Name: cultivation of heligmosomoides polygyrus: an immunomodulatory nematode parasite and its secreted products
Uploaded: 2015-04-06T13:00:00
Duration: 12 min 40 s
Description: Watch this Scientific Journal Video about Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products at JoVE.com

The authors are funded by the Wellcome Trust through the Edinburgh Clinical Academic Track (C.J.C.J.) and Programme Grant (Y.H., and R.M.M.) and Fellowship (J.R.G) funding, the BBSRC (G.C.), the American Asthma Foundation (E.R., H.J.McS. and R.M.M.), Asthma UK (H.J.McS.) and the Rainin Foundation (D.J.S. and R.M.M.).

NOTE: All procedures in this protocol are performed in accordance with guidelines set out by the United Kingdom Home Office and the University of Edinburgh Veterinary Services.
1. Infection of Mice by Gavage
<ol>
	<li>Store Heligmosomoides polygyrus L3 larvae in distilled water for up to six months at 4 &#176;C.</li>
	<li>Before use, wash L3 larvae three times in distilled water: centrifuge at 300 x g for 10 min (with brake), remove all but 500 &#956;l of water (to avoid disturbing pelleted L3 larvae) and resuspend the pellet each time.</li>
	<li>For the third wash, add water to an exact volume (typically 40 ml) and aspirate 20 &#181;l with a 200 &#181;l tip cut to widen its aperture. Place two 20 &#181;l samples on the surface of a 60 mm culture dish and count the L3 larvae (usually mobile, and best viewed under 50X magnification with a dissecting microscope). Resuspend the final pellet in distilled water to a concentration of 2,000 L3 larvae per ml.</li>
	<li>For life cycle production, infect 8 week-old F1 (C57BL/6xCBA) mice with 400 H. polygyrus L3 larvae in 200 &#956;l of distilled water by oral gavage (restrain mice in the upright position by the scruff of the neck and gently pass the blunt gavage needle through the mouth and esophagus into the stomach). Agitate thoroughly prior to each infection (larvae settle quickly in water) and aspirate 200 &#181;l in a 1 ml syringe; use a dedicated gavage needle with a rounded end. For experimental infection of younger mice (6-8 weeks old), or other inbred strains (e.g. C57BL/6 or BALBc), infect mice with 200 L3 larvae.</li>
</ol>
2. Propagation and Maintenance of H. polygyrus
<ol>
	<li>Place charcoal in the center of a large plastic tub and allow cold tap water to run over it for a minimum of 30 min (unwashed activated charcoal is toxic to L3 larvae). Drain the water from the tub and place the charcoal on two layers of absorbent paper, leaving it exposed to room air until completely dry.</li>
	<li>If eggs are required, scrape feces first out of the colon with forceps (and scissors if necessary). If a large number of L3 larvae is needed, place mice on a wire grid and collect fecal pellets over several days.</li>
	<li>Mix the feces with granulated charcoal at a ratio of at least a 1:1, to achieve a consistency just damp enough to adhere to filter paper. Smear a thin layer on the center of some dampened filter paper in a petri dish and place this in a humid box (add some damp paper towel and a dish of water) in the dark for 12-14 days.</li>
	<li>Remove L3 larvae from day 7 onwards, and collect them on at least two occasions before the paper is discarded. The larvae form a ring around the edge of the filter paper; lift the filter paper out of the petri dish and rinse the larvae that are left of the plate (using a pipette and 5 ml of sterile water per plate) into a 50 ml tube.</li>
	<li>Lift off the filter paper and harvest the remaining larvae left on the plate with distilled water into a 50 ml tube. Centrifuge the effluent solution at 300 x g for 10 min. Wash the larvae three times with distilled water and then store at 4 &#176;C in up to 50 ml of distilled water until required. 
	NOTE: Larvae remain viable and infective for at least 6 months.</li>
</ol>
3. Collection of Adult H. polygyrus Worms
<ol>
	<li>Prepare the modified Baermann Apparatus in advance as shown in Figure 1.</li>
	<li>Cull mice fourteen days after infection.</li>
	<li>Wash the abdomen with 70% ethanol. Cut the skin over the abdomen and pull back to reveal the anterior abdominal wall. Make midline incision to enter the peritoneal cavity.</li>
	<li>Remove the entire small and large intestine (from proximal duodenum to distal rectum). Place into a dry Petri dish.</li>
	<li>Straighten the gut along its entire length; excise the feces-containing colon; place this into a separate dish for egg preparations later.</li>
	<li>Excise the proximal 20 cm of small intestine that contains the adult worms &#8211;identified by the relatively thick wall of the duodenum and often a red appearance due to the intra-luminal worms. Place into a (100 mm diameter) Petri dish with 5 ml of Hanks&#8217; Solution, warmed to 37 &#176;C (two specimens per dish).</li>
	<li>Open the worm-filled proximal gut portion longitudinally with scissors (round-ended scissors are best for this), and scrape down inside of gut lining with two glass slides to remove the worms. Then discard the clean gut wall.</li>
	<li>Tip worms into small muslin bags, staple closed and secure with paperclips around the edge of the glass funnel (Figure 1).</li>
