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secretory products of adult Haemonchus contortus and Teladorsagia circumcincta which increase the permeability of Caco-2 cell monolayers are neutralised by antibodies from immune hosts
Accepted Manuscript Title: Excretory/secretory products of adult Haemonchus contortus and Teladorsagia circumcincta which increase the permeability of Caco-2 cell monolayers are neutralised by antibodies from immune hosts Author: Z.U. Rehman Q. Deng S. Umair M.S. Savoian J.S. Knight A. Pernthaner H.V. Simpson PII: DOI: Reference:
S0304-4017(16)30069-3 http://dx.doi.org/doi:10.1016/j.vetpar.2016.03.017 VETPAR 7947
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
14-12-2015 15-3-2016 19-3-2016
Please cite this article as: Rehman, Z.U., Deng, Q., Umair, S., Savoian, M.S., Knight, J.S., Pernthaner, A., Simpson, H.V., Excretory/secretory products of adult Haemonchus contortus and Teladorsagia circumcincta which increase the permeability of Caco-2 cell monolayers are neutralised by antibodies from immune hosts.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2016.03.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Excretory/secretory products of adult Haemonchus contortus and Teladorsagia circumcincta which increase the permeability of Caco-2 cell monolayers are neutralised by antibodies from immune hosts
Z.U. Rehmana, Q. Dengb, S. Umairb, M.S. Savoianc, J.S. Knightb, A. Pernthanerb and H.V. Simpsona*
a
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag
1-222, Palmerston North, New Zealand b
c
AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand
Institute of Fundamental Sciences, Massey University, Private Bag 1-222, Palmerston
North, New Zealand
* Corresponding author Prof. HV. Simpson, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand. Tel.: 64-6-356 9099; Fax: 64-6-350 5636; E-mail: [email protected]
Graphical Abstract
1
Highlights
ES products of H. contortus and T. circumcincta reduced TEER of Caco-2 cell monolayers Zona occludens-1 and occludin were internalised from disrupted junctions after exposure to ES products Increase in TEER partially blocked by phage displaying single chain antibodies derived from immune sheep and enriched by panning with H. contortus ES products
2
ABSTRACT
The onset of abomasal pathophysiology due to parasitism coincides with the presence of adult worms in the lumen, implicating worm excretory/secretory (ES) products acting on the surface mucosa. Caco-2 cell monolayers were grown to confluence on Transwell plates and exposed on the apical side to ES products of adult Haemonchus contortus and Teladorsagia circumcincta. ES products of both species significantly (p < 0.001) reduced transepithelial electrical resistance after 2 h to 81.1 ± 1.0% and 82.9 ± 1.1% respectively. Immunocytochemical staining of the Caco-2 monolayers for zona occludens-1 and occludin confirmed that the tight junctions remained intact in control medium, but these proteins were internalised from disrupted junctions after exposure to ES products. The components of H. contortus ES products responsible for increased epithelial permeability were partially blocked by phage displaying single chain antibodies derived from sheep immune to field infection and enriched by panning with H. contortus ES products. Immune hosts may therefore be able to reduce the effects of worm chemicals on the gastric epithelium. Permeabilisation of the abomasal surface mucosa by worm chemicals would also explain how cells deep in the gastric glands could rapidly be affected by parasites emerging from the glands or within a day of transplantation of adult worms into naïve hosts, resulting in the pathophysiology typically caused by abomasal nematode parasitism. Keywords: Haemonchus contortus; Teladorsagia circumcincta; ES products; Caco-2 cell TEER; epithelial permeability; phage display, single chain antibodies
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1. Introduction
The
onset
of
the
pathophysiology
(hypoacidity,
hypergastrinaemia
and
hyperpepsinogenaemia) and inflammatory responses associated with abomasal nematode parasitism coincides with the emergence of larvae or immature adult worms from the gastric glands into the lumen and also rapidly follows transplantation of adult worms into naïve recipients (McKellar et al., 1986; Lawton et al., 1996; Simpson et al., 1997). This strongly suggests the involvement of parasite excretory/secretory (ES) products in mediating the effects of luminal parasites, most likely through their action on the surface mucosa of the abomasum. The lack of overt effects of worm chemicals on the epithelial cells in glands containing developing larvae is not unexpected in light of the unusual properties of the apical membranes of gland cells, which make them resistant to chemical absorption or damage (Waisbren et al., 1994). ES products probably penetrate the surface epithelium of the abomasum and act on the basolateral membranes of the parietal and chief cells located deep in the glands. The increased leakage of plasma protein and pepsinogen into the gastric lumen of parasitised animals (Holmes and MacLean, 1971; McLeay et al., 1973) is consistent with increased paracellular permeability, which would also facilitate the entry of worm chemicals into host tissues. Infection with many pathogens, including nematodes, increases the permeability of the host gut (Scott et al., 2002; McDermott et al., 2003; Su et al., 2011); this may be through specific secreted molecules, but may also be reinforced by cytokines generated by inflammation which are known to increase gut permeability (Ma et al., 2005; Al-Sadi and Ma, 2007). Epithelial cell monolayers, such as Caco-2 cells, form tight junctions and are in vitro models frequently used to investigate changes in permeability induced by chemicals, infectious agents or inflammatory cytokines. Adjacent cells adhere to each other at junctional complexes, of which the tight junctions are considered to be responsible for intercellular sealing, permeability and selective
4
transport. Tight junctions are complex networks of transmembrane proteins, principally occludins, claudins and junctional adhesion molecules, with numerous peripheral proteins linking the tight junction to the actin cytoskeleton and to the intracellular signalling system (Gonzalez-Mariscal et al., 2008; Furuse, 2010). The permeability of tight junctions can be regulated by protein phosphorylation (Sawada et al., 2003) or by structural remodelling, which is continuous under physiological conditions, and involves exchange of protein with intracellular pools and altered expression of proteins (Ivanov et al., 2004, 2005; Shen et al., 2008). Occludin is an important determinant of the permeability of tight junctions, as the transepithelial resistance (TEER) decreases when interactions of occludin with other molecules are disrupted (Balda and Matter, 1998). The peripheral zona occludens proteins (ZO-1 and ZO-2) are essential for the polymerisation of the proteins of tight junctions (Umeda et al., 2006). As part of the cell invasion process, many pathogens disrupt tight junctions and increase epithelial permeability by a combination of effects on protein function, tight junction remodelling or interacting with integral and peripheral proteins of the cytoskeleton. The specific proteins targeted vary with the organism, often involving delocalisation of occludin and ZO-1 (Dickman et al., 2000; Nusrat et al., 2001; Boyle et al., 2006). Tissues collected from rodents infected with the helminths Heligmosomoides polygyrus or Hymenolepsis diminuta had reduced TEER in vitro ( KosikBogacka et al., 2010; Su et al., 2011) and occludin was relocated from tight junctions in the colon and ileum of infected rats from Days 8 - 40 p.i. (Kosik-Bogacka et al., 2011). In preliminary experiments, Büring (2009) observed that ES products of adult Haemonchus contortus, but not Teladorsagia circumcincta, reduced the TEER of Caco-2 cells at 6 and 24 hours and both internalised the tight junctional proteins ZO-1 and occludin, as well as causing cytoskeletal rearrangement in HeLa cells. The present experiments examined the time course of the effects of ES products of both adult and T. circumcincta and H. contortus on Caco-2 cell TEER and
5
internalisation of tight junction proteins. It was also established that the active components are released by the worms within one hour and for at least 24 hours in vitro.
Evidence that components of nematode ES products act in vivo as well as in in vitro model systems would be a demonstration that antibodies produced by immune sheep recognise and block the in vitro effects of ES products. Single chain antibody fragments (scFvs) libraries displayed on bacteriophage, created by coupling cloned light and heavy chain immunoglobulin-variable regioncoding DNA, are useful in identifying antibodies to target antigens (Burton and Barbas, 1994; Winter et al., 1994). Maass et al. (2007) developed a scFvs library derived from cells residing in lymph nodes draining the gastrointestinal tract of sheep which had become immune to field infections with nematodes including of H. contortus and T. circumcincta. Selective enrichment of this phage library for binding to ES products provided pools of antibodies for the present study to investigate host recognition and neutralisation of parasite chemicals involved in increased epithelial TEER.
2. MATERIALS AND METHODS
All chemicals were purchased from the Sigma Chemical Co. (MO, USA) unless stated otherwise. Use of experimental animals for culturing and harvesting adult worms was carried out under Massey University Animal Ethics Committee approval 09/11 and AgResearch animal ethics approval AE 13052.
2.1. Parasites
6
Pure cultures of H. contortus and T. circumcincta were maintained in the laboratory by regular passage through sheep. Adult worms of H. contortus or T. circumcincta were recovered from the abomasa of infected sheep on Day 21 or 28 post-infection (p.i.) respectively, as described previously (Umair et al., 2013). Briefly, abomasal contents were mixed 2:1 with 3% agar and the solidified agar blocks incubated at 37 °C in a saline bath. Clumps of parasites were collected from the saline soon after emergence and placed in RP2 incubation medium.
