activation through other pathways [26]. A two-factor analysis of variance showed no significant interaction between HCl treatment and bile treatment (F (1, 9) = 1.2357, P > 0.05).

After treatment with 1% HCl and 5% bile, incubation of A. ceylanicum iL3 larvae for 36 h with 21-day cultures of HT-29-MTX results in over 70% of the iL3 becoming activated (Fig. 2). This exceeds the percentage of A. ceylanicum iL3 activated by percutaneous infection of hamsters in vivo [19]. Although the larvae did activate, they did not continue development into L4.

Fig. 2
figure 2

Ancylostoma ceylanicum iL3 are activated by co-culture with IECs. Co-culture with HT-29-MTX for 36 h after 1% HCl and 5% porcine bile treatment activates A. ceylanicum iL3 (P = 0.00001). Error bars represent the standard error of the mean. Each bar represents the mean of three trials, each with n > 50. Abbreviation: SFM, serum-free media

Full size image

At 21-days, post-confluent cultures of HT-29-MTX are fully differentiated [25]. They express the differentiation-associated proteins dipeptidyl peptidase IV, carcinoembryonic antigen, and villin [23, 24] and have higher expression levels of the mucus protein-coding genes, mucs 1-5 [25, 27]. This led us to investigate the effect of age of culture on activation of A. ceylanicum iL3 larvae. Twenty one-day cultures of HT-29-MTX are significantly better at activating A. ceylanicum iL3 than 7-day or 14-day cultures, P = 0.0058 and P = 0.0266, respectively (Fig. 3). This difference may be due to the absence or decreased concentration of the activating signal in younger cultures.

Fig. 3
figure 3

Ancylostoma ceylanicum iL3 are maximally activated by differentiated IECs. Mature, 21-day-old cultures of HT-29-MTX maximally activate Ace iL3 compared to 7- and 14-day cultures (*P = 0.0058 and P = 0.0266, respectively). Direct contact with the cells induces greater activation than no direct contact (P = 0.014). Each bar represents the mean of three trials, each with n > 50. Error bars represent the standard error of the mean. Data from each trial was normalized to the percent activation observed in observed in cell-free controls

Full size image

The signal from HT-29-MTX that results in activation of may be secreted by the cells into the culture media, or may require direct contact of larvae with the cells. We used transwell inserts to separate larvae from the IECs to test these alternatives. Larvae were activated at a greater percentage with direct contact with the IECs, P = 0.014 (Fig. 3). The fact that the larvae were still activated to some extent in the absence of contact suggests that a soluble factor is at least partially responsible for activation of the worms. The reduced percentage of larvae activated without direct contact with the IECs could be due to the soluble factor being at lower concentration in the transwell than next to the cells, or due to some other contact-dependent factor playing a role in activation. A two-factor analysis of variance showed no significant interaction between age of cell culture and direct contact with the culture (F (4, 14) = 0.4501, P > 0.05).

To investigate the effect that the hookworm have on HT-29-MTX cells, we used RNA-Seq to analyze transcriptional changes in cells treated A. ceylanicum larvae. Nineteen genes were identified as differentially expressed, with a p-value of 0.00005 and a q-value of 0.03. These genes are listed in Table 1.

Table 1 Gene expression changes in IECs treated with A. ceylanicum iL3. Using RNA-Seq, gene expression was analyzed in HT-29-MTX cells treated with A. ceylanicum larvae and compared to controls (buffer treatment)
Full size table

Discussion

Identification of the host-specific factor responsible for promoting development of hookworm from iL3 to L4 has remained elusive. Similarities between the life cycle of Caenorhabditis elegans, a well-studied, free-living soil nematode which can enter an arrested stage of development called ‘dauer’ , and that of hookworm, which constitutively arrest development until entry into the host, has led researchers to look for parallels in the signaling mechanisms between dauer exit and the iL3-L4 transition [28].