	<li>Fill funnel with Hanks&#8217; Solution and add approximately 4 Petri dishes of worms into each funnel.</li>
	<li>Place apparatus in 37 &#176;C incubator for 1-2 hr, gently agitating half way through to dislodge debris from the gut preparation that may occlude the muslin filter. Take care to avoid spillage of debris outside the muslin bag &#8211; this will cause contamination of the final HES preparation. Adult worms should have slowly migrated through the muslin cloth and settled at the bottom of the glass test tube. Carefully detach the test tube from the connecting rubber hose over the sink (taking care to avoid losing worms at this point).</li>
	<li>Using a plastic pipette, transfer the worms into a 50 ml tube and wash six times with Hanks&#8217; Solution (allow worms to settle with gravity, remove media with a stripette, add 40 ml of Hanks&#8217; Solution and repeat five times). 
	NOTE: worm culture must be kept sterile from this point onwards.
	<ol>
		<li>Move to a laminar flow hood and wash another six times in sterile Hanks&#8217; Solution supplemented with 100 U/ml penicillin and 100 &#181;g/ml streptomycin, ready for in vitro culture.</li>
	</ol>
	</li>
	<li>Count the adult worms recovered by taking two samples of 20 &#181;l taken up with a yellow tip cut to widen its aperture; expect approximately 50% of the quantity of inoculated larvae.</li>
</ol>
4. Setting Up Cultures for HES: Medium Preparation, Washing Adult Worms
<ol>
	<li>Soak the worms from 3.11 in approximately 10 ml of RPMI supplemented with 10% Gentamycin for 20 min, leaving the tube resting at an angle to ensure worms are fully covered.</li>
	<li>Perform this in a laminar flow hood: wash again six times with Hanks&#39; Solution (supplemented with 5 U/ml penicillin and 5 &#956;g/ml streptomycin).</li>
	<li>Prepare H. polygrus media.</li>
	<li>Maintaining sterility in a laminar flow hood, to 500 ml of RPMI1640, add 11.1 ml of 45% glucose (final concentration 1.2% as RPMI1640 contains 0.2% glucose), 5 ml of 100x Peniclllin-Streptomycin (final concentration 5 U/ml penicillin, 5 &#956;g/ml streptomycin), 5 ml of L-glutamine (final concentration 2 mM), and 5 ml of Gentamycin (final concentration 1%). Do not add FCS.</li>
	<li>Aliquot worms into vented T25 flasks, approx. 1,000 worms in 15 ml H. polygyrus media per flask, and place upright in 37 &#176;C incubator (5% CO2) for 3 weeks.</li>
</ol>
5. Preparation of HES
<ol>
	<li>Collect HES-containing culture media from cultures at intervals of no longer than twice per week, setting aside the first collection after 24 hr of culture (due to high levels of LPS contamination and host proteins &#8211; can be processed separately or discarded). Keep each collection separate and clearly labeled with the date and batch number. Replace with an equal volume of H. polygyrus media on each occasion.</li>
	<li>Centrifuge HES-containing media at 400 x g for 5 min. Then filter sterilize through 0.2 &#181;m low-protein-binding filters into 50 ml tubes. Store in the -20 &#176;C freezer clearly labeled with date of worm harvest and date of HES collection.</li>
	<li>After 21 days of HES collection from culture, discard worms.</li>
	<li>Pool 500-1,000 ml of HES supernatant (usually from frozen stock, and not including the first 24 hr collection) and concentrate over a 3,000 MWCO filter in the ultrafiltration device under nitrogen pressure. 
	NOTE: Be very careful not to let the filter run dry.
	<ol>
		<li>To set up the filter device, first wash the 3 kDa membrane shiny side down in a 1 liter beaker with distilled water for 3 x 20 min whilst stirring. Assemble the ultrafiltration device as per manufacturer&#8217;s instructions with filter membrane shiny side up. Place in cabinet at 4 &#176;C and pass 50 ml of distilled water through before starting to concentrate pooled HES.</li>
		<li>Add each tube of HES into the filtration device as required (100-140 ml per day), until the volume is concentrated down to 2-5 ml.</li>
	</ol>
	</li>
	<li>In order to remove contaminants from the HES-containing culture media, add 50 ml of pyrogen-free PBS to the filtration device and and then concentrate down to approximately 2 ml. Repeat this step twice (150 ml of PBS in total). Transfer HES into a 15 ml tube, filter sterilize (with a 0.2 &#956;m filter) in a laminar flow hood and measure protein concentration using a spectrophotometer (E280 = 10) or by Bradford assay.</li>
	<li>Aliquot, label with batch number and date, and freeze at -80 &#176;C.</li>
	<li>Perform a chromogenic LAL assay according to the manufacturer&#8217;s protocols on each batch of HES prior to use. If LPS levels are greater than 1 U LPS per 1 &#181;g protein, consider not using this batch for in vivo experiments or in vitro cultures.</li>
	<li>Process the HES collected at 24 hr separately in the same manner; it will contain LPS and some host proteins and, while not suitable for functional experiments, it is a useful source of individual molecules that may be isolated by monoclonal antibody affinity purification.</li>
</ol>

The authors have no conflicts of interest.

