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.
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
2. Infection of mice
3. Isolation of lung, small intestine, mediastinal, and mesenteric lymph nodes
4. Preparation of single-cell suspensions from the small intestine, lung, and lymph nodes
NOTE: It is extremely important to process a maximum of six mice per person when preparing single-cell suspensions from small intestines, as cell viability decreases significantly with longer processing periods. This method was adapted from the Heligmosomoides polygyrus infection mouse model5.
5. Cell staining for flow cytometry (Figure 2 and Figure 3)
NOTE: Centrifuge the lymph node cell suspensions from step 4.11.3 at 450 x g for 5 min at 4 °C, and resuspend the cell pellet in 500 µL of FACS buffer (Table 1).
6. Determination of absolute numbers of cells in single-cell suspensions
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-infection for lymphocyte isolation and characterization of IL-9-producing populations. A total of 27 mice were used in two or more independent experiments, including nine controls and four to six N. brasiliensis-infected mice per time point analyzed. Sham-infected controls were included in every experiment to obtain basal numbers. Using this protocol, IL-9-producing CD4+ T cells (mostly Th9 cells in this model) can be isolated from lung, mesenteric, and mediastinal lymph nodes for quantification and further analysis.
Following the natural course of infection, we first assessed the frequencies of IL-9-producing CD4+ T lymphocytes (Th9) present in the lungs and mediastinal lymph nodes (gating strategy shown in Figure 2A and Supplementary Figure 3A). In both organs, Th9 frequencies increased, beginning at day 4, and reached a peak at day 7 post-infection (Figure 2C), an increment that was also observed in Th9 absolute numbers (Supplementary Figure 4A,B). In the mesenteric lymph nodes (gating strategy shown in Figure 2B), there was a significant increase in both the frequencies and absolute numbers of Th9 cells at days 7 and 10 post-infection (Figure 2C and Supplementary Figure 4C), in accordance with previous reports4. The presence of T cells and ILC2s was confirmed in the intraepithelial and lamina propria samples; however, no Th9 cells were observed in these intestinal compartments throughout the infection (Supplementary Figure 3B,C).
We then evaluated IL-9-expressing ILC2 cells in lymphoid and non-lymphoid tissues of infected mice (gating strategy shown in Figure 3A,B and Supplementary Figure 2). We observed a significant increase in the frequencies of ST2+ IL-9- and ST2+ IL-9+ expressing ILC2s in the lungs at day 7 post-infection (Figure 3C). At the same time, analysis of absolute numbers revealed a statistically significant increase only in the ST2+ IL-9+ population at day 7 post-infection (Supplementary Figure 5A). In the mediastinal lymph nodes (gating strategy shown in Supplementary Figure 2B), the numbers of IL-9-expressing ILC2 cells (ST2+ IL-9+) significantly increased at day 10 post-infection, while absolute numbers of ST2+ cells showed a trend to increase, starting at day 7 and continuing until day 10 post-infection (Supplementary Figure 6A,C).
IL-9+ ILC2 cells were also found in the lamina propria and intraepithelial compartments of the small intestine (gating strategy shown in Figure 3B and Supplementary Figure 2A). N. brasiliensis infection resulted in a statistically significant increase in the frequencies of ST2+ IL-9+ cells at day 4, and ST2- IL-9+ populations at days 7 and 10 in the lamina propria. In the intraepithelial compartment, a significant change was also observed in the frequencies of ST2+ IL9+ and ST2- IL-9+ cells at day 7 and day 10 post-infection, respectively (Figure 3C). Importantly, even with a relatively low number of larvae load, infection with the parasite caused extensive damage to the small intestine; hence, the analysis of IL-9-expressing ILC2s absolute numbers is technically unfeasible, hindering the accurate quantification of this population due to low yields of live cells (Supplementary Figure 5B,C). On the other hand, the analysis of mesenteric lymph nodes (gating strategy shown in Supplementary Figure 2C) unveiled a significant increase in absolute numbers of ST2+ IL-9-, ST2+ IL-9+, and ST2- IL-9+ ILC2 cells at day 10 post-infection (Supplementary Figure 6D), as previously reported4. Meanwhile, no difference was observed in the frequencies of these populations throughout the infection (Supplementary Figure 6B). These diverse subsets of ST2 and IL-9-expressing cells are intriguing populations with potentially differential roles in vivo, and might correspond to natural versus inflammatory ILC2s as recently described18,19,20,21. However, this needs to be addressed in future studies.