A bile acid-like, steroid hormone, dafachronic acid (DA), is required for dauer exit in C. elegans through its binding of the nuclear receptor, DAF-12 [26]. Ancylostoma ceylanicum lack a homologue of the cytochrome P450 enzyme, DAF-9, which is required for synthesis of DA, although they do have a DAF-12 homologue. Therefore, it has been speculated that a host-derived enzyme may play the role of DAF-9 for synthesis of the DAF-12 ligand [28]. Alternatively, a host-derived bile acid-like molecule could be the DAF-12 ligand. C. elegans DAF-12 ligands are capable of inducing feeding in iL3, although not development to L4 [28]. Non-endogenous ligands have been shown to be weak activators of C. elegans DAF-12 [29] and the two C. elegans DAF-12 ligands are required at higher concentration to activate parasite DAF-12 [28]. This suggests that in hookworm an alternative endogenous or host-derived ligand may better activate DAF-12 and result in development to L4. As the site in the host where the hookworm resume development is the small intestine, we hypothesized that a host bile-acid may be the ligand. To test this hypothesis, we tested if porcine bile acids would have any effect on activation of A. ceylanicum, as dog, hamster, and human bile acid are not commercially available. We found that treatment with porcine bile acid only marginally increased A. ceylanicum feeding. Since bile acids from a non-host species can induce some level of A. ceylanicum feeding, it is possible that host bile acids would be more effective and could contain a DAF-12 ligand.

Co-culture with HT-29-MTX IECs was effective in activating A. ceylanicum iL3 larvae, although it did not lead to development to L4. HT-29-MTX do express genes from the cytochrome P450 superfamily, however, they do no express all of the P450 cytochrome genes that are expressed in the human ileum or liver [30]. Therefore we cannot reject the hypothesis that a host P450 enzyme is responsible for the development of iL3 to L4.

As an alternative to the hypothesis of a conserved dauer exit/iL3-L4 signal, the signal for triggering development of iL3 to L4 could come from other cell types present in the intestine. The intestinal epithelium also contains enteroendocrine cells, Paneth cells, stem cells, M cells, and is home to all of the cell types found in the gut-associated lymphoid tissue. Any of these could produce the signal for the iL3-L4 transition. Preliminary work with intestinal organoids derived from human pluripotent stem cells showed that incubation of iL3 larvae with the organoids did not promote development to L4 (data not shown), although further work could be done to optimize conditions.

Ancylostoma larvae can infect via a cutaneous or oral route. When infecting via the oral route, larvae do not activate prior to development through L4 [19]. Only when larvae infect via the cutaneous route do they activate and resume feeding prior to arrival in the intestine. It is thought that when larvae infect percutaneously they need to resume feeding to provide energy for their journey to the intestine [19]. In our assay, we aimed to replicate the oral route of infection; however, larvae resumed feeding but did not develop to L4. This suggests that signal for development to L4 is missing from the HT-29-MTX culture and that the larvae perceive the intestinal epithelium provided in our model more generally as epithelium.

Despite this, the model we have described here can be used to further study activation of iL3. Larvae activated in the presence of an epithelium may more closely resemble larvae activated in vivo than those activated by serum and reduced glutathione alone. This model provides an opportunity to study the signaling events occurring in the hookworm and the epithelium as they interact. We have taken a first step in this direction by analyzing the transcriptional changes that occur in HT-29-MTX cells treated with A. ceylanicum larvae. Of the 19 differentially expressed genes that we identified, 14 have been previously shown to play some role in immunity, including in parasitic infection, bacterial infection, and epithelial barrier function, suggesting that our in vitro system is representative of at least some in vivo events of infection. References for these previous studies are listed in Table 1.

Ht-29 has been used in other immunological studies, both of innate and adaptive immunity [31,32,33,34,35,36,37]. There has been some evidence that HT-29 cells respond differently to certain stimuli than do differentiated short-term primary cultures and intestinal epithelial cells in vivo [36, 37] and that HT-29 cells exposed to inflammatory mediators and cytokines produced by cells of the adaptive immune system show behavioral changes [31, 38,39,40]. Use of an immortalized cell line and lack of feedback from cells of the adaptive immune system may account for why we did not observe large changes in gene expression of HT-29 cells upon exposure to activated hookworm larvae.