2.2. Preparation of ES products
H. contortus ES products (HcES) and T. circumcincta ES products (TcES) were prepared by incubating adult worms (approximately 50 mg/mL) at 37o C in RP2 medium (RPMI 1640 (Life Technologies, Auckland, NZ) supplemented with 2 g/L NaHCO3, 5.958 g/L HEPES, 1 g/L penicillin and 1 g/L streptomycin) in an atmosphere of 5% CO2 and 95% humidified air. Worm viability was monitored during the incubation. ES products were harvested and replaced with fresh medium for T. circumcincta at 1, 2, 4, 12 and 24 h and for H. contortus at 1, 2, 4, 12 and 24 h (batch 1) or 1, 4 and 24 h (batch 2). ES products were filtered through 0.2 µm filters (Minisart, Sartorius, Gottingen, Germany) into sterile tubes and aliquots were frozen at –80oC. Each batch of ES products was confirmed to be negative for LPS using PyrosatR endotoxin kits (Cape Cod Inc, MA, USA) with a sensitivity level of 0.25 EU/mL. The control solution was incubation medium that was treated similarly to the ES products. The protein content of the ES products, measured with a NanoDrop A-280 spectrophotometer (Thermo Scientific, Australia) with reference settings based on a 1 mg/mL protein solution, decreased from 0.1 mg/mL in preparations at 2 and 4 h to 0.9 mg/mL at 24 h.
2.3. Panning with HcES of an ovine single chain antibody library displayed on phage
7
Anti-HcES single chain antibody fragments (scFvs) were obtained by panning an ovine scFvs library, which was prepared from B cell mRNA obtained from abomasal and mesenteric lymph nodes of two year-old Romney sheep naturally infected with a poly-generic nematode challenge (Maass et al., 2007). These animals were refractory to Trichostrongylus colubriformis, T. circumcincta and H. contortus infections. Selection was carried out by 3 rounds of panning of scFv-displayed phage libraries by binding to HcES. Four mL HcES (an equal mixture of 1 and 4 h collections) was immobilised on immunotubes (Nunc) overnight at 4 ⁰C. The tubes were then washed 3 times with PBS, filled completely with 2% skim milk powder in PBS (PBSM) and incubated at 37 ⁰C for 2 h. After rinsing the tube 3 times with PBS, 10¹²-10¹³ colony forming units (CFU) of phage was added in 4 mL 2% PBSM and incubated for 2h at room temperature with inversion. The tube was washed 10 times with PBS containing 0.1% Tween 20 (PBST), then 10 times with PBS. Bound phage was eluted by continuous inversion with 1 mL 100mM glycine (pH 3.0) for 10 min, the neutralised immediately with 0.5 mL 1 M Tris-HCl, pH 7.4. 0.75 mL of eluted phage was used to infect 9.25 mL of a culture of log-phase E.coli TG1 cells. After incubating for 30 min, the cells were spun down, the pellet was re-suspended in a small amount of medium and spread on 2xYT plates containing 2% glucose and 100 µg/mL ampicillin and incubated overnight at 30 ⁰C. Phage was prepared from the recovered colonies as described by Maass et al. (2007), then second and third panning performed as above except the washing steps before elution were increased to 20 times with PBST and PBS. The titre of eluted phage was determined by diluting the eluted phage, mixing each dilution with exponentially growing TG1 cells and incubating at 37 ⁰C for 30 min. The infected cells were plated on 2xYT/Glu/Amp(100) plates, incubated overnight at 30 ⁰C and counted the next day to determine the phage titre.
8
2.4. Caco-2 cell monolayers
Caco-2 cells were maintained in culture in DMEM (Life Technologies), supplemented with 10% foetal bovine serum (FBS) (Gibco, Auckland, NZ), 1% non-essential amino acid (100x solution, Gibco) and 1% antibiotic solution (PSN 100x (Gibco)). To establish confluent monolayers, Caco-2 cells were seeded with a suspension of 3.105 cells/mL on polyester membrane transwell-clear inserts (Corning Clear 12-well Transwell plates; 12 mm diameter, 0.4 μm pore size) until the TEER (adjusted for blank reading), measured with an epithelial volt-ohm meter (EVOM; World Precision Instruments, Inc., Sarasota, FL, USA), became stable at 400 - 600 Ω/cm2.
2.5. Effects of ES products on TEER of Caco-2 cell monolayers
Culture medium in the apical and basolateral chambers was changed 24 h prior to testing for the effect of ES products. Test solutions were pre-warmed to 37 oC and equilibrated in 5% CO2 for 30 min to ensure the pH was 7.2- 7.4. After the culture medium on the apical side was discarded and replaced with 500 µl of test solution, the monolayers were incubated for 24 h and TEER measured at 0, 2, 4, 6, 8 and 24 h. The test solutions were: (1) HcES (batch 1, collected 2 - 4 h) (N = 16) were compared with control medium (same medium incubated without worms) (N = 18); (2) TcES (collected 2 - 4 h) (N = 52) were compared with control medium (both N = 12); (3) TcES (collected from 0 - 1, 1 - 2, 4 - 12 and 12 - 24 h of incubation) (N = 10, except N = 6 for 4 - 12 h collection) were compared with control medium (N = 12). Data from TcES collected from 2 4 h in (2) above were included for comparison (N = 52).
2.6. Immunocytochemical staining of tight junction proteins
9
Plates were set up with control medium and undiluted H. contortus or T. circumcincta ES products on the apical side, as described in 2.5. The H. contortus ES products were batch 1, collected from 2 - 4 h, and the T. circumcincta ES products were collected from 2 - 4 h. TEER was measured at time zero and at 4 h, when the TEER had decreased by 2% for control medium and 24.5% for H. contortus and 19.4% for T. circumcincta ES products respectively. The membranes with Caco-2 monolayers were fixed in situ in 4% paraformaldehyde for 30 min at room temperature, washed 3 times with PBS, and stored in 0.02% sodium azide in PBS at 4 o
C until stained. The filter membranes were carefully removed from the transwells and the sodium
azide was removed with a PBS wash prior to permeabilisation with 0.2% Triton-X-100 in PBS for 15 min at room temperature. The detergent solution was thoroughly removed from the membranes by 3 washes with PBS for 5 min each. Membranes were incubated with blocking buffer (1% BSA, 2% goat serum and 0.05% Tween-20 in PBS) for 60 min at 37 oC. The primary Abs were rabbit anti-ZO-1 and mouse anti-occludin, raised against synthetic peptides encoded by the human N terminus and
C terminus of each protein respectively (Invitrogen, Life Technologies). The
secondary Abs were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (2 mg/mL) (Life Technologies). Each primary Ab was diluted with blocking buffer to a final concentration of ~2 µg/mL prior to use. Secondary Ab was diluted 1:500 in blocking buffer and applied successively for 60 min with washing 3 times between with blocking buffer. Membranes were mounted with SlowFade Gold antifade reagent supplemented with DAPI (Life Technologies) and the slides observed by scanning confocal microscopy (Leica SP5 DM6000B, Wetzlar, Germany). Each channel was acquired sequentially as a z-series using a 63x NA 1.4 lens with an optical zoom of 3 and a step size of 0.5 µm. Data sets were imported into ImageJ to generate maximum intensity projections. Figures of projections were processed and constructed using Adobe Photoshop software.
10
2.7. Effect of anti-HcES scFvs on TEER of Caco-2 cell monolayers
TEER was measured during incubation at 0, 2, 4, 6, 8 and 24 h after replacing the apical solution with a test solution which was pre-incubated for 30 min at 37oC in 5% CO2: (1) HcES (undiluted batch 1, collected 2 - 4 h) (N = 3); (2) HcES (batch 1, collected 2 - 4 h) with the pool of anti-HcES scFvs obtained from the third panning at final concentrations of 1015, 1014, 1013 or 1012 CFU/mL (N = 3); (3) HcES (batch 2, collected 1 - 4 h) with the pool of anti-HcES scFvs obtained from the third panning at final concentrations of 1015, 1014 or 1013 CFU/mL (N = 3); (4) Pool of anti-HcES scFvs obtained from the third panning at final concentrations of 1015, 1014, 1013 or 1012 CFU/mL (N = 3).
2.8. Data analysis
TEER data for each well at successive time points were normalised to time zero (100%). Replicate data are presented as mean ± SEM. Graphpad Prism v5 was used to plot graphs and to analyse data using repeated measures two-way ANOVA with Bonferri post-tests to compare data at successive time points.
3. RESULTS
3.1. Effect of H. contortus or T. circumcincta ES products on Caco-2 cell TEER
11
Apical exposure to ES products, collected from 2 - 4 h incubation of adult worms of both species, significantly reduced the TEER of Caco-2 cell monolayers (p < 0.05 to p < 0.001) from 2 24 h, compared with the effect of control incubate, treated similarly, but without worms in the incubate (Fig. 1). The greatest mean reductions were seen at 2 h, with slow, incomplete recovery over 24 h.
