The authors wish to acknowledge Jos&#233; Luis Ramos-Balderas for his technical support. This work was supported by the following grant to PLL from CONACYT (FORDECYT-PRONACE-303027). OM-P and EO-M received a fellowship from CONACYT (736162 and 481437, respectively). MCM-M received a fellowship from CONACYT (Estancias Posdoctorales por M&#233;xico 2022 (3)).

All animal experiments described here were approved by the Internal Committee for Animal Handling (CICUAL) of the Institute of Cellular Physiology, National Autonomous University of Mexico.
NOTE: A flowchart of the entire protocol is shown in Figure 1.
1. Housing of mice
<ol>
	<li>Use 8-10-week-old, female or male groups of mice, housed in animal facilities with constant temperature and humidity in 12 h light/dark cyc...

<ol>
	<li>Centers for Disease Control and Prevention. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Parasites - Hookworm. , (2022).</li><li>Camberis, M., Le Gros, G., Urban, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+model+of+Nippostrongylus+brasiliensis+and+Heligmosomoides+polygyrus.">Animal model of Nippostrongylus brasiliensis and Heligmosomoides polygyrus.</a> Current Protocols in Immunology. , (2003).</li><li>Mearns, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-4-promoted+T+helper+2+responses+enhance+Nippostrongylus+brasiliensis-induced+pulmonary+pathology.">Interleukin-4-promoted T helper 2 responses enhance Nippostrongylus brasiliensis-induced pulmonary pathology.</a> Infection and Immunity. 76 (12), 5535-5542 (2008).</li><li>Licona-Limon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th9+cells+drive+host+immunity+against+gastrointestinal+worm+infection.">Th9 cells drive host immunity against gastrointestinal worm infection.</a> Immunity. 39 (4), 744-757 (2013).</li><li>Ferrer-Font, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-dimensional+analysis+of+intestinal+immune+cells+during+helminth+infection.">High-dimensional analysis of intestinal immune cells during helminth infection.</a> Elife. 9, 51678 (2020).</li><li>Kharwadkar, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+efficiency+of+multiple+IL9+reporter+alleles+Is+determined+by+cell+lineage.">Expression efficiency of multiple IL9 reporter alleles Is determined by cell lineage.</a> Immunohorizons. 4 (5), 282-291 (2020).</li><li>Wilhelm, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+IL-9+fate+reporter+demonstrates+the+induction+of+an+innate+IL-9+response+in+lung+inflammation.">An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation.</a> Nature Immunology. 12 (11), 1071-1077 (2011).</li><li>Gerlach, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TH9+cells+that+express+the+transcription+factor+PU.1+drive+T+cell-mediated+colitis+via+IL-9+receptor+signaling+in+intestinal+epithelial+cells.">TH9 cells that express the transcription factor PU.1 drive T cell-mediated colitis via IL-9 receptor signaling in intestinal epithelial cells.</a> Nature Immunology. 15 (7), 676-686 (2014).</li><li>Olson, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+IL-2+is+required+for+optimal+type+2+effector+cytokine+production.">Paracrine IL-2 is required for optimal type 2 effector cytokine production.</a> Journal of Immunology. 198 (11), 4352-4359 (2017).</li><li>Cold Spring Harbor Protocols. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphate-buffered+saline+(PBS).">Phosphate-buffered saline (PBS).</a> Cold Spring Harbor Protocols. , (2006).</li><li>Pinto, M. E. S., Licona-Limon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th9+cells+and+parasitic+inflammation:+Use+of+Nippostrongylus+and+Schistosoma+models.">Th9 cells and parasitic inflammation: Use of Nippostrongylus and Schistosoma models.</a> Methods in Molecular Biology. 1585, 223-245 (2017).