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

Summary

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.

Abstract

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.

Introduction

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-Limón 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 reported^3,4. 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.

Protocol

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 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…

Representative Results

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-…

Discussion

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…

Materials

ACK buffer	Homemade
Attune Nxt cytometer	Thermofisher
B220	Biolegend	103204
CD11b	Biolegend	101204
CD11c	Biolegend	117304
CD19	Biolegend	115504
CD4	Biolegend	100404
CD4 (BV421)	Biolegend	100443
CD45.2	Biolegend	109846
CD8	Biolegend	100703
CD90.2	Biolegend	105314
Collagenase D	Roche	11088866001
DNAse I	Invitrogen	18068015	Specific activity: ≥10 000 units/mg
Facs ARIA II sorter	BD Biosciences
FACS Melody cell sorter	BD Biosciences
Fc-Block	Biolegend	101320
FcεRI	eBioscience	13589885
Fetal bovine serum	Gibco	26140079
FlowJo	FlowJo		Flow cytometry analysis data software
Gr-1	Tonbo	305931
Hanks Balanced Salt Solution (HBSS)	Homemade
IL-9	biolegend	514103
NK1.1	Biolegend	108704
Nylon mesh	‎ lba	B07HYHHX5V
OptiPrep Density Gradient Medium	Sigma	D1556
Phosphate-buffered saline	Homemade
RPMI	Gibco	11875093
Siglec F	Biolegend	155512
Streptavidin	Biolegend	405206
TCR-β	Biolegend	109203
TCR-β (PE/Cy7)	Biolegend	109222
TCR-γδ	Biolegend	118103
Ter119	Biolegend	116204
Tricine buffer	Homemade
Zombie Aqua Fixable Viability Dye	Biolegend	423101