3.1. Effect of time of collection of T. circumcincta ES products on Caco-2 cell TEER
Apical exposure to T. circumcincta ES products, collected in sequential, but lengthening worm incubation periods to 24 h, reduced the TEER of Caco-2 cell monolayers (Fig. 2) to mean values at 2 h to 79 - 82% of that at zero time. The relative TEER at each time point for these incubates is shown in Table 1, together with the level of significant difference from the TEER produced by exposure to control solution. The TEER was significantly lower than that produced by the control incubate at 2 and 4 h for all incubates (p < 0.05 to p < 0.001) and also later in some cases (Table 1).
3.4. Tight junction proteins
Immunocytochemical staining of the tight junction proteins ZO-1 and occludin showed that exposure to ES products, but not control medium, caused internalisation of these proteins from the tight junction after 4 h exposure. This was more pronounced for occludin than for ZO-1. The intracellular punctate staining of these proteins following exposure to ES products contrasted with the location in the cell membrane after exposure to control medium (Fig. 3).
3.5. Panning of an ovine anti-HcES scFvs against HcES
12
Three sets of in vitro panning against immobilised HcES resulted in an approximate 100fold increase in elution phage titres from the second to the third panning (Table 2).
3.6. Effect of anti-HcES scFvs on TEER of Caco-2 cell monolayers
In 2 separate experiments with different batches of H. contortus ES products, phage displaying anti-HcES scFvs at a concentration of 1015 CFU/mL, but not at lower concentrations, significantly blocked the effect of HcES at 2 to 8 h (p < 0.01 to 0.001) (Fig. 4 A, B). No concentration of phage alone had any effect on TEER (Fig. 4 C).
4. DISCUSSION
These experiments have shown that ES products derived from two species of adult nematode parasites of the abomasum are capable of increasing the permeability of a model epithelium and the likelihood of this also occurring in vivo, as immune sheep recognise the chemicals involved and generate antibodies which can neutralise them in vitro. This implicates worm chemicals in the pathophysiology typically produced by luminal stages of the parasites, but not those developing in gastric glands (McKellar et al., 1986; Lawton et al., 1996; Simpson et al., 1997). Parietal cells located within the gastric glands are known targets of abomasal parasites, despite their apical membranes being resistant to chemical absorption or damage (Waisbren et al., 1994). Permeabilisation of the surface epithelium by worm chemicals would allow parietal cell inhibitors to enter the gastric tissues and act on the basolateral membranes of gland cells, resulting in both reduced acid secretion and altered also through modifying the release of growth factors which regulate the populations of gastric cells (Simpson, 2000).
13
Increased paracellular permeability of the epithelium of the parasitised abomasum is not unexpected as it has been inferred from increased leakage of plasma protein and pepsinogen into the gastric lumen (Holmes and MacLean, 1971; McLeay et al., 1973) and intestinal permeability is similarly increased by nematode (Kosik-Bogacka et al., 2010, 2011; Su et al., 2011) and unicellular parasites (Scott et al., 2002). Cultured epithelial cells also develop increased permeability upon exposure to bacteria and protozoa (Maia-Brigagão et al., 2012). This could result from direct effects of the organism or be secondary to host immune responses. In the present experiments, a direct role for worm chemicals in opening tight junctions is supported by the rapid fall in TEER of Caco-2 cell monolayers on exposure to adult H. contortus or T. circumcincta ES products and histological evidence of internalisation of the tight junctional proteins occludin and ZO-1 to a lesser extent (Fig. 3). The effects were rapid and recovery of the TEER continued over 24 hours (Fig. 1). The identity and the exact mode of action of the active constituents of ES products remain to be elucidated, but could be any of the 200 proteins or other chemicals known to be present in these ES products of sheep parasites, including glycolytic and other metabolic enzymes, proteases and structural components (Yatsuda et al., 2003; Craig et al., 2006; Kiel et al., 2007). The potency of T. circumcincta ES products was high from the beginning of the collection in vitro and maintained for at least 24 hours (Fig. 2). The collection periods for the ES products were increased from 1 hour to 12 hours, suggesting the rate of release of the active chemicals probably decreased over time. It is unlikely that excreted ammonia contributes to opening of tight junctions, as adult worm ES products usually contain only around 100 µM ammonia after 4 hours of incubation (Simpson et al., 2009), which is much less than the 50 mM ammonia used to produce a 20% decrease in TEER of cultured MDCK cells (Vastag et al., 2005) or the 10 - 15 mM ammonia which acted similarly on Caco-2 cells exposed to Helicobacter pylori (Lytton et al., 2005). Exposure to ES products of either adult H. contortus or T. circumcincta (Fig. 3) resulted in disruption of tight junctions with internalisation of the tight junctional proteins ZO-1 and occludin,
14
as previously observed by Büring (2009). Similarly, tissues from rodents infected with H. polygyrus or H. diminuta had reduced TEER in vitro ( Kosik-Bogacka et al., 2010; Su et al., 2011) and occludin was relocated from tight junctions in the colon and ileum of infected rats from Days 8 - 40 p.i. (Kosik-Bogacka et al., 2011). Many pathogens increase epithelial permeability using a variety of specific molecules which result in delocalisation of occludin and ZO-1 (Dickman et al., 2000; Nusrat et al., 2001; Boyle et al., 2006). Disrupting the interactions of occludin with other tight junction molecules is frequently seen, as this increases the permeability of tight junctions (Balda and Matter, 1998). How nematode ES products reversibly cause dissociation of occludin and ZO-1 from tight junctions remains to be determined. There may be specific molecules released by the parasites which act on either the tight junctions or the attached cytoskeleton, however, cytokine release from Caco-2 cells cannot be ruled out, as cultured epithelial cells both release endogenous cytokines when exposed to pathogens (Jung et al., 1995) and are responsive to exogenous cytokines (Van De Walle et al., 2010). Inflammatory mediators may be involved in vivo, if parasites also target the epithelium in this way to allow their ES products to penetrate the mucosa. The active components of abomasal nematode ES products were recognised by sheep immune to field infection and selected phage displaying anti-HcEs scFvs blocked the in vitro effects of HcES on Caco-2 cell permeability. Anti-HcES scFvs (1015 CFU/mL) significantly blocked the effect of HcES from 2 - 8 h (p < 0.001), whereas phage alone had no effect on the TEER (Fig. 4). Single chain antibody fragment libraries displayed on bacteriophage are used to identify antibodies to defined target antigens (Burton and Barbas, 1994; Winter et al., 1994) or to study antibody responses to immunogens in many vertebrates, including sheep (Li et al., 2000) and cattle (O'Brien et al., 1999). The scFvs library used in these experiments was constructed from mRNA from abomasal and mesenteric lymph nodes from two year-old sheep refractory to T. colubriformis, T. circumcincta and H. contortus infections and originally used to characterise larval surface antigens (Maass et al., 2007). Selective panning of this phage library with H. contortus ES products provided antibodies to
15
investigate host recognition of parasite chemicals involved in increased epithelial TEER. The blocking of the TEER by selected phage showed the potential for immune sheep to neutralise the permeabilising chemicals produced by the parasite, which would be expected to limit the pathophysiology and perhaps restrict colonisation of the abomasum. These experiments confirmed the ability of adult H. contortus and T. circumcincta ES products to disrupt tight junctions of cultured epithelial cells. The reduction in TEER was greatest after exposure for 2 hours, the first time point studied and slowly increased again over 24 hours. It was also shown that the immune host recognised and produced blocking antibodies against ES products responsible for permeabilisation of epithelia. Production of these antibodies indicates that there is exposure in vivo to key epitopes on the active ES components.
Acknowledgments Meat and Wool New Zealand, the E. and C. Thoms Bequest, AgResearch Ltd and Massey University are thanked for financial support. Imaging was performed at the Manawatu Microscopy and Imaging Centre, Massey University. Dr R. Anderson is thanked for helpful comments on the manuscript.
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Kiel, M., Josh, P., Jones, A., Windon, R., Hunt, P., Kongsuwan, K., 2007. Identification of immune-reactive proteins from a sheep gastrointestinal nematode, Trichostrongylus colubriformis, using two-dimensional electrophoresis and mass spectrometry. Int. J. Parasitol. 37, 1419-1429. Kosik-Bogacka, D.I., Baranowska-Bosiacka, I., Salamatin, R., 2010. Hymenolepis diminuta: Effect of infection on ion transport in colon and blood picture of rats. Exp. Parasitol. 124, 285-294. Kosik-Bogacka, D.I., Kolasa, A., Baranowska-Bosiacka, I., Marchlewicz, M., 2011. Hymenolepis diminuta: The effects of infection on transepithelial ion transport and tight junctions in rat intestines. Exp. Parasitol. 127, 398-404. Lawton, D.E.B., Reynolds, G.W., Hodgkinson, S.M., Pomroy, W.E., Simpson, H.V., 1996. Infection of sheep with adult and larval Ostertagia circumcincta: Effects on abomasal pH and serum gastrin and pepsinogen. Int. J. Parasitol. 26, 1063-1074. Li, Y., Kilpatrick, J., Whitelam, G.C., 2000. Sheep monoclonal antibody fragments generated using a phage display system. J. Immunol. Meth. 236, 133-146 Lytton, S.D., Fischer, W., Nagel, W., Haas, R., Beck, F.X., 2005. Production of ammonium by Helicobacter pylori mediates occludin processing and disruption of tight junctions in Caco-2 cells. Microbiology 151, 3267-3276. Ma, T.Y., Boivin, M.A., Ye, D., Pedram, A., Said, H.M., 2005. Mechanism of TNF-α modulation of Caco-2 intestinal epithelial tight junction barrier: role of myosin light-chain kinase protein expression. Am. J. Physiol. 288, G422-G430. Maass, D.R., Harrison, G.B., Grant, W.N., Shoemaker, C.B., 2007. Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep. Int. J. Parasitol. 37, 953-962.