The life cycle of H. polygyrus proceeds in a reliably consistent fashion. Following natural ingestion or oral gavage of L3 larvae on day 0, cysts begin to form under the duodenal serosa by day 5, progressing to larval moults and then emerging as adult worms into the gut lumen from day 10, eggs can be seen in feces from day 14 and granulomas are visible on the duodenal serosal surface from day 21. The protocol described above (and summarized in Figure 6) allows for high-throughput production of H. polygyrus excretory-secretory products (HES), in addition to reliable recovery of viable L3 larvae for future experimental and life cycle infections.
H. polygyrus&#160;infection has been shown to be protective in models of asthma dependent on OVA or Der p 1 (House dust mite allergen)14. Furthermore, suppression of airway inflammation could be transferred with CD4+CD25+ regulatory T cells 14&#160;or CD19+CD23hi regulatory B cells 24 from non-sensitised,&#160;H. polygyrus-infected mice. In models of autoimmunity, H. polygyrus&#160;infection has been shown to be suppressive in the&#160;experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis 9, and suppression can be transferred with either CD4+ T cells or CD19+ B cells from infected mice 24.
Heligmosomoides polygyrus excretory-secretory products (HES) modulate the host immune response and suppress Th2-mediated inflammation by a number of mechanisms (outlined in Figure 7), including: a) Inhibition of dendritic cell responsiveness to stimulation 25, b) induction of CD4+Foxp3+ regulatory T cells 18 . and c) blockade of IL-33 production 20. HES has been shown to be protective when administered at sensitization or challenge in the OVA-alum model of asthma 19 and also when administered intranasally with Alternaria extract allergen 20, through suppression of early IL-33 release. Avoidance of LPS contamination of HES is often crucial for the success of future immunological experiments. In the protocol outlined here, the critical steps in achieving this are to ensure that debris contained within the muslin bags of the Baermann Apparatus does not enter the final HES preparation (see 2.10) and to set aside ES solution from the first 24 hr of culture (see 5.1).
Over recent years, HES has been thoroughly characterized at the proteomic level with over 370 distinct proteins identified 26,27. In addition, ES from the 4th stage larvae has been subjected to proteomic analysis 28. Ongoing work includes characterizing the major glycan components of HES, known to be strongly immunogenic 3, and secreted micro-RNAs that are encapsulated in 50-100 nm vesicles or exosomes (Buck et al., submitted for publication). With the establishment of reproducible protocols for the collection of ES from this highly immunomodulatory parasite, and with extensive proteomic data identifying the molecular components of HES, the stage is now set for the identification and therapeutic testing of individual molecules from H. polygyrus that can mediate key effects on the host immune system.

Heligmosomoides polygyrus is a natural murine gastrointestinal helminth that is closely related to highly-prevalent human nematode parasites 1. In contrast to other nematode models such as Nippostrongylus brasiliensis, H. polygyrus consistently establishes chronic infection in mice as a direct result of multiple powerful immunomodulatory mechanisms it employs to suppress the host immune response 2.
H. polygyrus has a direct life cycle: infective L3 larvae are ingested by feco-oral transmission (or administered by oral gavage in the laboratory setting), whereupon they migrate to the subserosal layer of the duodenum and encyst before returning to the intestinal lumen as adult worms approximately eight days after initial infection. Mating and egg production occurs by day 10 and it is possible to harvest adult worms for culture and collection of excretory-secretory products from day 14 onwards 3. H. polygyrus also interacts with the commensal microflora, with increased Lactobacilli present in infected susceptible mice 4-6, and increased levels of H. polygyrus infection following exposure of mice to Lactobacilli 6.
Active infection with H. polygyrus has been shown to protect against immunopathology in many animal models of autoimmunity 7-10, colitis 11,12 and allergy 13-16. There has consequently been great interest in the potential of Excretory-Secretory molecules from this parasite (&#8220;HES&#8221;) to down-modulate pathology in vivo 17,18. Indeed, protective effects are seen following treatment of mice with HES products 19 through pathways which are now being identified 18,20. Here we describe a protocol for the reliable production of Heligmosomoides polygyrus and recovery of its secreted products that can be further utilized for a range of functional biochemical and immunological investigations.