In summary, we adjusted this protocol for the recovery of IL-9-expressing cells from N. brasiliensis-infected intestine, while still being able to detect them in the lung and lymphoid tissue. Retrieving immune cells from parasite-infected guts has proven to be technically difficult. Here, an adjusted number of larvae used for infection resulted in improved intestinal tissue integrity, that allowed the recovery of an IL-9+ population. To our knowledge, this is the first protocol developed specifically for the analysis of IL-9-expressing cells in the gut during N. brasiliensis infection. Nevertheless, other immune cells can also be isolated following these steps. The data presented here show that most tissue resident IL-9-expressing cells in the small intestine are ILC2s.
Figure 1: Flowchart of the described protocol. Schematic diagram with the main steps used to process different organs and the expected IL-9-expressing cell subsets obtained. Please click here to view a larger version of this figure.
Figure 2: Th9 cells are found in lungs, mediastinal, and mesenteric lymph nodes throughout N. brasiliensis infection. Representative flow cytometry analysis and gating strategy used for Th9 cell identification from (A) lungs and (B) mesenteric lymph nodes. Single-cell suspensions from each organ were stained with a fixable viability dye, fluorescently labeled anti-CD45 for the identification of hematopoietic cells, and anti-T-cell receptor (TCR) β and anti-CD4 for identification of CD4+ T lymphocytes. The GFP+ signal indicates the percentage of Th9 cells present. The first gate shows side scatter (SSC)-A/forward scatter (FSC)-A properties with the classical morphology of lymphoid cells. Single-cell events were selected, followed by live cells and then CD45+ cells. Within this population, we focused on TCR-β and CD4 double-positive cells, from which GFP+ cells were identified as Th9 cells. The staining of lymph nodes did not include the anti-CD45 antibody, since the entire population is expected to be positive. (C) Frequencies of Th9 cells found in lungs, mediastinal, and mesenteric lymph nodes at the indicated times post-infection. Data represent the mean ± SEM of one or two mice analyzed per group, from three independent experiments, per time point; Unpaired T-test; *p≤ 0.05, **p≤ 0.001, ***p≤ 0.0001. Please click here to view a larger version of this figure.
Figure 3: IL-9-producing ILC2 cells are found in non-lymphoid organs. Representative flow cytometry analysis and gating strategy used for the identification of IL-9+ ILC2 cells from (A) lung and (B) small intestine lamina propria. Single-cell suspensions from each organ were marked with biotinylated antibodies to identify lineage cells (anti-B220, anti-CD11b, anti-FcεRI, anti-TCR-β, anti-TCR-γδ, anti-Siglec F, anti-CD4, anti-CD11c, anti-Gr-1, anti-CD8, anti-CD19, anti-NK1.1, and anti-Ter119), and Fc-Block. Cell suspensions were then stained with fixable viability dye, fluorochrome-labeled streptavidin, anti-CD45 for the identification of hematopoietic cells, and anti-CD90 for the identification of ILC2 cells. The first gate shows scatter (SSC)-A/side scatter (FSC)-A properties with the classical morphology of lymphoid cells. Single-cell events were selected, followed by live cells and then CD45+ cells. Within this population, we focused on CD90+ lin- cells; from this group, we evaluated ST2 and GFP expression to identify IL-9+ ILC2 cells. (C) Frequencies of ILC2 cells found in lungs, lamina propria, and the intraepithelial cell compartment at the indicated times points post-infection. Data represent the mean ± SEM of one or two mice analyzed per group, from three independent experiments, per time point; unpaired T-test; *p ≤ 0.05. Please click here to view a larger version of this figure.
Table 1: List of chemical solutions and their compositions. Please click here to download this Table.
Table 2: Antibody cocktail to identify IL-9-producing ILC2 cells. Please click here to download this Table.
Table 3: Antibody cocktail to identify IL-9-producing T lymphocytes. Please click here to download this Table.
Supplementary Figure 1: Schematic representation of the mediastinal lymph node location. Created in BioRender.com Please click here to download this File.