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McDermott, J.R., Bartram, R.E., Knight, P.A., Miller, H.R.P., Garrod, D.R., Grencis, R.K., 2003. Mast cells disrupt epithelial barrier function during enteric nematode function. Proc. Natl. Acad. Sci. USA 100, 7761-7766. McLeay, L.M., N. Anderson, J.B. Bingley, D.A. Titchen, D.A., 1973. Effects on abomasal function of Ostertagia circumcincta infections in sheep. Parasitology 66, 241–257. McKellar, Q., Duncan, J.L., Armour, J, McWilliam, P., 1986. Response to transplanted adult Ostertagia ostertagi in calves. Res. Vet. Sci. 40, 367-371. Maia-Brigagão, C., Morgado-Diaz, J.A., De Souza, W., 2012. Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells. Parasitol. Int. 61, 280287. Nusrat, A., Von Eichel-Streiber, C., Turner, J.R., Verkade, P., Madara, J.L., Parkos, C.A., 2001. Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect. Immun. 69, 1329-1336. O'Brien, P.M., Aitken, R., O'Neil, B.W., Campo, M.S., 1999. Generation of native bovine mAbs by phage display. Proc. Natl. Acad. Sci. USA 96, 640-645 Sawada, N., Murata, M., Kikuchi, K., Osanai, M., Tobioka, H., Kojima, T., Chiba, H., 2003. Tight junctions and human diseases. Med. Elect. Microscopy 36, 147-156. Scott, K.G., Meddings, J.B., Kirk, D.R., Lees-Miller, S.P., Buret, A.G., 2002. Intestinal function with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology 123, 1179-1190. Shen, L., Weber, C.R., Turner, J.R., 2008. The tight junction protein complex undergoes rapid and continuous molecular remodelling at steady state. J. Cell Biol. 181, 683-695. Simpson, H.V., 2000. Pathophysiology of abomasal parasitism: is the host or parasite responsible? Vet J. 160, 177-191.
19
Simpson, H.V., Lawton, D.E.B., Simcock, D.C., Reynolds, G.W., Pomroy, W.E., 1997. Effects of adult and larval Haemonchus contortus on abomasal secretion. Int. J. Parasitol. 27, 825-831. Simpson, H.V., Muhamad, N., Walker, L.R., Simcock, D.C., Brown, S., Pedley, K.C., 2009. Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta. Exp. Parasitol. 123, 17-23. Su, C., Cao, Y., Kaplan, J., Zhang, M., Li, W., Conroy, M., Walker, W.A., Shi, H.N., 2011. Duodenal helminth infection alters barrier function of the colonic epithelium via adaptive immune activation. Infect. Immun. 79, 2285-2294. Umair, S., Knight, J.S., Bland, R.J., Simpson, H.V., 2013. Molecular and biochemical characterisation of arginine kinases in Haemonchus contortus and Teladorsagia circumcincta. Exp. Parasitol. 134, 362-367. Umeda, K., Ikenouchi, J., Katahira-Tayama, S., Furuse, K., Sasaki, H., Nakayama, M., Matsui, T., Tsukita, S., Furuse, M., 2006. ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation. Cell 126, 741-754. Van De Walle, J., Hendrickx, A., Romier, B., Laondelle, Y., Schneider, Y.-J., 2010. Inflammatory parameters in Caco-2 cells: Effect of stimuli nature, concentration and cell differentiation. Toxicol. In Vitro 24, 1441-1449. Vastag, M., Neuhofer, W., Nagel, W., Beck, F.X., 2005. Ammonium affects tight junctions and the cytoskeleton in MDCK cells. Pflüg. Arch. 449, 384-391. Waisbren, S.J., Geibel, J.P., Modlin, I.M., Boron, W.F., 1994. Unusual permeability properties of gastric glands. Nature 368, 332-335. Winter, G., Griffiths, A.D., Hawkins, R.E., Hoogenboom, H.R., 1994. Making antibodies by phage display technology. Ann. Rev. Immunol. 12, 433-455. Yatsuda, A.P., Krijgsveld, J., Cornelissen, A.W.C.A., Heck, A.J.R., De Vries, E., 2003. Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals
20
extensive sequence variation and differential immune recognition. J. Biol. Chem. 278, 1694116951.
Figure Legends
Fig. 1. Effect of apical membrane exposure to ES products, collected from 2-4 h of incubation of adult Haemonchus contortus (N = 16) (top) or Teladorsagia circumcincta (N = 52) (bottom) or control incubate (N = 16 or 14), on the transepithelial resistance (TEER) (mean ± SEM), normalised to zero time, of Caco-2 cell monolayers. Symbols: (■) ES products; (▲) control incubate. Significant differences at each time point are shown: ⃰ : p < 0.05; ⃰ ⃰ : p < 0.01; ⃰ ⃰ ⃰ ⃰ : p < 0.001.
Fig. 2. Effects of exposure to Teladorsagia circumcincta ES products collected over four incubation periods on the normalised trans-epithelial resistance (TEER) (mean ± SEM) of Caco-2 cell monolayers. Symbols: (x) control medium (N = 12); (■): 0 - 1 h (N = 10); (▲): 1 - 2 h (N = 10); (■): 2 – 4 h (N = 52); (): 4 - 12 h (N = 6); (): 12 - 24 h (N = 10).
Fig. 3. Effect of apical membrane exposure to control incubate (top row) or ES products of adult Haemonchus contortus (middle row) or Teladorsagia circumcincta (bottom row) on the location of ZO-1 (left column) or occludin (centre column) in the tight junctions of Caco-2 cell monolayers. The merged images are also shown (ZO-1 in green and occludin in red; regions of overlap appear yellow). DNA was stained with DAPI (blue) to reveal the positions of the nuclei. Scale bar: 10µm.
Fig. 4. Effect on the transepithelial resistance (TEER) (mean ± SEM, N = 3), normalised to zero time, of Caco-2 cell monolayers of apical membrane exposure to two different batches of ES 21
products of adult Haemonchus contortus pre-incubated with anti-HcES single chain antibody fragments (scFvs) (A, B) or scFvs in control incubate (C). Symbols: (■) no scFvs; (▲) 1015 CFU/mL; (■) 1014 CFU/mL; (♦) 1013 CFU/mL and (●) 1012 CFU/mL. Significant differences at each time point are shown: ⃰ ⃰ : p < 0.01; ⃰ ⃰ ⃰ ⃰ : p < 0.001.
22
Table 1. Transepithelial electrical resistance (TEER) (normalised to time zero) (mean ± SEM) of Caco-2 cell monolayers after exposure to ES products collected over sequential time periods of incubation of Teladorsagia circumcincta adult worms. Significant differences of ES products from control medium are shown: ⃰ : p < 0.5; ⃰ ⃰ : p < 0.01; ⃰ ⃰ ⃰ : p < 0.001.
Incubate
Normalised TEER (% zero time) 2h
4h
6h
8h
24 h
Control, N = 12
95.3 ± 1.3
101.3 ± 1.4
106.2 ± 1.8
107.2 ± 2.2
130.4 ± 2.3
0 - 1 h, N = 10
81.7 ± 4.5 ⃰ ⃰ ⃰
86.6 ± 2.3 ⃰ ⃰ ⃰
92.6 ± 1.8 ⃰ ⃰ ⃰
99.5 ± 1.8
117.7 ± 4.2 ⃰ ⃰ ⃰
1 - 2 h, N = 10
80.0 ± 4.4 ⃰ ⃰ ⃰
83.1 ± 4.2 ⃰ ⃰ ⃰
92.9 ± 3.7 ⃰ ⃰
97.8 ± 3.3.