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	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Material Name</td>
			<td style="width:83px;">Company</td>
			<td style="width:143px;">Catalogue Number</td>
			<td style="width:157px;">Comments</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Amicon concentrator</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:143px;">5112</td>
			<td style="width:157px;">Model 8050 50 ml 
			http://www.emdmillipore.com/INTL/en/product/Stirred-Cell-Model-8050,-50%C2%A0mL,MM 
			_NF-5122</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Hanks&#8217; buffered salt solution</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">H9394</td>
			<td style="width:157px;">500 ml 
			http://www.sigmaaldrich.com/catalog/product/sigma/h9394?lang=en&#38;region=GB</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Penicillin streptomycin</td>
			<td style="width:83px;">Invitrogen</td>
			<td style="width:143px;">15070-063</td>
			<td style="width:157px;">100 ml 
			http://www.lifetechnologies.com/order/catalog/product/15070063</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">L-glutamine</td>
			<td style="width:83px;">Invitrogen</td>
			<td style="width:143px;">25030-081</td>
			<td style="width:157px;">200 MM 100ml 
			https://www.lifetechnologies.com/order/catalog/product/25030081?ICID=search-25030081</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Activated charcoal</td>
			<td style="width:83px;">Merck</td>
			<td style="width:143px;">1.09624.0500</td>
			<td style="width:157px;">18-35 mesh 
			http://www.merckmillipore.com/GB/en/product/Charcoal-activated,MDA_CHEM-109624</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Glucose solution</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">G8769</td>
			<td style="width:157px;">100 ml 45% solution 
			http://www.sigmaaldrich.com/catalog/product/sigma/g8769?lang=en&#38;region=GB</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Falcon tubes</td>
			<td style="width:83px;">Sarstedt</td>
			<td style="width:143px;">62.547.254</td>
			<td style="width:157px;">50 ml 
			http://www.scribd.com/doc/124320105/Sarstedt-Falcon</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">T25 Flasks</td>
			<td style="width:83px;">Sigma</td>
			<td style="width:143px;">CLS430639&#160;</td>
			<td style="width:157px;">25cm vented flask 
			http://www.sigmaaldrich.com/catalog/product/sigma/cls430639?lang=en&#38;region=GB</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Chromogenic LAL assay</td>
			<td style="width:83px;">Lonza</td>
			<td style="width:143px;">50-647U</td>
			<td style="width:157px;">QCL-1000 assay 
			http://www.lonza.com/products-services/pharma-biotech/endotoxin-detection/endotoxin-detection-assays/endpoint-chromogenic-lal-assay.aspx</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:209px;">Gentamicin</td>
			<td style="width:83px;">Gibco</td>
			<td style="width:143px;">15710-049</td>
			<td style="width:157px;">100 ml 
			http://www.lifetechnologies.com/order/catalog/product/15710072</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:209px;">Roswell Park Memorial Institute (RPMI) 1640</td>
			<td style="width:83px;">Gibco</td>
			<td align="right">11875093</td>
			<td style="width:157px;">500 ml; without L-glutamine 
			http://www.lifetechnologies.com/order/catalog/product/11875093</td>
		</tr>
	</tbody>
</table>

cultivation of heligmosomoides polygyrus: an immunomodulatory nematode parasite and its secreted products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that employs multiple immunomodulatory mechanisms to establish chronic infection in mice and closely resembles prevalent human helminth infections. H. polygyrus has been studied extensively in the field of helminth-derived immune regulation and has been found to potently suppress experimental models of allergy and autoimmunity (both with active infection and isolated secreted products). The protocol described in this paper outlines management of the H. polygyrus life cycle for consistent production of L3 larvae, recovery of adult parasites, and collection of their excretory-secretory products (HES).