Conclusions

This work is the first to show that human IECs are capable of inducing feeding of iL3 human hookworm larvae. Larvae are maximally activated by direct contact with fully-differentiated HT-29-MTX cells. Incubation with HT-29-MTX cells may provide a better model of iL3 activation than the current model, since the larvae are exposed to a more complex signaling environment than they encounter during activation by serum and reduced glutathione alone. Additionally, this model provides the opportunity to study the interactions between the activated parasite and the host tissue.

Abbreviations

DA:

Dafachronic acid

HT-29-MTX:

Methotrexate-adapted HT-29 cells

IECs:

Intestinal epithelial cells

iL3:

Infective third-stage larvae

L4:

Fourth-stage larvae

PBS:

Phosphate-buffered saline

SFM:

Serum-free media

References

  1. Hotez PJ, Brindley PJ, Bethony JM, King CH, Pearce EJ, Jacobson J. Helminth infections: the great neglected tropical diseases. J Clin Invest. 2008;118(4):1311–21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Crompton DW, Nesheim MC. Nutritional impact of intestinal helminthiasis during the human life cycle. Annu Rev Nutr. 2002;22:35–59.

    Article  CAS  PubMed  Google Scholar 

  3. Hotez PJ, Molyneux DH, Fenwick A, Ottesen E, Ehrlich Sachs S, Sachs JD. Incorporating a rapid-impact package for neglected tropical diseases with programs for HIV/AIDS, tuberculosis, and malaria. PLoS Med. 2006;3(5):e102.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Brooker S, Bethony J, Hotez PJ. Human hookworm infection in the 21st century. Adv Parasitol. 2004;58:197–288.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Inpankaew T, Schar F, Dalsgaard A, Khieu V, Chimnoi W, Chhoun C, et al. High prevalence of Ancylostoma ceylanicum hookworm infections in humans, Cambodia, 2012. Emerg Infect Dis. 2014;20(6):976–82.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Ngui R, Lim YA, Traub R, Mahmud R, Mistam MS. Epidemiological and genetic data supporting the transmission of Ancylostoma ceylanicum among human and domestic animals. PLoS Negl Trop Dis. 2012;6(2):e1522.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Traub RJ. Ancylostoma ceylanicum, a re-emerging but neglected parasitic zoonosis. Int J Parasitol. 2013;43(12-13):1009–15.

    Article  PubMed  Google Scholar 

  8. Hu Y, Ellis BL, Yiu YY, Miller MM, Urban JF, Shi LZ, et al. An extensive comparison of the effect of anthelmintic classes on diverse nematodes. PLoS One. 2013;8(7):e70702.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Dryanovski DI, Dowling C, Gelmedin V, Hawdon JM. RNA and protein synthesis is required for Ancylostoma caninum larval activation. Vet Parasitol. 2011;179(1-3):137–43.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Goud GN, Zhan B, Ghosh K, Loukas A, Hawdon J, Dobardzic A, et al. Cloning, yeast expression, isolation, and vaccine testing of recombinant Ancylostoma-secreted protein (ASP)-1 and ASP-2 from Ancylostoma ceylanicum. J Infect Dis. 2004;189(5):919–29.

    Article  CAS  PubMed  Google Scholar 

  11. Hawdon JM, Narasimhan S, Hotez PJ. Ancylostoma secreted protein 2: cloning and characterization of a second member of a family of nematode secreted proteins from Ancylostoma caninum. Mol Biochem Parasitol. 1999;99(2):149–65.

  12. Hawdon JM, Schad GA. Ancylostoma caninum: reduced glutathione stimulates feeding by third-stage infective larvae. Exp Parasitol. 1992;75(1):40–6.

    Article  CAS  PubMed  Google Scholar 

  13. Schwarz EM, Hu Y, Antoshechkin I, Miller MM, Sternberg PW, Aroian RV. The genome and transcriptome of the zoonotic hookworm Ancylostoma ceylanicum identify infection-specific gene families. Nat Genet. 2015;47(4):416–22.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Arasu P. In vitro reactivation of Ancylostoma caninum tissue-arrested third-stage larvae by transforming growth factor-Beta. J Parasitol. 2001;87(4):733–8.