119.0 ± 4.8 ⃰
2 - 4 h, N = 52
81.0 ± 1.9 ⃰ ⃰ ⃰
85.8 ± 2.1 ⃰ ⃰ ⃰
93.7 ± 2.0 ⃰ ⃰
100.4 ± 1.8
119.7 ± 2.4 ⃰
4 - 12 h, N = 6
81.7 ± 1.8 ⃰ ⃰ ⃰
90.0 ±3.0 ⃰ ⃰
99.5 ± 3.8
109.7 ± 3.4
137.1 ± 4.6
12 - 24 h, N = 10
78.6 ± 5.9 ⃰
82.7 ± 6.4 ⃰ ⃰
92.1 ± 6.1
97.8 ± 4.9
116.9 ± 6.7
23
Table 2. Titres of ovine scFv phage (colony forming units/mL) in the original library and in eluted samples and after amplification.
scFv library Eluted (CFU/ml) Amplified (CFU/mL) 3.1013 original 1st panning
5.107
not tested
2nd panning
2.106
5.1016
3rd panning
3.108
1.1017
24
Graphical Abstract
TEER (% zero time)
110
ES + phage 1015 CFU/ml
100
ES + phage 1012 CFU/ml
ES only
90
80
70 0
4
8
12
16
Time (hours)
20
24
Figure 1
TEER (% zero time)
110
100 90
80
70 0
4
8
12
16
20
24
TEER (% zero time)
140
120
100
80
60
0
4
8
12
Time (hours)
16
20
24
Figure 2
TEER (% zero time)
140
120
100
80
60 0
4
8
12
Time (hours)
16
20
24
Figure 3 Click here to download high resolution image
Figure 4
TEER (% zero time)
110
A
100
90
80
70 0
TEER (% zero time)
120
4
8
12
16
20
24
12
16
20
24
12
16
20
24
B
100
80
60
TEER (% zero time)
0
105
4
8
4
8
C
100 95 90 0
Time (hours)
S0304-4017(16)30069-3 http://dx.doi.org/doi:10.1016/j.vetpar.2016.03.017 VETPAR 7947
To appear in:
Veterinary Parasitology
Received date: Revised date: Accepted date:
14-12-2015 15-3-2016 19-3-2016
Please cite this article as: Rehman, Z.U., Deng, Q., Umair, S., Savoian, M.S., Knight, J.S., Pernthaner, A., Simpson, H.V., Excretory/secretory products of adult Haemonchus contortus and Teladorsagia circumcincta which increase the permeability of Caco-2 cell monolayers are neutralised by antibodies from immune hosts.Veterinary Parasitology http://dx.doi.org/10.1016/j.vetpar.2016.03.017 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Excretory/secretory products of adult Haemonchus contortus and Teladorsagia circumcincta which increase the permeability of Caco-2 cell monolayers are neutralised by antibodies from immune hosts
Z.U. Rehmana, Q. Dengb, S. Umairb, M.S. Savoianc, J.S. Knightb, A. Pernthanerb and H.V. Simpsona*
a
Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag
1-222, Palmerston North, New Zealand b
c
AgResearch Ltd, Private Bag 11-008, Palmerston North, New Zealand
Institute of Fundamental Sciences, Massey University, Private Bag 1-222, Palmerston
North, New Zealand
* Corresponding author Prof. HV. Simpson, Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Private Bag 11-222, Palmerston North 4442, New Zealand. Tel.: 64-6-356 9099; Fax: 64-6-350 5636; E-mail: [email protected]
Graphical Abstract
1
Highlights
ES products of H. contortus and T. circumcincta reduced TEER of Caco-2 cell monolayers Zona occludens-1 and occludin were internalised from disrupted junctions after exposure to ES products Increase in TEER partially blocked by phage displaying single chain antibodies derived from immune sheep and enriched by panning with H. contortus ES products
2
ABSTRACT
The onset of abomasal pathophysiology due to parasitism coincides with the presence of adult worms in the lumen, implicating worm excretory/secretory (ES) products acting on the surface mucosa. Caco-2 cell monolayers were grown to confluence on Transwell plates and exposed on the apical side to ES products of adult Haemonchus contortus and Teladorsagia circumcincta. ES products of both species significantly (p < 0.001) reduced transepithelial electrical resistance after 2 h to 81.1 ± 1.0% and 82.9 ± 1.1% respectively. Immunocytochemical staining of the Caco-2 monolayers for zona occludens-1 and occludin confirmed that the tight junctions remained intact in control medium, but these proteins were internalised from disrupted junctions after exposure to ES products. The components of H. contortus ES products responsible for increased epithelial permeability were partially blocked by phage displaying single chain antibodies derived from sheep immune to field infection and enriched by panning with H. contortus ES products. Immune hosts may therefore be able to reduce the effects of worm chemicals on the gastric epithelium. Permeabilisation of the abomasal surface mucosa by worm chemicals would also explain how cells deep in the gastric glands could rapidly be affected by parasites emerging from the glands or within a day of transplantation of adult worms into naïve hosts, resulting in the pathophysiology typically caused by abomasal nematode parasitism. Keywords: Haemonchus contortus; Teladorsagia circumcincta; ES products; Caco-2 cell TEER; epithelial permeability; phage display, single chain antibodies
3
1. Introduction
The
onset
of
the
pathophysiology
(hypoacidity,
hypergastrinaemia
and
hyperpepsinogenaemia) and inflammatory responses associated with abomasal nematode parasitism coincides with the emergence of larvae or immature adult worms from the gastric glands into the lumen and also rapidly follows transplantation of adult worms into naïve recipients (McKellar et al., 1986; Lawton et al., 1996; Simpson et al., 1997). This strongly suggests the involvement of parasite excretory/secretory (ES) products in mediating the effects of luminal parasites, most likely through their action on the surface mucosa of the abomasum. The lack of overt effects of worm chemicals on the epithelial cells in glands containing developing larvae is not unexpected in light of the unusual properties of the apical membranes of gland cells, which make them resistant to chemical absorption or damage (Waisbren et al., 1994). ES products probably penetrate the surface epithelium of the abomasum and act on the basolateral membranes of the parietal and chief cells located deep in the glands. The increased leakage of plasma protein and pepsinogen into the gastric lumen of parasitised animals (Holmes and MacLean, 1971; McLeay et al., 1973) is consistent with increased paracellular permeability, which would also facilitate the entry of worm chemicals into host tissues. Infection with many pathogens, including nematodes, increases the permeability of the host gut (Scott et al., 2002; McDermott et al., 2003; Su et al., 2011); this may be through specific secreted molecules, but may also be reinforced by cytokines generated by inflammation which are known to increase gut permeability (Ma et al., 2005; Al-Sadi and Ma, 2007). Epithelial cell monolayers, such as Caco-2 cells, form tight junctions and are in vitro models frequently used to investigate changes in permeability induced by chemicals, infectious agents or inflammatory cytokines. Adjacent cells adhere to each other at junctional complexes, of which the tight junctions are considered to be responsible for intercellular sealing, permeability and selective
4
transport. Tight junctions are complex networks of transmembrane proteins, principally occludins, claudins and junctional adhesion molecules, with numerous peripheral proteins linking the tight junction to the actin cytoskeleton and to the intracellular signalling system (Gonzalez-Mariscal et al., 2008; Furuse, 2010). The permeability of tight junctions can be regulated by protein phosphorylation (Sawada et al., 2003) or by structural remodelling, which is continuous under physiological conditions, and involves exchange of protein with intracellular pools and altered expression of proteins (Ivanov et al., 2004, 2005; Shen et al., 2008). Occludin is an important determinant of the permeability of tight junctions, as the transepithelial resistance (TEER) decreases when interactions of occludin with other molecules are disrupted (Balda and Matter, 1998). The peripheral zona occludens proteins (ZO-1 and ZO-2) are essential for the polymerisation of the proteins of tight junctions (Umeda et al., 2006). As part of the cell invasion process, many pathogens disrupt tight junctions and increase epithelial permeability by a combination of effects on protein function, tight junction remodelling or interacting with integral and peripheral proteins of the cytoskeleton. The specific proteins targeted vary with the organism, often involving delocalisation of occludin and ZO-1 (Dickman et al., 2000; Nusrat et al., 2001; Boyle et al., 2006). Tissues collected from rodents infected with the helminths Heligmosomoides polygyrus or Hymenolepsis diminuta had reduced TEER in vitro ( KosikBogacka et al., 2010; Su et al., 2011) and occludin was relocated from tight junctions in the colon and ileum of infected rats from Days 8 - 40 p.i. (Kosik-Bogacka et al., 2011). In preliminary experiments, Büring (2009) observed that ES products of adult Haemonchus contortus, but not Teladorsagia circumcincta, reduced the TEER of Caco-2 cells at 6 and 24 hours and both internalised the tight junctional proteins ZO-1 and occludin, as well as causing cytoskeletal rearrangement in HeLa cells. The present experiments examined the time course of the effects of ES products of both adult and T. circumcincta and H. contortus on Caco-2 cell TEER and
5
internalisation of tight junction proteins. It was also established that the active components are released by the worms within one hour and for at least 24 hours in vitro.
Evidence that components of nematode ES products act in vivo as well as in in vitro model systems would be a demonstration that antibodies produced by immune sheep recognise and block the in vitro effects of ES products. Single chain antibody fragments (scFvs) libraries displayed on bacteriophage, created by coupling cloned light and heavy chain immunoglobulin-variable regioncoding DNA, are useful in identifying antibodies to target antigens (Burton and Barbas, 1994; Winter et al., 1994). Maass et al. (2007) developed a scFvs library derived from cells residing in lymph nodes draining the gastrointestinal tract of sheep which had become immune to field infections with nematodes including of H. contortus and T. circumcincta. Selective enrichment of this phage library for binding to ES products provided pools of antibodies for the present study to investigate host recognition and neutralisation of parasite chemicals involved in increased epithelial TEER.
2. MATERIALS AND METHODS
All chemicals were purchased from the Sigma Chemical Co. (MO, USA) unless stated otherwise. Use of experimental animals for culturing and harvesting adult worms was carried out under Massey University Animal Ethics Committee approval 09/11 and AgResearch animal ethics approval AE 13052.