Susceptibility to infection with H. polygyrus is controlled to a large degree by the genetic background of the mouse strain (Table 1); C57BL/6 and CBA mice are highly susceptible 21,22. For maintenance of the parasite life cycle, the F1 hybrid between these two strains has been chosen for its ability to withstand much higher worm burdens without morbidity (excessive intestinal epithelial damage) compared to either parental strain. Oral gavage of 400 L3 larvae is used to maintain the life cycle in F1 mice (resulting in adult worm burdens shown in Figure 2), whilst a dose of 200 L3 larvae is generally used for experiments in homozygous inbred strains (e.g., C57BL/6 or BALBc). However, this dosage may need to be reduced depending on environmental co-factors that may differ between animal facilities, such as variations in gut flora.
Batches of HES have proven reproducible efficacy in functional assays and in protein composition; moreover, when supernatants from each successive week of culture were analysed, up to a total of 4 weeks, the protein profiles were found to be very similar (Figure 3). When HES concentration is measured by Bradford assay (see 5.5), the total protein is usually approximately 1 mg/ml (Figure 4).
An alternative method of concentrating HES is with a centrifugal concentrator (eg Vivaspin 3-kDa cut-off membrane), in which samples are spun at up to 4,000 g in a swing-out rotor centrifuge, removing buffer salts and low molecular weight components. Centrifugal concentration is best suited to small processing volumes (1-10 ml) and are limited to a maximum concentration factor of approximately 30x.
When collecting HES, avoidance of contamination is critical. To avoid contamination with host molecules, we discard HES-containing culture media from the first 24 hr after adult worm harvest from the mouse intestine. We also quantify the level of LPS contamination in each batch of HES with a Chromogenic LAL assay (see 5.7). 1 U of LPS equates to ~100 pg LPS and levels below this are considered negligible 23. In our hands, most batches of HES are significantly below this limit, the mean concentration of LPS in HES being 0.23 U/&#181;g (Figure 5). The effects of LPS in in vivo models of pathology (for instance the suppression of asthmatic responses) requires at least 10 ng of LPS 23. Hence in vivo administration of 5 &#181;g HES from a batch with 100 pg LPS/&#181;g HES will include 500 pg LPS, well below the effective concentration where LPS becomes a problem.
<img alt="Figure 1" src="/files/ftp_upload/52412/52412fig1highres.jpg" /> 
Figure 1: Baermann Apparatus Baermann Apparatus setup for collection of adult H. polygyrus (as described in section 2).
<img alt="Figure 2" src="/files/ftp_upload/52412/52412fig2highres.jpg" /> 
Figure 2: Mean Worm Burden 14 Days After Infection with 400 L3 Larvae Mean worm burdens 14 days after infection with 400 L3 larvae. Data points shown are from 19 separate rounds of infection of C57BL/6xCBA mice. Mean &#177; SEM shown.
<img alt="Figure 3" src="/files/ftp_upload/52412/52412fig3highres.jpg" /> 
Figure 3: Protein Profiles of HES From Successive Weeks in Culture SDS-PAGE profile of HES proteins collected in successive weeks of culture.
<img alt="Figure 4" src="/files/ftp_upload/52412/52412fig4highres.jpg" /> 
Figure 4: Final Quantity of HES from 500 ml ES-Containing Media Yield of HES protein from 11 different batches derived from approximately 500 ml of culture supernatant. Mean &#177; SEM shown.
<img alt="Figure 5" src="/files/ftp_upload/52412/52412fig5highres.jpg" /> 
Figure 5: LPS Contamination of HES Levels of LPS contamination in 41 batches of HES measured by the Limulus ameobocyte assay. Median LPS concentration = 86 U per mg of HES.
<img alt="Figure 6" src="/files/ftp_upload/52412/52412fig6highres.jpg" /> 
Figure 6: Animated Schematic of H. polygyrus Life Cycle Summary of key life cycle stages from oral gavage of L3 larvae, through recovery of larvae and adult worms to isolation of HES.
<img alt="Figure 7" src="/files/ftp_upload/52412/52412fig7highres.jpg" /> 
Figure 7: Animated Schematic of HES and its Functions Key immunomodulatory effects of soluble mediators and exosomes contained within HES.
<table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always">
	<tbody>
		<tr>
			<td>Genotype (and background strain)</td>
			<td>Primary Infection Phenotype</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Inbred strains</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A/J, CBA, C3H&#160;</td>
			<td>Highly susceptible&#160;</td>
			<td>22,29</td>
		</tr>
		<tr>
			<td>C57BL/6 and C57BL/10&#160;</td>
			<td>Susceptible&#160;</td>
			<td>22,30</td>
		</tr>
		<tr>
			<td>BALB/c, DBA/2, 129/J&#160;</td>
			<td>Intermediate&#160;</td>
			<td>22,31</td>
		</tr>
		<tr>
			<td>NIH, SJL, SWR</td>
			<td>Low susceptibility&#160;</td>
			<td>22,32</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Transgenic for cytokines or cytokine receptors</td>
			<td></td>
		</tr>
		<tr>
			<td>IL-1&#946;-/-</td>
			<td>More susceptible</td>
			<td>33</td>
		</tr>
		<tr>
			<td rowspan="2">IL-1R-/-</td>
			<td rowspan="2">Less susceptible (a); no change in susceptibility but increased granulomas (b)</td>
			<td>(a) 33</td>
		</tr>
		<tr>
			<td>(b) 34</td>
		</tr>
		<tr>
			<td>IL-2R&#946; Transgenic (C57BL/6)</td>
			<td>Resistant&#160;</td>
			<td>35</td>
		</tr>
		<tr>
			<td>IL-4-/-</td>
			<td>Higher fecundity</td>
			<td>36</td>
		</tr>
		<tr>
			<td>IL-4R-/- (C57BL/6 or BALB/c)</td>
			<td>Highly susceptible</td>
			<td>22</td>
		</tr>
		<tr>
			<td>IL-6&#8211;/&#8211; (BALB/c or C57BL/6)</td>
			<td>Highly Resistant&#160;</td>
			<td>37</td>
		</tr>
		<tr>
			<td>IL-9 Transgenic (FVB)&#160;</td>
			<td>Resistant&#160;</td>
			<td>38</td>
		</tr>
		<tr>
			<td>IL-21R &#8211;/&#8211; (C57BL/6)</td>
			<td>Deficient Th2, decreased granuloma gormation</td>
			<td>39,40</td>
		</tr>
		<tr>
			<td>IL-25-/- (BALB/c)</td>
			<td>More susceptible</td>
			<td>33</td>
		</tr>
		<tr>
			<td>IL-33R (T1/ST2) -/- (BALB/c)</td>
			<td>No change in susceptibility</td>
			<td>33</td>
		</tr>
		<tr>
			<td>TGF&#946;RIIdn (C57BL/6)</td>
			<td>High Th1, Increased susceptibility</td>
			<td>41,42</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Transgenic for T cell markers</td>
			<td></td>
		</tr>
		<tr>
			<td>CD28&#8211;/&#8211; (BALB/c)&#160;</td>
			<td>Marginally higher fecundity</td>
			<td>43</td>
		</tr>
		<tr>
			<td>CD86 (B7-2)&#8211;/&#8211; (BALB/c)&#160;</td>
			<td>Higher fecundity&#160;</td>
			<td>44</td>
		</tr>
		<tr>
			<td>OX40L&#8211;/&#8211; (BALB/c)&#160;</td>
			<td>Higher fecundity&#160;</td>
			<td>45</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="2">Transgenic for innate Immune loci&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Type 1 interferon receptor (IFNAR)-/- (C57BL/6)&#160;</td>
			<td>Higher fecundity, more granulomas</td>
			<td>34</td>
		</tr>
		<tr>
			<td>MyD88&#8211;/&#8211; (C57BL/6)&#160;</td>
			<td>More resistant, more granulomas</td>
			<td>34</td>
		</tr>
		<tr>
			<td>C-KitW/Wv (WBB6)&#160;</td>
			<td>More susceptible</td>
			<td>46,47</td>
		</tr>
	</tbody>
</table>
Table 1: Primary infections with H. polygyrus in genetically different and gene-targetted mouse strains.