    CAS  PubMed  Google Scholar 

  15. Loukas A, Prociv P. Immune responses in hookworm infections. Clin Microbiol Rev. 2001;14(4):689–703.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Hawdon JM, Schad GA. Serum-stimulated feeding in vitro by third-stage infective larvae of the canine hookworm Ancylostoma caninum. J Parasitol. 1990;76(3):394–8.

  17. Hawdon JM, Volk SW, Pritchard DI, Schad GA. Resumption of feeding in vitro by hookworm third-stage larvae: a comparative study. J Parasitol. 1992;78(6):1036–40.

    Article  CAS  PubMed  Google Scholar 

  18. Datu BJ, Gasser RB, Nagaraj SH, Ong EK, O'Donoghue P, McInnes R, et al. Transcriptional changes in the hookworm, Ancylostoma caninum, during the transition from a free-living to a parasitic larva. PLoS Negl Trop Dis. 2008;2(1):e130.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Hawdon JM, Volk SW, Rose R, Pritchard DI, Behnke JM, Schad GA. Observations on the feeding behaviour of parasitic third-stage hookworm larvae. Parasitology. 1993;106(Pt 2):163–9.

    Article  PubMed  Google Scholar 

  20. Garside P, Behnke JM. Ancylostoma ceylanicum in the hamster: observations on the host-parasite relationship during primary infection. Parasitology. 1989;98(Pt 2):283–9.

    Article  PubMed  Google Scholar 

  21. Hawdon JM, Schad GA. Long-term storage of hookworm infective larvae in buffered saline solution maintains larval responsiveness to host signals. J Helminthol Soc Wash. 1991;58(1):140–2.

  22. Li CK, Seth R, Gray T, Bayston R, Mahida YR, Wakelin D. Production of proinflammatory cytokines and inflammatory mediators in human intestinal epithelial cells after invasion by Trichinella spiralis. Infect Immun. 1998;66(5):2200–6.

  23. Lesuffleur T, Barbat A, Dussaulx E, Zweibaum A. Growth adaptation to methotrexate of HT-29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus-secreting cells. Cancer Res. 1990;50(19):6334–43.

    CAS  PubMed  Google Scholar 

  24. Lievin-Le Moal V, Servin AL. Pathogenesis of human enterovirulent bacteria: lessons from cultured, fully differentiated human colon cancer cell lines. Microbiol Mol Biol Rev. 2013;77(3):380–439.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Leteurtre E, Gouyer V, Rousseau K, Moreau O, Barbat A, Swallow D, et al. Differential mucin expression in colon carcinoma HT-29 clones with variable resistance to 5-fluorouracil and methotrexate. Biol Cell. 2004;96(2):145–51.

    Article  CAS  PubMed  Google Scholar 

  26. Gerisch B, Rottiers V, Li D, Motola DL, Cummins CL, Lehrach H, et al. A bile acid-like steroid modulates Caenorhabditis elegans lifespan through nuclear receptor signaling. Proc Natl Acad Sci USA. 2007;104(12):5014–9.

  27. Valeri M, Rossi Paccani S, Kasendra M, Nesta B, Serino L, Pizza M, et al. Pathogenic E. coli exploits SslE mucinase activity to translocate through the mucosal barrier and get access to host cells. PLoS One. 2015;10(3):e0117486.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Wang Z, Zhou XE, Motola DL, Gao X, Suino-Powell K, Conneely A, et al. Identification of the nuclear receptor DAF-12 as a therapeutic target in parasitic nematodes. Proc Natl Acad Sci USA. 2009;106(23):9138–43.

  29. Motola DL, Cummins CL, Rottiers V, Sharma KK, Li T, Li Y, et al. Identification of ligands for DAF-12 that govern dauer formation and reproduction in C. elegans. Cell. 2006;124(6):1209–23.

    Article  CAS  PubMed  Google Scholar 

  30. Carriere V, Lesuffleur T, Barbat A, Rousset M, Dussaulx E, Costet P, et al. Expression of cytochrome P-450 3A in HT29-MTX cells and Caco-2 clone TC7. FEBS Lett. 1994;355(3):247–50.