2.1. Parasites
6
Pure cultures of H. contortus and T. circumcincta were maintained in the laboratory by regular passage through sheep. Adult worms of H. contortus or T. circumcincta were recovered from the abomasa of infected sheep on Day 21 or 28 post-infection (p.i.) respectively, as described previously (Umair et al., 2013). Briefly, abomasal contents were mixed 2:1 with 3% agar and the solidified agar blocks incubated at 37 °C in a saline bath. Clumps of parasites were collected from the saline soon after emergence and placed in RP2 incubation medium.
2.2. Preparation of ES products
H. contortus ES products (HcES) and T. circumcincta ES products (TcES) were prepared by incubating adult worms (approximately 50 mg/mL) at 37o C in RP2 medium (RPMI 1640 (Life Technologies, Auckland, NZ) supplemented with 2 g/L NaHCO3, 5.958 g/L HEPES, 1 g/L penicillin and 1 g/L streptomycin) in an atmosphere of 5% CO2 and 95% humidified air. Worm viability was monitored during the incubation. ES products were harvested and replaced with fresh medium for T. circumcincta at 1, 2, 4, 12 and 24 h and for H. contortus at 1, 2, 4, 12 and 24 h (batch 1) or 1, 4 and 24 h (batch 2). ES products were filtered through 0.2 µm filters (Minisart, Sartorius, Gottingen, Germany) into sterile tubes and aliquots were frozen at –80oC. Each batch of ES products was confirmed to be negative for LPS using PyrosatR endotoxin kits (Cape Cod Inc, MA, USA) with a sensitivity level of 0.25 EU/mL. The control solution was incubation medium that was treated similarly to the ES products. The protein content of the ES products, measured with a NanoDrop A-280 spectrophotometer (Thermo Scientific, Australia) with reference settings based on a 1 mg/mL protein solution, decreased from 0.1 mg/mL in preparations at 2 and 4 h to 0.9 mg/mL at 24 h.
2.3. Panning with HcES of an ovine single chain antibody library displayed on phage
7
Anti-HcES single chain antibody fragments (scFvs) were obtained by panning an ovine scFvs library, which was prepared from B cell mRNA obtained from abomasal and mesenteric lymph nodes of two year-old Romney sheep naturally infected with a poly-generic nematode challenge (Maass et al., 2007). These animals were refractory to Trichostrongylus colubriformis, T. circumcincta and H. contortus infections. Selection was carried out by 3 rounds of panning of scFv-displayed phage libraries by binding to HcES. Four mL HcES (an equal mixture of 1 and 4 h collections) was immobilised on immunotubes (Nunc) overnight at 4 ⁰C. The tubes were then washed 3 times with PBS, filled completely with 2% skim milk powder in PBS (PBSM) and incubated at 37 ⁰C for 2 h. After rinsing the tube 3 times with PBS, 10¹²-10¹³ colony forming units (CFU) of phage was added in 4 mL 2% PBSM and incubated for 2h at room temperature with inversion. The tube was washed 10 times with PBS containing 0.1% Tween 20 (PBST), then 10 times with PBS. Bound phage was eluted by continuous inversion with 1 mL 100mM glycine (pH 3.0) for 10 min, the neutralised immediately with 0.5 mL 1 M Tris-HCl, pH 7.4. 0.75 mL of eluted phage was used to infect 9.25 mL of a culture of log-phase E.coli TG1 cells. After incubating for 30 min, the cells were spun down, the pellet was re-suspended in a small amount of medium and spread on 2xYT plates containing 2% glucose and 100 µg/mL ampicillin and incubated overnight at 30 ⁰C. Phage was prepared from the recovered colonies as described by Maass et al. (2007), then second and third panning performed as above except the washing steps before elution were increased to 20 times with PBST and PBS. The titre of eluted phage was determined by diluting the eluted phage, mixing each dilution with exponentially growing TG1 cells and incubating at 37 ⁰C for 30 min. The infected cells were plated on 2xYT/Glu/Amp(100) plates, incubated overnight at 30 ⁰C and counted the next day to determine the phage titre.
8
2.4. Caco-2 cell monolayers
Caco-2 cells were maintained in culture in DMEM (Life Technologies), supplemented with 10% foetal bovine serum (FBS) (Gibco, Auckland, NZ), 1% non-essential amino acid (100x solution, Gibco) and 1% antibiotic solution (PSN 100x (Gibco)). To establish confluent monolayers, Caco-2 cells were seeded with a suspension of 3.105 cells/mL on polyester membrane transwell-clear inserts (Corning Clear 12-well Transwell plates; 12 mm diameter, 0.4 μm pore size) until the TEER (adjusted for blank reading), measured with an epithelial volt-ohm meter (EVOM; World Precision Instruments, Inc., Sarasota, FL, USA), became stable at 400 - 600 Ω/cm2.
2.5. Effects of ES products on TEER of Caco-2 cell monolayers
Culture medium in the apical and basolateral chambers was changed 24 h prior to testing for the effect of ES products. Test solutions were pre-warmed to 37 oC and equilibrated in 5% CO2 for 30 min to ensure the pH was 7.2- 7.4. After the culture medium on the apical side was discarded and replaced with 500 µl of test solution, the monolayers were incubated for 24 h and TEER measured at 0, 2, 4, 6, 8 and 24 h. The test solutions were: (1) HcES (batch 1, collected 2 - 4 h) (N = 16) were compared with control medium (same medium incubated without worms) (N = 18); (2) TcES (collected 2 - 4 h) (N = 52) were compared with control medium (both N = 12); (3) TcES (collected from 0 - 1, 1 - 2, 4 - 12 and 12 - 24 h of incubation) (N = 10, except N = 6 for 4 - 12 h collection) were compared with control medium (N = 12). Data from TcES collected from 2 4 h in (2) above were included for comparison (N = 52).
2.6. Immunocytochemical staining of tight junction proteins
9
Plates were set up with control medium and undiluted H. contortus or T. circumcincta ES products on the apical side, as described in 2.5. The H. contortus ES products were batch 1, collected from 2 - 4 h, and the T. circumcincta ES products were collected from 2 - 4 h. TEER was measured at time zero and at 4 h, when the TEER had decreased by 2% for control medium and 24.5% for H. contortus and 19.4% for T. circumcincta ES products respectively. The membranes with Caco-2 monolayers were fixed in situ in 4% paraformaldehyde for 30 min at room temperature, washed 3 times with PBS, and stored in 0.02% sodium azide in PBS at 4 o
C until stained. The filter membranes were carefully removed from the transwells and the sodium
azide was removed with a PBS wash prior to permeabilisation with 0.2% Triton-X-100 in PBS for 15 min at room temperature. The detergent solution was thoroughly removed from the membranes by 3 washes with PBS for 5 min each. Membranes were incubated with blocking buffer (1% BSA, 2% goat serum and 0.05% Tween-20 in PBS) for 60 min at 37 oC. The primary Abs were rabbit anti-ZO-1 and mouse anti-occludin, raised against synthetic peptides encoded by the human N terminus and
C terminus of each protein respectively (Invitrogen, Life Technologies). The
secondary Abs were Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 647 goat anti-mouse IgG (2 mg/mL) (Life Technologies). Each primary Ab was diluted with blocking buffer to a final concentration of ~2 µg/mL prior to use. Secondary Ab was diluted 1:500 in blocking buffer and applied successively for 60 min with washing 3 times between with blocking buffer. Membranes were mounted with SlowFade Gold antifade reagent supplemented with DAPI (Life Technologies) and the slides observed by scanning confocal microscopy (Leica SP5 DM6000B, Wetzlar, Germany). Each channel was acquired sequentially as a z-series using a 63x NA 1.4 lens with an optical zoom of 3 and a step size of 0.5 µm. Data sets were imported into ImageJ to generate maximum intensity projections. Figures of projections were processed and constructed using Adobe Photoshop software.
10
2.7. Effect of anti-HcES scFvs on TEER of Caco-2 cell monolayers
TEER was measured during incubation at 0, 2, 4, 6, 8 and 24 h after replacing the apical solution with a test solution which was pre-incubated for 30 min at 37oC in 5% CO2: (1) HcES (undiluted batch 1, collected 2 - 4 h) (N = 3); (2) HcES (batch 1, collected 2 - 4 h) with the pool of anti-HcES scFvs obtained from the third panning at final concentrations of 1015, 1014, 1013 or 1012 CFU/mL (N = 3); (3) HcES (batch 2, collected 1 - 4 h) with the pool of anti-HcES scFvs obtained from the third panning at final concentrations of 1015, 1014 or 1013 CFU/mL (N = 3); (4) Pool of anti-HcES scFvs obtained from the third panning at final concentrations of 1015, 1014, 1013 or 1012 CFU/mL (N = 3).
2.8. Data analysis
TEER data for each well at successive time points were normalised to time zero (100%). Replicate data are presented as mean ± SEM. Graphpad Prism v5 was used to plot graphs and to analyse data using repeated measures two-way ANOVA with Bonferri post-tests to compare data at successive time points.