Watch this Scientific Journal Video about Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products at JoVE.com

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus is a murine nematode with powerful immunomodulatory capabilities that closely resemble those of highly-prevalent human helminth infection. Here we describe a protocol for the long-term maintenance of the H. polygyrus lifecycle.

Cultivation of Heligmosomoides Polygyrus: An Immunomodulatory Nematode Parasite and its Secreted Products

Heligmosomoides polygyrus (formerly known as Nematospiroides dubius, and also referred to by some as H. bakeri) is a gastrointestinal helminth that ...

cultivation-heligmosomoides-polygyrus-an-immunomodulatory-nematode

Research

JoVE Journal

Immunology and Infection

Intravital Microscopy of the Spleen: Quantitative Analysis of Parasite Mobility and Blood Flow

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. Thus, in vivo imaging of malaria parasites during pre-erythrocytic stages have revealed the active entrance of parasites into skin lymph nodes 3, the complete development of the parasite in the skin 4, and the formation of a hepatocyte-derived merosome to assure migration and release of merozoites into the blood stream 5. Moreover, the development of individual parasites in erythrocytes has been recently documented using 4D imaging and challenged our current view on protein export in malaria 6. Thus, intravital imaging has radically changed our view on key events in Plasmodium development. Unfortunately, studies of the dynamic passage of malaria parasites through the spleen, a major lymphoid organ exquisitely adapted to clear infected red blood cells are lacking due to technical constraints.
Using the murine model of malaria Plasmodium yoelii in Balb/c mice, we have implemented intravital imaging of the spleen and reported a differential remodeling of it and adherence of parasitized red blood cells (pRBCs) to barrier cells of fibroblastic origin in the red pulp during infection with the non-lethal parasite line P.yoelii 17X as opposed to infections with the P.yoelii 17XL lethal parasite line 7. To reach these conclusions, a specific methodology using ImageJ free software was developed to enable characterization of the fast three-dimensional movement of single-pRBCs. Results obtained with this protocol allow determining velocity, directionality and residence time of parasites in the spleen, all parameters addressing adherence in vivo. In addition, we report the methodology for blood flow quantification using intravital microscopy and the use of different colouring agents to gain insight into the complex microcirculatory structure of the spleen. 
Ethics statement
All the animal studies were performed at the animal facilities of University of Barcelona in accordance with guidelines and protocols approved by the Ethics Committee for Animal Experimentation of the University of Barcelona CEEA-UB (Protocol No DMAH: 5429). Female Balb/c mice of 6-8 weeks of age were obtained from Charles River Laboratories.

We show the method for performing intravital microscopy of the spleen using GFP transgenic malaria parasites and the quantification of parasite mobility and blood flow within this organ.

The advent of intravital microscopy in experimental rodent malaria models has allowed major advances to the knowledge of parasite-host interactions 1,2. ...

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

Leishmaniasis is one of the world's most neglected diseases, largely affecting the poorest of the poor, mainly in developing countries. Over 350 million people are considered at risk of contracting leishmaniasis, and approximately 2 million new cases occur yearly1. Leishmania donovani is the causative agent for visceral leishmaniasis (VL), the most fatal form of the disease. The choice of drugs available to treat leishmaniasis is limited 2;current treatments provide limited efficacy and many are toxic at therapeutic doses. In addition, most of the first line treatment drugs have already lost their utility due to increasing multiple drug resistance 3. The current pipeline of anti-leishmanial drugs is also severely depleted. Sustained efforts are needed to enrich a new anti-leishmanial drug discovery pipeline, and this endeavor relies on the availability of suitable in vitro screening models.
In vitro promastigotes 4 and axenic amastigotes assays5 are primarily used for anti-leishmanial drug screening however, may not be appropriate due to significant cellular, physiological, biochemical and molecular differences in comparison to intracellular amastigotes. Assays with macrophage-amastigotes models are considered closest to the pathophysiological conditions of leishmaniasis, and are therefore the most appropriate for in vitro screening. Differentiated, non-dividing human acute monocytic leukemia cells (THP1) (make an attractive) alternative to isolated primary macrophages and can be used for assaying anti-leishmanial activity of different compounds against intracellular amastigotes.
Here, we present a parasite-rescue and transformation assay with differentiated THP1 cells infected in vitro with Leishmania donovani for screening pure compounds and natural products extracts and determining the efficacy against the intracellular Leishmania amastigotes. The assay involves the following steps: (1) differentiation of THP1 cells to non-dividing macrophages, (2) infection of macrophages with L. donovani metacyclic promastigotes, (3) treatment of infected cells with test drugs, (4) controlled lysis of infected macrophages, (5) release/rescue of amastigotes and (6) transformation of live amastigotes to promastigotes. The assay was optimized using detergent treatment for controlled lysis of Leishmania-infected THP1 cells to achieve almost complete rescue of viable intracellular amastigotes with minimal effect on their ability to transform to promastigotes. Different macrophage:promastigotes ratios were tested to achieve maximum infection. Quantification of the infection was performed through transformation of live, rescued Leishmania amastigotes to promastigotes and evaluation of their growth by an alamarBlue fluorometric assay in 96-well microplates. This assay is comparable to the currently-used microscopic, transgenic reporter gene and digital-image analysis assays. This assay is robust and measures only the live intracellular amastigotes compared to reporter gene and image analysis assays, which may not differentiate between live and dead amastigotes. Also, the assay has been validated with a current panel of anti-leishmanial drugs and has been successfully applied to large-scale screening of pure compounds and a library of natural products fractions (Tekwani et al. unpublished).