</li><li>Lawrance, C. C., Lucas, E. A., Clarke, S. L., Smith, B. J., Kuvibidila, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+effects+of+isoflurane+and+CO2+inhalation+on+plasma+levels+of+inflammatory+markers+associated+with+collagen-induced+arthritis+in+DBA+mice.">Differential effects of isoflurane and CO2 inhalation on plasma levels of inflammatory markers associated with collagen-induced arthritis in DBA mice.</a> International Immunopharmacology. 9 (7-8), 807-809 (2009).</li><li>Boivin, G. P., Bottomley, M. A., Schiml, P. A., Goss, L., Grobe, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiologic,+behavioral,+and+histologic+responses+to+various+euthanasia+methods+in+C57BL/6Ntac+male+mice.">Physiologic, behavioral, and histologic responses to various euthanasia methods in C57BL/6Ntac male mice.</a> Journal of the American Association for Laboratory Animal Science. 56 (1), 69-78 (2017).</li><li>old Spring Harbor Protocols. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hank's+balanced+salt+solution+(HBSS)+without+phenol+red.">Hank's balanced salt solution (HBSS) without phenol red.</a> Cold Spring Harbor Protocols. , (2006).</li><li>Bielecki, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin-resident+innate+lymphoid+cells+converge+on+a+pathogenic+effector+state.">Skin-resident innate lymphoid cells converge on a pathogenic effector state.</a> Nature. 592 (7852), 128-132 (2021).</li><li>Sanjabi, S., Mosaheb, M. M., Flavell, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposing+effects+of+TGF-beta+and+IL-15+cytokines+control+the+number+of+short-lived+effector+CD8++T+cells.">Opposing effects of TGF-beta and IL-15 cytokines control the number of short-lived effector CD8+ T cells.</a> Immunity. 31 (1), 131-144 (2009).</li><li>Liu, H., Li, M., Wang, Y., Piper, J., Jiang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+single-cell+encapsulation+efficiency+and+reliability+through+neutral+buoyancy+of+suspension.">Improving single-cell encapsulation efficiency and reliability through neutral buoyancy of suspension.</a> Micromachines. 11 (1), 94 (2020).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-25-responsive,+lineage-negative+KLRG1(hi)+cells+are+multipotential+'inflammatory'+type+2+innate+lymphoid+cells.">IL-25-responsive, lineage-negative KLRG1(hi) cells are multipotential 'inflammatory' type 2 innate lymphoid cells.</a> Nature Immunology. 16 (2), 161-169 (2015).</li><li>Huang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S1P-dependent+interorgan+trafficking+of+group+2+innate+lymphoid+cells+supports+host+defense.">S1P-dependent interorgan trafficking of group 2 innate lymphoid cells supports host defense.</a> Science. 359 (6371), 114-119 (2018).</li><li>Flamar, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-33+induces+the+enzyme+Tryptophan+hydroxylase+1+to+promote+inflammatory+group+2+innate+lymphoid+cell-mediated+immunity.">Interleukin-33 induces the enzyme Tryptophan hydroxylase 1 to promote inflammatory group 2 innate lymphoid cell-mediated immunity.</a> Immunity. 52 (4), 606-619 (2020).</li><li>Olgu&#237;n-Mart&#237;nez, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IL-33+and+the+PKA+pathway+regulate+ILC2+populations+expressing+IL-9+and+ST2.">IL-33 and the PKA pathway regulate ILC2 populations expressing IL-9 and ST2.</a> Frontiers in Immunology. 13, 787713 (2022).</li><li>Olguin-Martinez, E., Ruiz-Medina, B. E., Licona-Limon, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue-specific+molecular+markers+and+heterogeneity+in+type+2+innate+lymphoid+cells.">Tissue-specific molecular markers and heterogeneity in type 2 innate lymphoid cells.</a> Frontiers in Immunology. 12, 757967 (2021).</li><li>Noelle, R. J., Nowark, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+sources+and+immune+functions+of+interleukin-9.">Cellular sources and immune functions of interleukin-9.</a> Nature Reviews. Immunology. 10 (10), 683-687 (2010).</li></ol>