3. RESULTS
3.1. Effect of H. contortus or T. circumcincta ES products on Caco-2 cell TEER
11
Apical exposure to ES products, collected from 2 - 4 h incubation of adult worms of both species, significantly reduced the TEER of Caco-2 cell monolayers (p < 0.05 to p < 0.001) from 2 24 h, compared with the effect of control incubate, treated similarly, but without worms in the incubate (Fig. 1). The greatest mean reductions were seen at 2 h, with slow, incomplete recovery over 24 h.
3.1. Effect of time of collection of T. circumcincta ES products on Caco-2 cell TEER
Apical exposure to T. circumcincta ES products, collected in sequential, but lengthening worm incubation periods to 24 h, reduced the TEER of Caco-2 cell monolayers (Fig. 2) to mean values at 2 h to 79 - 82% of that at zero time. The relative TEER at each time point for these incubates is shown in Table 1, together with the level of significant difference from the TEER produced by exposure to control solution. The TEER was significantly lower than that produced by the control incubate at 2 and 4 h for all incubates (p < 0.05 to p < 0.001) and also later in some cases (Table 1).
3.4. Tight junction proteins
Immunocytochemical staining of the tight junction proteins ZO-1 and occludin showed that exposure to ES products, but not control medium, caused internalisation of these proteins from the tight junction after 4 h exposure. This was more pronounced for occludin than for ZO-1. The intracellular punctate staining of these proteins following exposure to ES products contrasted with the location in the cell membrane after exposure to control medium (Fig. 3).
3.5. Panning of an ovine anti-HcES scFvs against HcES
12
Three sets of in vitro panning against immobilised HcES resulted in an approximate 100fold increase in elution phage titres from the second to the third panning (Table 2).
3.6. Effect of anti-HcES scFvs on TEER of Caco-2 cell monolayers
In 2 separate experiments with different batches of H. contortus ES products, phage displaying anti-HcES scFvs at a concentration of 1015 CFU/mL, but not at lower concentrations, significantly blocked the effect of HcES at 2 to 8 h (p < 0.01 to 0.001) (Fig. 4 A, B). No concentration of phage alone had any effect on TEER (Fig. 4 C).
4. DISCUSSION
These experiments have shown that ES products derived from two species of adult nematode parasites of the abomasum are capable of increasing the permeability of a model epithelium and the likelihood of this also occurring in vivo, as immune sheep recognise the chemicals involved and generate antibodies which can neutralise them in vitro. This implicates worm chemicals in the pathophysiology typically produced by luminal stages of the parasites, but not those developing in gastric glands (McKellar et al., 1986; Lawton et al., 1996; Simpson et al., 1997). Parietal cells located within the gastric glands are known targets of abomasal parasites, despite their apical membranes being resistant to chemical absorption or damage (Waisbren et al., 1994). Permeabilisation of the surface epithelium by worm chemicals would allow parietal cell inhibitors to enter the gastric tissues and act on the basolateral membranes of gland cells, resulting in both reduced acid secretion and altered also through modifying the release of growth factors which regulate the populations of gastric cells (Simpson, 2000).
13
Increased paracellular permeability of the epithelium of the parasitised abomasum is not unexpected as it has been inferred from increased leakage of plasma protein and pepsinogen into the gastric lumen (Holmes and MacLean, 1971; McLeay et al., 1973) and intestinal permeability is similarly increased by nematode (Kosik-Bogacka et al., 2010, 2011; Su et al., 2011) and unicellular parasites (Scott et al., 2002). Cultured epithelial cells also develop increased permeability upon exposure to bacteria and protozoa (Maia-Brigagão et al., 2012). This could result from direct effects of the organism or be secondary to host immune responses. In the present experiments, a direct role for worm chemicals in opening tight junctions is supported by the rapid fall in TEER of Caco-2 cell monolayers on exposure to adult H. contortus or T. circumcincta ES products and histological evidence of internalisation of the tight junctional proteins occludin and ZO-1 to a lesser extent (Fig. 3). The effects were rapid and recovery of the TEER continued over 24 hours (Fig. 1). The identity and the exact mode of action of the active constituents of ES products remain to be elucidated, but could be any of the 200 proteins or other chemicals known to be present in these ES products of sheep parasites, including glycolytic and other metabolic enzymes, proteases and structural components (Yatsuda et al., 2003; Craig et al., 2006; Kiel et al., 2007). The potency of T. circumcincta ES products was high from the beginning of the collection in vitro and maintained for at least 24 hours (Fig. 2). The collection periods for the ES products were increased from 1 hour to 12 hours, suggesting the rate of release of the active chemicals probably decreased over time. It is unlikely that excreted ammonia contributes to opening of tight junctions, as adult worm ES products usually contain only around 100 µM ammonia after 4 hours of incubation (Simpson et al., 2009), which is much less than the 50 mM ammonia used to produce a 20% decrease in TEER of cultured MDCK cells (Vastag et al., 2005) or the 10 - 15 mM ammonia which acted similarly on Caco-2 cells exposed to Helicobacter pylori (Lytton et al., 2005). Exposure to ES products of either adult H. contortus or T. circumcincta (Fig. 3) resulted in disruption of tight junctions with internalisation of the tight junctional proteins ZO-1 and occludin,
14
as previously observed by Büring (2009). Similarly, tissues from rodents infected with H. polygyrus or H. diminuta had reduced TEER in vitro ( Kosik-Bogacka et al., 2010; Su et al., 2011) and occludin was relocated from tight junctions in the colon and ileum of infected rats from Days 8 - 40 p.i. (Kosik-Bogacka et al., 2011). Many pathogens increase epithelial permeability using a variety of specific molecules which result in delocalisation of occludin and ZO-1 (Dickman et al., 2000; Nusrat et al., 2001; Boyle et al., 2006). Disrupting the interactions of occludin with other tight junction molecules is frequently seen, as this increases the permeability of tight junctions (Balda and Matter, 1998). How nematode ES products reversibly cause dissociation of occludin and ZO-1 from tight junctions remains to be determined. There may be specific molecules released by the parasites which act on either the tight junctions or the attached cytoskeleton, however, cytokine release from Caco-2 cells cannot be ruled out, as cultured epithelial cells both release endogenous cytokines when exposed to pathogens (Jung et al., 1995) and are responsive to exogenous cytokines (Van De Walle et al., 2010). Inflammatory mediators may be involved in vivo, if parasites also target the epithelium in this way to allow their ES products to penetrate the mucosa. The active components of abomasal nematode ES products were recognised by sheep immune to field infection and selected phage displaying anti-HcEs scFvs blocked the in vitro effects of HcES on Caco-2 cell permeability. Anti-HcES scFvs (1015 CFU/mL) significantly blocked the effect of HcES from 2 - 8 h (p < 0.001), whereas phage alone had no effect on the TEER (Fig. 4). Single chain antibody fragment libraries displayed on bacteriophage are used to identify antibodies to defined target antigens (Burton and Barbas, 1994; Winter et al., 1994) or to study antibody responses to immunogens in many vertebrates, including sheep (Li et al., 2000) and cattle (O'Brien et al., 1999). The scFvs library used in these experiments was constructed from mRNA from abomasal and mesenteric lymph nodes from two year-old sheep refractory to T. colubriformis, T. circumcincta and H. contortus infections and originally used to characterise larval surface antigens (Maass et al., 2007). Selective panning of this phage library with H. contortus ES products provided antibodies to
15
investigate host recognition of parasite chemicals involved in increased epithelial TEER. The blocking of the TEER by selected phage showed the potential for immune sheep to neutralise the permeabilising chemicals produced by the parasite, which would be expected to limit the pathophysiology and perhaps restrict colonisation of the abomasum. These experiments confirmed the ability of adult H. contortus and T. circumcincta ES products to disrupt tight junctions of cultured epithelial cells. The reduction in TEER was greatest after exposure for 2 hours, the first time point studied and slowly increased again over 24 hours. It was also shown that the immune host recognised and produced blocking antibodies against ES products responsible for permeabilisation of epithelia. Production of these antibodies indicates that there is exposure in vivo to key epitopes on the active ES components.
Acknowledgments Meat and Wool New Zealand, the E. and C. Thoms Bequest, AgResearch Ltd and Massey University are thanked for financial support. Imaging was performed at the Manawatu Microscopy and Imaging Centre, Massey University. Dr R. Anderson is thanked for helpful comments on the manuscript.