A Parasite Rescue and Transformation Assay for Antileishmanial Screening Against Intracellular Leishmania donovani Amastigotes in THP1 Human Acute Monocytic Leukemia Cell Line

A parasite-rescue and transformation assay with THP1 cells infected in vitro with Leishmania donovani has been optimized for anti-leishmanial drug screening. The assay involves differentiation of THP1 cells, infection with promastigotes, treatment with test drugs, controlled lysis of the infected macrophages, rescue of amastigotes, transformation to promastigotes and monitoring promastigote growth and proliferation with a fluorometric assay.

Leishmaniasis is  one of the world's most neglected diseases, largely affecting the  poorest of the poor, mainly in developing countries. Over 350 million ...

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on the amber suppression technology that was originally developed by Peter Schultz and colleagues1. An amber mutation is first introduced at a specific codon of the gene encoding the protein of interest. The amber mutant is then expressed in E. coli together with genes encoding an amber suppressor tRNA and an amino acyl-tRNA synthetase derived from Methanococcus jannaschii. Using this system, the photo activatable amino acid analog p-benzoylphenylalanine (Bpa) is incorporated at the amber codon. Cells are then irradiated with ultraviolet light to covalently link the Bpa residue to proteins that are located within 3-8 &#197;. Photocrosslinking is performed in combination with pulse-chase labeling and immunoprecipitation of the protein of interest in order to monitor changes in protein-protein interactions that occur over a time scale of seconds to minutes. We optimized the procedure to study the assembly of a bacterial virulence factor that consists of two independent domains, a domain that is integrated into the outer membrane and a domain that is translocated into the extracellular space, but the method can be used to study many different assembly processes and biological pathways in both prokaryotic and eukaryotic cells. In principle interacting factors and even specific residues of interacting factors that bind to a protein of interest can be identified by mass spectrometry.

This article illustrates the use of pulse-chase radio labeling in combination with site-specific photocrosslinking to monitor interactions between a protein of interest and other factors in E. coli. Unlike traditional chemical cross-linking methods, this approach generates high resolution &#8220;snapshots&#8221; of an ordered assembly pathway in a living cell.

This article describes a method to detect and analyze dynamic interactions between a protein of interest and other factors in vivo. Our method is based on ...

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

The immunomodulatory properties of human mesenchymal stem cells (MSC) appear increasingly relevant for clinical application. Using a co-culture system of MSCs and peripheral blood leukocytes pre-stained with the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE), we describe the in vitro assessment of MSC immunomodulation on effector leukocyte proliferation and specific subpopulations.

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. Regulatory systems that utilize intracellular cyclic di-GMP (c-di-GMP) as a second messenger are one such class of target. c-di-GMP is a signaling molecule found in almost all bacteria that acts to regulate an extensive range of processes including antibiotic resistance, biofilm formation and virulence. The understanding of how c-di-GMP signaling controls aspects of antibiotic resistant biofilm development has suggested approaches whereby alteration of the cellular concentrations of the nucleotide or disruption of these signaling pathways may lead to reduced biofilm formation or increased susceptibility of the biofilms to antibiotics. We describe a simple high-throughput bioreporter protocol, based on green fluorescent protein (GFP), whose expression is under the control of the c-di-GMP responsive promoter cdrA, to rapidly screen for small molecules with the potential to modulate c-di-GMP cellular levels in Pseudomonas aeruginosa (P. aeruginosa). This simple protocol can screen upwards of 3,500 compounds within 48 hours and has the ability to be adapted to multiple microorganisms.

Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa

This article describes the high-throughput assay that has been successfully established to screen large libraries of small molecules for their potential ability to manipulate cellular levels of cyclic di-GMP in Pseudomonas aeruginosa, providing a new powerful tool for antibacterial drug discovery and compound testing.

Bacterial resistance to traditional antibiotics has driven research attempts to identify new drug targets in recently discovered regulatory pathways. ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...

Rapid Quantification of Mitogen-induced Blastogenesis in T Lymphocytes for Identifying Immunomodulatory Drugs

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., immunosuppressive or immunostimulatory) chemical compounds and biologics. One of the earliest steps during mitogenesis is cell enlargement or blastogenic transformation, whereupon the cell volume increases before division. It is usually detectable in the first several hours of T-lymphocyte stimulation. Here, we describe a rapid method to quantify blastogenesis in T lymphocytes isolated from mouse spleens and human peripheral blood mononuclear cells (PBMCs) using an automated cell counter. Various commonly used proliferation assays for the most part are laborious and only reflect the overall population effect rather than individual cellular effects within a population. In contrast, the presented automated cell counter assay provides rapid, direct, and precise measurements of cell diameters that can be used for assessing the effectiveness of various mitogens and immunomodulatory drugs in vitro.