The authors have nothing to disclose.

A complete understanding of intestinal parasite-host interactions and immune responses to helminth infection requires the identification and analysis of the different cell populations and effector molecules that are key for the induction of tissue remodeling and worm expulsion. Soil-transmitted helminth infections represent a big problem in developing countries throughout the world. However, until recently, a protocol that allowed for the analysis of rare cell populations present in the small intestine, the main organ af...

Hookworms are intestinal parasites that infect approximately 700 million people worldwide, mostly in tropical areas in underdeveloped countries. High-intensity infections with Ancylostoma duodenale and Necator americanus, the most common hookworm parasites in humans, cause anemia and protein deficiency that can result in delayed growth and mental development1. N. americanus and the rodent parasite Nippostrongylus brasiliensis induce a prototypical type 2 immune response in their host and share similarities in their life cycle. Hence, the infection of mice with N. brasiliensis is the most commonly used model for human hookworm infections. Stage 3 (L3) N. brasiliensis infective larvae move from the skin to the lung in the first few hours post-infection. Once in the lung, they become L4 and migrate up the trachea to get swallowed, pass through the stomach, and reach the gut to become adults (L5) within 4-5 days. In the gut, L5 worms lay eggs that are excreted in the feces to restart the parasite life cycle2.
The immune response induced by N. brasiliensis is characterized by an increase in several type 2 cytokines, including IL-4, IL-5, IL-9, IL-10, and IL-13, along with eosinophilia, basophilia, goblet and mast cell hyperplasia, and heightened IgG1 and IgE production. Most of the studies trying to identify and define the immune responses elicited upon N. brasiliensis infection are centered on the role of IL-4 or IL-13 in this model3. However, the identification and characterization of IL-9-expressing cells and the function of this cytokine had been largely overlooked, until Licona-Lim&#243;n et al. published the first study demonstrating a critical role for IL-9 in the immune response against N. brasiliensis. Using reporter mice, this study described T cells (mostly T helper 9) and type 2 innate lymphoid cells (ILC2s) as the main cellular subsets expressing IL-9 upon infection4.
Isolation and characterization of immune cells from helminth-infected lungs is feasible, and has been extensively reported3,4. However, because of the inherent tissue remodeling and mucus production, to do so in the infected gut proved to be a technical challenge, until the recent publication of Ferrer-Font et al.5. The group outlined a protocol to isolate and analyze single-cell suspensions of immune subsets from Heligmosomoides polygyrus-infected murine intestines. Based on it , we have now standardized a protocol for isolation and cytometric analysis of IL-9-expressing lymphoid cells from the N. brasiliensis infected gut. In addition, we have established IL-9 kinetics from different cellular sources and anatomical locations throughout the infection.
Characterizing the distinct cell populations involved in this infection is vital for a wider understanding of the immune response to the parasite and its interaction with the host. This comprehensive protocol provides a clear route to isolate and analyze IL-9-producing cells from desired organs at disease stages of interest, allowing for a sharp improvement of the knowledge about the role of these cells in N. brasiliensis infection and parasite infections in general.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACK buffer</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Attune Nxt cytometer</td>
			<td>Thermofisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>B220</td>
			<td>Biolegend</td>
			<td>103204</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11b</td>
			<td>Biolegend</td>
			<td>101204</td>
			<td></td>
		</tr>
		<tr>
			<td>CD11c&#160;</td>
			<td>Biolegend</td>
			<td>117304</td>
			<td></td>
		</tr>
		<tr>
			<td>CD19&#160;</td>
			<td>Biolegend</td>
			<td>115504</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4</td>
			<td>Biolegend</td>
			<td>100404</td>
			<td></td>
		</tr>
		<tr>
			<td>CD4 (BV421)</td>
			<td>Biolegend</td>
			<td>100443</td>
			<td></td>
		</tr>
		<tr>
			<td>CD45.2</td>
			<td>Biolegend</td>
			<td>109846</td>
			<td></td>
		</tr>
		<tr>
			<td>CD8&#160;</td>
			<td>Biolegend</td>
			<td>100703</td>
			<td></td>
		</tr>
		<tr>
			<td>CD90.2</td>
			<td>Biolegend</td>
			<td>105314</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase D</td>
			<td>Roche</td>
			<td>11088866001</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAse I</td>
			<td>Invitrogen</td>
			<td>18068015</td>
			<td>Specific activity: &#8805;10 000 units/mg&#160;&#160;&#160;</td>
		</tr>
		<tr>
			<td>Facs ARIA II sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Melody cell sorter</td>
			<td>BD Biosciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fc-Block</td>
			<td>Biolegend</td>
			<td>101320</td>
			<td></td>
		</tr>
		<tr>
			<td>Fc&#949;RI</td>
			<td>eBioscience</td>
			<td>13589885</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>26140079</td>
			<td></td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo</td>
			<td></td>
			<td>Flow cytometry analysis data software</td>
		</tr>
		<tr>
			<td>Gr-1</td>
			<td>Tonbo</td>
			<td>305931</td>
			<td></td>
		</tr>
		<tr>
			<td>Hanks Balanced Salt Solution (HBSS)</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IL-9</td>
			<td>biolegend</td>
			<td>514103</td>
			<td></td>
		</tr>
		<tr>
			<td>NK1.1&#160;</td>
			<td>Biolegend</td>
			<td>108704</td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon mesh</td>
			<td>&#160;&#8206; lba</td>
			<td>B07HYHHX5V</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiPrep Density Gradient Medium</td>
			<td>Sigma</td>
			<td>D1556</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline&#160;</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>11875093</td>
			<td></td>
		</tr>
		<tr>
			<td>Siglec F&#160;</td>
			<td>Biolegend</td>
			<td>155512</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin</td>
			<td>Biolegend</td>
			<td>405206</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#946;&#160;</td>
			<td>Biolegend</td>
			<td>109203</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#946; (PE/Cy7)</td>
			<td>Biolegend</td>
			<td>109222</td>
			<td></td>
		</tr>
		<tr>
			<td>TCR-&#947;&#948;&#160;</td>
			<td>Biolegend</td>
			<td>118103</td>
			<td></td>
		</tr>
		<tr>
			<td>Ter119</td>
			<td>Biolegend</td>
			<td>116204</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricine buffer&#160;</td>
			<td>Homemade</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zombie Aqua Fixable Viability Dye</td>
			<td>Biolegend</td>
			<td>423101</td>
			<td></td>
		</tr>
	</tbody>
</table>

