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.
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.
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 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.
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…
The authors have nothing to disclose.
The authors wish to acknowledge José 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 México 2022 (3)).
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 |