T-lymphocyte mitogenesis is accompanied by blastogenic transformation, whereupon the cell volume enlarges before cell division. Here, we describe a method to quantify blastogenesis in T lymphocytes using an automated cell counter with the capability of measuring cell diameters.

Lymphocyte proliferation in response to antigenic or mitogenic stimulation is a readily quantifiable phenomenon useful for testing immunomodulatory (i.e., ...

Generation of Monoclonal Antibodies Against Natural Products

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional Chinese medicine and food safety/toxicology. Many of the classical analysis techniques require expensive equipment and/or expertise. Notably, enzyme-linked immunosorbent assays (ELISAs) have become an emerging method for the analysis of foods and natural products. This method is based on antibody-mediated detection of the target components. However, as many of the bioactive components in natural products are small (&lt;1,000 Da) and do not induce an immune response, creating monoclonal antibodies (mAbs) against them is often difficult. In this protocol, we provide a detailed explanation of the steps required to generate mAbs against target molecules as well as those needed to create the associated indirect competitive (ic)ELISA for the rapid analysis of the compound in multiple samples. The procedure describes the synthesis of the artificial antigen (i.e., the hapten-carrier conjugate), immunization, cell fusion, monoclonal hybridoma preparation, characterization of the mAb, and the ELISA-based application of the mAb. The hapten-carrier conjugate was synthesized by the sodium periodate method and evaluated by MALDI-TOF-MS. After immunization, splenocytes were isolated from the immunized mouse with the highest antibody titer and fused with the hypoxanthine-aminopterin-thymidine (HAT)-sensitive mouse myeloma cell line Sp2/0 -Ag14 using a polyethylene glycol (PEG)-based method. The hybridomas secreting mAbs reactive to the target antigen were screened by icELISA for specificity and cross-reactivity. Furthermore, the limiting dilution method was applied to prepare monoclonal hybridomas. The final mAbs were further characterized by icELISA and then utilized in an ELISA-based application for the rapid and convenient detection of the example hapten (naringin (NAR)) in natural products.

This article provides a detailed protocol for the preparation and evaluation of monoclonal antibodies against natural products for use in various immunoassays. This procedure includes immunization, cell fusion, indirect competitive ELISA for positive clone screening, and monoclonal hybridoma preparation. The specifications for antibody characterization using MALDI-TOF-MS and ELISA analyses are also provided.

The analysis of the bioactive components present in foods and natural products has become a popular area of study in many fields, including traditional ...

Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular components responsible for supplying cell energy; however, excess mitochondria also produce reactive oxygen species (ROS) that could cause cell death. Therefore, the number of mitochondria must constantly be adjusted to fit the needs of the cells. This dynamic regulation is achieved in part through the function of lysosomes that remove surplus/damaged organelles and macromolecules. Hence, cellular mitochondrial and lysosomal contents are key indicators to evaluate the metabolic adjustment of cells. With the development of probes for organelles, well-characterized lysosome or mitochondria-specific dyes have become available in various formats to label cellular lysosomes and mitochondria. Multicolor flow cytometry is a common tool to profile cell phenotypes, and has the capability to be integrated with other assays. Here, we present a detailed protocol of how to combine organelle-specific dyes with surface markers staining to measure the amount of lysosomes and mitochondria in different T cell populations on a flow cytometer.

This article illustrates a powerful method to quantify mitochondria or lysosomes in living cells. The combination of lysosome- or mitochondria-specific dyes with fluorescently conjugated antibodies against surface markers allows the quantification of these organelles in mixed cell populations, like primary cells harvested from tissue samples, by using multicolor flow cytometry.

T cells utilize different metabolic programs to match their functional needs during differentiation and proliferation. Mitochondria are crucial cellular ...

Concomitant Isolation of Primary Astrocytes and Microglia for Protozoa Parasite Infection

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central nervous system (CNS). The increasing evidences of their involvement in the control of infectious diseases justify the emerging interest in the improvement of methodologies to isolate primary astrocytes and microglia in order to evaluate their responses to infections that affect the CNS. Considering the impact of Trypanosoma cruzi (T. cruzi) and Toxoplasma gondii (T. gondii) infection in the CNS, here we provide a method to extract, maintain, dissociate and infect murine astrocytes and microglia cells with protozoa parasites. Extracted cells from newborn cortices are maintained in vitro for 14 days with periodic differential media replacement. Astrocytes and microglia are obtained from the same extraction protocol by mechanical dissociation. After phenotyping by flow cytometry, cells are infected with protozoa parasites. The infection rate is determined by fluorescence microscopy at different time points, thus enabling the evaluation of differential ability of glial cells to control protozoan invasion and replication. These techniques represent simple, cheap and efficient methods to study the responses of astrocytes and microglia to infections, opening the field for further neuroimmunology analysis.

The overall goal of this protocol is to instruct how to extract, maintain, and dissociate murine astrocyte and microglia cells from the central nervous system, followed by infection with protozoa parasites.

Astrocytes and microglia are the most abundant glial cells. They are responsible for physiological support and homeostasis maintenance in the central ...