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McDermott, J.R., Bartram, R.E., Knight, P.A., Miller, H.R.P., Garrod, D.R., Grencis, R.K., 2003. Mast cells disrupt epithelial barrier function during enteric nematode function. Proc. Natl. Acad. Sci. USA 100, 7761-7766. McLeay, L.M., N. Anderson, J.B. Bingley, D.A. Titchen, D.A., 1973. Effects on abomasal function of Ostertagia circumcincta infections in sheep. Parasitology 66, 241–257. McKellar, Q., Duncan, J.L., Armour, J, McWilliam, P., 1986. Response to transplanted adult Ostertagia ostertagi in calves. Res. Vet. Sci. 40, 367-371. Maia-Brigagão, C., Morgado-Diaz, J.A., De Souza, W., 2012. Giardia disrupts the arrangement of tight, adherens and desmosomal junction proteins of intestinal cells. Parasitol. Int. 61, 280287. Nusrat, A., Von Eichel-Streiber, C., Turner, J.R., Verkade, P., Madara, J.L., Parkos, C.A., 2001. Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect. Immun. 69, 1329-1336. O'Brien, P.M., Aitken, R., O'Neil, B.W., Campo, M.S., 1999. Generation of native bovine mAbs by phage display. Proc. Natl. Acad. Sci. USA 96, 640-645 Sawada, N., Murata, M., Kikuchi, K., Osanai, M., Tobioka, H., Kojima, T., Chiba, H., 2003. Tight junctions and human diseases. Med. Elect. Microscopy 36, 147-156. Scott, K.G., Meddings, J.B., Kirk, D.R., Lees-Miller, S.P., Buret, A.G., 2002. Intestinal function with Giardia spp. reduces epithelial barrier function in a myosin light chain kinase-dependent fashion. Gastroenterology 123, 1179-1190. Shen, L., Weber, C.R., Turner, J.R., 2008. The tight junction protein complex undergoes rapid and continuous molecular remodelling at steady state. J. Cell Biol. 181, 683-695. Simpson, H.V., 2000. Pathophysiology of abomasal parasitism: is the host or parasite responsible? Vet J. 160, 177-191.
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Simpson, H.V., Lawton, D.E.B., Simcock, D.C., Reynolds, G.W., Pomroy, W.E., 1997. Effects of adult and larval Haemonchus contortus on abomasal secretion. Int. J. Parasitol. 27, 825-831. Simpson, H.V., Muhamad, N., Walker, L.R., Simcock, D.C., Brown, S., Pedley, K.C., 2009. Nitrogen excretion by the sheep abomasal parasite Teladorsagia circumcincta. Exp. Parasitol. 123, 17-23. Su, C., Cao, Y., Kaplan, J., Zhang, M., Li, W., Conroy, M., Walker, W.A., Shi, H.N., 2011. Duodenal helminth infection alters barrier function of the colonic epithelium via adaptive immune activation. Infect. Immun. 79, 2285-2294. Umair, S., Knight, J.S., Bland, R.J., Simpson, H.V., 2013. Molecular and biochemical characterisation of arginine kinases in Haemonchus contortus and Teladorsagia circumcincta. Exp. Parasitol. 134, 362-367. Umeda, K., Ikenouchi, J., Katahira-Tayama, S., Furuse, K., Sasaki, H., Nakayama, M., Matsui, T., Tsukita, S., Furuse, M., 2006. ZO-1 and ZO-2 independently determine where claudins are polymerized in tight-junction strand formation. Cell 126, 741-754. Van De Walle, J., Hendrickx, A., Romier, B., Laondelle, Y., Schneider, Y.-J., 2010. Inflammatory parameters in Caco-2 cells: Effect of stimuli nature, concentration and cell differentiation. Toxicol. In Vitro 24, 1441-1449. Vastag, M., Neuhofer, W., Nagel, W., Beck, F.X., 2005. Ammonium affects tight junctions and the cytoskeleton in MDCK cells. Pflüg. Arch. 449, 384-391. Waisbren, S.J., Geibel, J.P., Modlin, I.M., Boron, W.F., 1994. Unusual permeability properties of gastric glands. Nature 368, 332-335. Winter, G., Griffiths, A.D., Hawkins, R.E., Hoogenboom, H.R., 1994. Making antibodies by phage display technology. Ann. Rev. Immunol. 12, 433-455. Yatsuda, A.P., Krijgsveld, J., Cornelissen, A.W.C.A., Heck, A.J.R., De Vries, E., 2003. Comprehensive analysis of the secreted proteins of the parasite Haemonchus contortus reveals
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extensive sequence variation and differential immune recognition. J. Biol. Chem. 278, 1694116951.
Figure Legends
Fig. 1. Effect of apical membrane exposure to ES products, collected from 2-4 h of incubation of adult Haemonchus contortus (N = 16) (top) or Teladorsagia circumcincta (N = 52) (bottom) or control incubate (N = 16 or 14), on the transepithelial resistance (TEER) (mean ± SEM), normalised to zero time, of Caco-2 cell monolayers. Symbols: (■) ES products; (▲) control incubate. Significant differences at each time point are shown: ⃰ : p < 0.05; ⃰ ⃰ : p < 0.01; ⃰ ⃰ ⃰ ⃰ : p < 0.001.
Fig. 2. Effects of exposure to Teladorsagia circumcincta ES products collected over four incubation periods on the normalised trans-epithelial resistance (TEER) (mean ± SEM) of Caco-2 cell monolayers. Symbols: (x) control medium (N = 12); (■): 0 - 1 h (N = 10); (▲): 1 - 2 h (N = 10); (■): 2 – 4 h (N = 52); (): 4 - 12 h (N = 6); (): 12 - 24 h (N = 10).
Fig. 3. Effect of apical membrane exposure to control incubate (top row) or ES products of adult Haemonchus contortus (middle row) or Teladorsagia circumcincta (bottom row) on the location of ZO-1 (left column) or occludin (centre column) in the tight junctions of Caco-2 cell monolayers. The merged images are also shown (ZO-1 in green and occludin in red; regions of overlap appear yellow). DNA was stained with DAPI (blue) to reveal the positions of the nuclei. Scale bar: 10µm.
Fig. 4. Effect on the transepithelial resistance (TEER) (mean ± SEM, N = 3), normalised to zero time, of Caco-2 cell monolayers of apical membrane exposure to two different batches of ES 21
products of adult Haemonchus contortus pre-incubated with anti-HcES single chain antibody fragments (scFvs) (A, B) or scFvs in control incubate (C). Symbols: (■) no scFvs; (▲) 1015 CFU/mL; (■) 1014 CFU/mL; (♦) 1013 CFU/mL and (●) 1012 CFU/mL. Significant differences at each time point are shown: ⃰ ⃰ : p < 0.01; ⃰ ⃰ ⃰ ⃰ : p < 0.001.
22
Table 1. Transepithelial electrical resistance (TEER) (normalised to time zero) (mean ± SEM) of Caco-2 cell monolayers after exposure to ES products collected over sequential time periods of incubation of Teladorsagia circumcincta adult worms. Significant differences of ES products from control medium are shown: ⃰ : p < 0.5; ⃰ ⃰ : p < 0.01; ⃰ ⃰ ⃰ : p < 0.001.
Incubate
Normalised TEER (% zero time) 2h
4h
6h
8h
24 h
Control, N = 12
95.3 ± 1.3
101.3 ± 1.4
106.2 ± 1.8
107.2 ± 2.2
130.4 ± 2.3
0 - 1 h, N = 10
81.7 ± 4.5 ⃰ ⃰ ⃰
86.6 ± 2.3 ⃰ ⃰ ⃰
92.6 ± 1.8 ⃰ ⃰ ⃰
99.5 ± 1.8
117.7 ± 4.2 ⃰ ⃰ ⃰
1 - 2 h, N = 10
80.0 ± 4.4 ⃰ ⃰ ⃰
83.1 ± 4.2 ⃰ ⃰ ⃰
92.9 ± 3.7 ⃰ ⃰
97.8 ± 3.3.
119.0 ± 4.8 ⃰
2 - 4 h, N = 52
81.0 ± 1.9 ⃰ ⃰ ⃰
85.8 ± 2.1 ⃰ ⃰ ⃰
93.7 ± 2.0 ⃰ ⃰
100.4 ± 1.8
119.7 ± 2.4 ⃰
4 - 12 h, N = 6
81.7 ± 1.8 ⃰ ⃰ ⃰
90.0 ±3.0 ⃰ ⃰
99.5 ± 3.8
109.7 ± 3.4
137.1 ± 4.6
12 - 24 h, N = 10
78.6 ± 5.9 ⃰
82.7 ± 6.4 ⃰ ⃰
92.1 ± 6.1
97.8 ± 4.9
116.9 ± 6.7
23
Table 2. Titres of ovine scFv phage (colony forming units/mL) in the original library and in eluted samples and after amplification.
scFv library Eluted (CFU/ml) Amplified (CFU/mL) 3.1013 original 1st panning
5.107
not tested
2nd panning
2.106
5.1016
3rd panning
3.108
1.1017
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Graphical Abstract
TEER (% zero time)
110
ES + phage 1015 CFU/ml
100
ES + phage 1012 CFU/ml
ES only
90
80
70 0
4
8
12
16
Time (hours)
20
24
Figure 1
TEER (% zero time)
110
100 90
80
70 0
4
8
12
16
20
24
TEER (% zero time)
140
120
100
80
60
0
4
8
12
Time (hours)
16
20
24
Figure 2
TEER (% zero time)
140
120
100
80
60 0
4
8
12
Time (hours)
16
20
24
Figure 3 Click here to download high resolution image
Figure 4
TEER (% zero time)
110
A
100
90
80
70 0
TEER (% zero time)
120
4
8
12
16
20
24
12
16
20
24
12
16
20
24
B
100
80
60
TEER (% zero time)
0
105
4
8
4
8
C
100 95 90 0
Time (hours)