    Article  CAS  PubMed  Google Scholar 

  31. Bruno ME, Kaetzel CS. Long-term exposure of the HT-29 human intestinal epithelial cell line to TNF causes sustained up-regulation of the polymeric Ig receptor and proinflammatory genes through transcriptional and posttranscriptional mechanisms. J Immunol. 2005;174(11):7278–84.

    Article  CAS  PubMed  Google Scholar 

  32. Duary RK, Batish VK, Grover S. Immunomodulatory activity of two potential probiotic strains in LPS-stimulated HT-29 cells. Genes Nutr. 2014;9(3):398.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Gao Q, Qi L, Wu T, Wang J. An important role of interleukin-10 in counteracting excessive immune response in HT-29 cells exposed to Clostridium butyricum. BMC Microbiol. 2012;12:100.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Lopez P, Gonzalez-Rodriguez I, Sanchez B, Ruas-Madiedo P, Suarez A, Margolles A, et al. Interaction of Bifidobacterium bifidum LMG13195 with HT29 cells influences regulatory-T-cell-associated chemokine receptor expression. Appl Environ Microbiol. 2012;78(8):2850–7.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Mack DR, Ahrne S, Hyde L, Wei S, Hollingsworth MA. Extracellular MUC3 mucin secretion follows adherence of Lactobacillus strains to intestinal epithelial cells in vitro. Gut. 2003;52(6):827–33.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Sanchez B, Gonzalez-Rodriguez I, Arboleya S, Lopez P, Suarez A, Ruas-Madiedo P, et al. The effects of Bifidobacterium breve on immune mediators and proteome of HT29 cells monolayers. Biomed Res Int. 2015;2015:479140.

    PubMed  PubMed Central  Google Scholar 

  37. Pedersen G. Development, validation and implementation of an in vitro model for the study of metabolic and immune function in normal and inflamed human colonic epithelium. Dan Med J. 2015;62(1):B4973.

    PubMed  Google Scholar 

  38. Li YY, Hsieh LL, Tang RP, Liao SK, Yeh KY. Interleukin-6 (IL-6) released by macrophages induces IL-6 secretion in the human colon cancer HT-29 cell line. Hum Immunol. 2009;70(3):151–8.

    Article  CAS  PubMed  Google Scholar 

  39. Shioya M, Nishida A, Yagi Y, Ogawa A, Tsujikawa T, Kim-Mitsuyama S, et al. Epithelial overexpression of interleukin-32alpha in inflammatory bowel disease. Clin Exp Immunol. 2007;149(3):480–6.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Wright K, Kolios G, Westwick J, Ward SG. Cytokine-induced apoptosis in epithelial HT-29 cells is independent of nitric oxide formation. Evidence for an interleukin-13-driven phosphatidylinositol 3-kinase-dependent survival mechanism. J Biol Chem. 1999;274(24):17193–201.

    Article  CAS  PubMed  Google Scholar 

  41. Prado-Montes de Oca E. Human beta-defensin 1: a restless warrior against allergies, infections and cancer. Int J Biochem Cell Biol. 2010;42(6):800–4.

    Article  CAS  PubMed  Google Scholar 

  42. Ariza AC, Deen PM, Robben JH. The succinate receptor as a novel therapeutic target for oxidative and metabolic stress-related conditions. Front Endocrinol. 2012;3:22.

    Article  Google Scholar 

  43. Liehl P, Meireles P, Albuquerque IS, Pinkevych M, Baptista F, Mota MM, et al. Innate immunity induced by Plasmodium liver infection inhibits malaria reinfections. Infect Immun. 2015;83(3):1172–80.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Hu J, Wang G, Liu X, Zhou L, Jiang M, Yang L. Polo-like kinase 1 (PLK1) is involved in toll-like receptor (TLR)-mediated TNF-alpha production in monocytic THP-1 cells. PLoS One. 2013;8(10):e78832.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Aune TM, Spurlock CF III. Long non-coding RNAs in innate and adaptive immunity. Virus Res. 2016;212:146–60.