a strategy for the study of il-9-producing lymphoid cells in the nippostrongylus brasiliensis infection model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response against helminth infections, where it plays an important role in the expulsion of the parasite. In a murine model of Nippostrongylus brasiliensis infection, IL-9 is produced mainly by CD4+ T lymphocytes and innate lymphoid cells found in the lung, small intestine, and draining lymph nodes. Given the technical difficulties involved in the intracellular staining of IL-9, as well as the complexity of isolating hematopoietic cells from the small intestine upon infection, there is a pressing need for a comprehensive but straightforward protocol to analyze the expression of IL-9 in different lymphoid and non-lymphoid tissues in this model. The protocol described here outlines the kinetics of IL-9 produced by CD4+ T cells and innate lymphoid cells in the lung and small intestine, the main organs targeted by N. brasiliensis, as well as in the mediastinal and mesenteric lymph nodes, throughout the infection. In addition, it details the number of larvae needed for infection, depending on the cell type and organ of interest. This protocol aims to assist in the standardization of assays to save time and resources by offering the opportunity to focus on the specific cells, organs, and disease stages of interest in the N. brasiliensis infection model.

Mice were subcutaneously injected with 200 L3 stage N. brasiliensis larvae, or with PBS for sham controls. The number of larvae used in this protocol was adjusted in order to isolate viable cells from the lungs, lymphoid tissue, and the small intestine, unlike previous reports where higher loads of worms were used to detect cells in lymphoid tissues and lungs only4. Lungs, mediastinal lymph nodes, mesenteric lymph nodes, and the small intestine were harvested at days 0, 4, 7, and 10 post-...

Watch this Scientific Journal Video about A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model at JoVE.com

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9-expressing T and ILC2 cells are induced during N. brasiliensis infection, yet their characterization has been largely overlooked in the infected intestine due to their low frequency and differential kinetics. This protocol describes the isolation of these cells from different target organs and confirmation of their identity via flow cytometry at different infection stages.

A Strategy for the Study of IL-9-Producing Lymphoid Cells in the Nippostrongylus brasiliensis Infection Model

IL-9 is a pleiotropic cytokine associated with various processes, including antitumor immunity, induction of allergic pathologies, and the immune response ...

This protocol allows for the isolation and characterization of lymphoid cells expressing IL-9 in N.brasiliensis non-lymphoid infected tissues including the infected cut which recently was technically invisible.This protocol also provides an alternative to analyze populations of cells from peripheral tissues previously unappreciated in infection due to the intrinsic difficulties of isolating cells from these anatomical sites.This method allows the study of difficult-to-isolate populations from N.brasiliensis infected cut and could be applied to other helminthic infections, broadening our understanding of hookworm infections in humans.Demonstrating the procedure will be Ofelia Mu

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Immunology and Infection

1.2K 조회수.  Universidad Nacional Autónoma de México. This protocol allows for the isolation and characterization of lymphoid cells expressing IL-9 in N.brasiliensis non-lymphoid infected tissues including the infected cut which recently was technically invisible.This protocol also provides an alternative to analyze populations of cells from peripheral tissues previously unappreciated in infection due to the intrinsic difficulties of isolating cells from these anatomical sites.This method allows the study of difficult-to-isolate populations from...