Supplementary Figure 2: Representative flow cytometry and gating strategy used to identify IL-9+ ILC2s within intraepithelial cells from the small intestine and lymph nodes. (A) Single-cell suspensions from small intestine intraepithelial cells were first incubated with biotin-labeled antibodies to select lineage cells (anti-B220, anti-CD11b, anti-FcεRI, anti-TCR-β, anti-TCR-γδ, anti-Siglec F, anti-CD4, anti-CD11c, anti-Gr-1, anti-CD8, anti-CD19, anti-NK1.1, and anti-Ter119), and Fc-Block. This was followed by staining with a fixable viability dye, fluorochrome-labeled streptavidin, anti-CD45 for the identification of hematopoietic cells, and anti-CD90 for the identification of ILC2 cells. The first gate shows side scatter (SSC)-A/forward scatter (FSC)-A properties with the classical morphology of lymphoid cells. Single-cell events were selected, followed by live cells and then CD45+ cells. Within this population, we focused on CD90+ lin- cells; from this group, GFP+ cells were identified as IL-9+ ILC2 cells. (B,C) Representative flow cytometry and gating strategy for (B) mediastinal lymph nodes and (C) mesenteric lymph nodes to identify IL-9+ ILC2 cells. Staining of lymph nodes did not include the anti-CD45 antibody, since the entire population was expected to be positive. Please click here to download this File.
Supplementary Figure 3: Representative flow cytometry analysis and gating strategy used to identify Th9 cells in mediastinal lymph nodes and the small intestine. Single-cell suspensions from (A) mediastinal lymph nodes were stained with a fixable viability dye and fluorescently labeled anti-TCR-β and anti-CD4 antibodies. The first gate shows side scatter (SSC)-A/forward scatter (FSC)-A properties with the classical morphology of lymphoid cells. Single-cell events were selected, followed by live cells and then TCR-β and CD4 double-positive cells; from this group, GFP+ cells were identified as Th9 cells. A similar flow cytometry strategy was used for (B) lamina propria and (C) intraepithelial cells from the small intestine, except that CD45+ cells were selected after the live cells, followed by TCR-β and CD4 double-positive cells, from where GFP+ cells were identified as Th9 cells. Please click here to download this File.
Supplementary Figure 4: Absolute numbers of Th9 cells significantly change throughout the infection. Absolute numbers of IL-9+ cells from TCR-β and CD4 double-positive cells were determined at days 0, 4, 7, and 10 post-infection in (A) lung, (B) mediastinal lymph nodes, and (C) mesenteric lymph nodes. Data represent the mean ± SEM of one or two mice analyzed per group, from three independent experiments, per time point; unpaired T-test *p ≤ 0.05, **p ≤ 0.001, ***P ≤ 0.0001. Please click here to download this File.
Supplementary Figure 5: Absolute numbers of IL-9+ ILC2 cells differ throughout the infection. Absolute numbers of IL-9+ ILC2 cells from the lin- CD90+ population at days 0, 4, 7, and 10 post-infection were determined in (A) lung, (B) lamina propria, and (C) intraepithelial cells from the small intestine. Data represent the mean ± SEM of one or two mice analyzed per group, from three independent experiments, per time point; unpaired T-test *p ≤ 0.05. Please click here to download this File.
Supplementary Figure 6: Frequencies and absolute numbers of IL-9+ ILC2 cells in lymph nodes significantly increase during infection. (A) Frequency and (C) absolute numbers of IL-9+ ILC2 cells from mediastinal lymph nodes. (B) Frequency and (D) absolute numbers of IL-9+ ILC2 cells from mesenteric lymph nodes. Values were determined as IL-9+ ILC2 cells within the lin- CD90+ population at days 0, 4, 7, and 10 post-infection. Data represent the mean ± SEM of one or two mice analyzed per group, from three independent experiments, per time point;unpaired T-test *p ≤ 0.05, ***p ≤ 0.0001. Please click here to download this File.