    Article  CAS  PubMed  Google Scholar 

  46. Kelly B, O'Neill LA. Metabolic reprogramming in macrophages and dendritic cells in innate immunity. Cell Res. 2015;25(7):771–84.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Li M, Schwerbrock NM, Lenhart PM, Fritz-Six KL, Kadmiel M, Christine KS, et al. Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta. J Clin Invest. 2013;123(6):2408–20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lang R, Hammer M, Mages J. DUSP meet immunology: dual specificity MAPK phosphatases in control of the inflammatory response. J Immunol. 2006;177(11):7497–504.

    Article  CAS  PubMed  Google Scholar 

  49. Labzin LI, Schmidt SV, Masters SL, Beyer M, Krebs W, Klee K, et al. ATF3 is a key regulator of macrophage IFN responses. J Immunol. 2015;195(9):4446–55.

    Article  CAS  PubMed  Google Scholar 

  50. Tan JB, Xu K, Cretegny K, Visan I, Yuan JS, Egan SE, et al. Lunatic and manic fringe cooperatively enhance marginal zone B cell precursor competition for delta-like 1 in splenic endothelial niches. Immunity. 2009;30(2):254–63.

    Article  PubMed  Google Scholar 

  51. Martinez Rodriguez NR, Eloi MD, Huynh A, Dominguez T, Lam AH, Carcamo-Molina D, et al. Expansion of Paneth cell population in response to enteric Salmonella enterica serovar typhimurium infection. Infect Immun. 2012;80(1):266–75.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Oshiumi H, Miyashita M, Okamoto M, Morioka Y, Okabe M, Matsumoto M, et al. DDX60 is involved in RIG-I-dependent and independent antiviral responses, and its function is attenuated by virus-induced EGFR activation. Cell Rep. 2015;11(8):1193–207.

    Article  CAS  PubMed  Google Scholar 

  53. Liu D, Rhebergen AM, Eisenbarth SC. Licensing adaptive immunity by NOD-like receptors. Front Immunol. 2013;4:486.

    PubMed  PubMed Central  Google Scholar 

  54. Nakatsukasa M, Kawasaki S, Yamasaki K, Fukuoka H, Matsuda A, Tsujikawa M, et al. Tumor-associated calcium signal transducer 2 is required for the proper subcellular localization of claudin 1 and 7: implications in the pathogenesis of gelatinous drop-like corneal dystrophy. Am J Pathol. 2010;177(3):1344–55.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Melissa Keaney and Caitlyn Leasure for parasite maintenance.

Funding

The project was supported by grant R21AI103531 from the National Institute of Allergy and Infectious Diseases and grant 1-DP20D007155-01 from the Office of the Director of the National Institutes of Health and by grant HDTRA1-13-1-0037 from the Defense Threat Reduction Agency. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of Allergy and Infectious Diseases nor the Office of the Director of the National Institutes of Health. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Availability of data and materials

The datasets supporting the conclusions of this article are available from the NCBI Gene Expression Omnibus (GEO) and are accessible through the GEO Series accession number GSE93620 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE93620).

Author information

Authors and Affiliations

  1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, NY, USA

    Caitlin M. Feather & John C. March

  2. Department of Microbiology, Immunology and Tropical Medicine, George Washington University, Washington, D.C., USA

    John M. Hawdon

Authors
  1. Caitlin M. Feather
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. John M. Hawdon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. John C. March
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

JMH provided iL3 A. ceylanicum larvae. CMF acquired the data. JMH, CMF and JCM made contributions to the design, analysis, and interpretation of the data. CMF drafted the manuscript and JMH and JCM revised it critically for intellectual content. All authors read and approved the final manuscript.

Corresponding author

Correspondence to John C. March.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Feather, C.M., Hawdon, J.M. & March, J.C. Ancylostoma ceylanicum infective third-stage larvae are activated by co-culture with HT-29-MTX intestinal epithelial cells. Parasites Vectors 10, 606 (2017). https://doi.org/10.1186/s13071-017-2513-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13071-017-2513-x

Keywords

Parasites & Vectors

ISSN: 1756-3305

Contact us