Supplementary Figure 7: Flow cytometry analysis of IL-9 expressing ILC2 cells from N. brasiliensis infected gut. Representative plots of ILC2 cells isolated from the intestine of IL-9-reporter mice at day 7 post-infection. IL-9 expression was assessed in ILC2 cells freshly isolated from the intraepithelial or lamina propria compartments of infected reporter mice (IL-9 REP) versus following the previously described IL-9 intracellular staining protocol9 (IL-9 AB, using an IL-9 antibody clone RM9A4, BioLegend). To identify IL-9-expressing ILC2s, single cell suspensions were first incubated with biotin labeled antibodies to select lineage cells (anti-B220, anti-CD11b, anti-FcεRI, anti-TCR-β, anti-TCR-γδ, anti-Siglec F, anti-CD4, anti-CD11c, anti-Gr-1, anti-CD8, anti-CD19, anti-NK1.1, and anti-Ter119) and Fc-Block, followed by staining with flexible viability dye, fluorochrome-labeled streptavidin, anti-CD45 and anti-CD90. The plots show IL-9-expressing ILC2 cells gated on the lin-CD45+CD90+ population present in the intraepithelial (A,B) and lamina propria (C,D) compartments, detected via the reporter signal from freshly isolated cells (A,C) or following intracellular staining of IL-9 (B,D). Control mice were injected with PBS. Please click here to download this File.
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 affected by this infection, was not available5. This protocol covers previously described methods that allow the flow cytometric analysis of IL-9-expressing lymphoid cells in the lung, mediastinal, and mesenteric lymph nodes during N. brasiliensis infection, with the additional advantage of the identification of this population for the first time in the small intestine.
The success of this protocol is highly dependent on tissue processing times. It is imperative to process a maximum of six small intestine samples, per person, at once. If different organs are to be processed, we recommend it be done in parallel by another person. By using a relatively low parasite load (200 L3 larvae), this protocol allows for the isolation of lymphoid cells from the N. brasiliensis-infected intestine while maintaining the ability to isolate these cells from other organs. The use of higher parasite loads jeopardizes the quality and quantity of cells isolated from the small intestine (data not shown); however, it could result in a significant increase of IL-9-expressing lymphoid cells in other organs4. When processing the intestine and mesenteric lymph nodes, it is critical to remove the adhered fat tissue, as failure to do so results in increased cell death. The data support the observation that the natural course of infection negatively impacts the absolute number of lymphoid cells recovered from the small intestine. In the studies described here, the frequencies of IL-9+ ILC2 cells remain consistent, while absolute numbers show high variability in each independent experiment. Hence, conclusions drawn from absolute numbers could be inaccurate and should be avoided. In addition, given the low frequency of IL-9-expressing T cells in tissues, the acquisition and staining of the highest possible number of cells is recommended.
A limitation of this ILC2 analysis is the use of a discrete combination of markers to define this population. Depending on the tissue of interest and phase of infection, additional markers, such as CD25, Klrg1, and ST2 among others, could be used for a more detailed phenotypic characterization of ILC2 cells22. We acknowledge that the protocol presented here is based on the INFER reporter mouse strain4, and could represent an advantage for the identification of IL-9-expressing cells. However, we expect that the results found here could be obtained using alternative strategies, including the use of other mouse reporter strains and intracellular IL-9 staining6,7,8,9. It is important to mention that using conventional mice and performing intracellular staining of IL-9 in intraepithelial and lamina propria cells may result in losing any detectable signal and compromising the data obtained (Supplementary Figure 7). This could be due to the long-term manipulation required to stain a subset that is already hard to isolate and maintain ex vivo. Therefore, we recommend the use of reporter mice strains to reduce processing times and ensure a reliable analysis of IL-9 expression in vivo. We also acknowledge the fact that Th9 cells are not the only T cells expressing IL-923; however, in this parasitic model, we would not expect other subsets to express it. Nevertheless, to discard the presence of other IL-9-expressing subsets in the described context, a complete characterization of type 2 cytokines along with the use of transcriptional markers is feasible.
The quantification of IL-9-expressing lymphoid cells before day 5 post-infection has been previously reported; however, it results in very low cell frequencies4. The protocol in this study covers a large part of N. brasiliensis infection kinetics up until the beginning of parasite clearance2. However, we noticed that some of the cell populations in this study did not return to basal levels within the timeframe analyzed, suggesting that the study could benefit from extended time points to have a full perspective on the behavior of these cells in a more advanced stage of the infection in vivo. Regardless, we believe that the work presented here could serve as a reference guide for researchers interested in studying IL-9-expressing lymphoid cells in parasite infection models, allowing them to choose the ideal phase of infection for detection of the specific cell types from tissues of interest. Altogether, the methods described here will be useful to assess the phenotype and function of T lymphocytes and ILC2 cells that express IL-9 during a physiologically relevant model of parasite infection, widening our knowledge of these cells and potentially improving our understanding of them in other pathological conditions.
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 |