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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

Protokół

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

1. 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 cycles, with ad libitum access to water and food.
   NOTE: This protocol uses an IL-9 reporter mouse strain in C57BL/6 background, as previously described⁴; however, other IL-9 reporter strains could be used^6,7,8_, as well as intracellular IL-9 staining, with variable results⁹.

2. Infection of mice

1. Shave the back of the mice from the middle of the body to near the base of the tail 1 day prior to infection. The use of anesthetics is not essential.
2. Subcutaneously inoculate each mouse with 200 viable third-stage N. brasiliensis larvae (L3) in 100 µL of phosphate buffered saline (PBS)¹⁰ in the lower back, as previously described^2,11, or inject 100 µL of PBS alone as a control. Sacrifice the animals on day 4, day 7, or day 10 post-infection.

3. Isolation of lung, small intestine, mediastinal, and mesenteric lymph nodes

1. Euthanize the mouse by cervical dislocation. Place the mouse on its back and spray it with 70% ethanol. Make a midline incision using scissors and open the skin to expose the abdominal and thoracic areas.
   NOTE: Isoflurane can be used as an alternative euthanasia method¹². Carbon dioxide chambers are not recommended, as CO₂ leads to lung tissue damage and hemorrhage¹³.
2. Isolation of mediastinal lymph nodes and lungs
   1. Make an incision at the sternum and cut in a "V" shape to remove the ribs and thoracic muscles. Once the thoracic cavity is exposed, locate the mediastinal lymph nodes next to the esophagus, below the heart (see Supplementary Figure 1).
   2. Extract the mediastinal lymph nodes and collect them in 1 mL of R-10 medium (Table 1) in a 12-well culture plate. Keep on ice, protected from light until processing.
      NOTE: The samples should be protected from light, to avoid decreasing the fluorescent signal from the reporter mice used in these experiments.
   3. Extract the lungs and collect them in 1 mL of R-10 medium in a 12-well culture plate per mouse. Keep on ice, protected from light until processing.
3. Isolation of mesenteric lymph node chains and small intestines
   1. Expose the peritoneal cavity, and carefully move the small intestine to the right to expose the mesenteric lymph node (MLN) chain along the colon.
   2. Using forceps, remove the MLN, roll it gently on a paper towel, and pull the fat off.
   3. Transfer the MLN to 1 mL of R-10 medium in a 12-well culture plate. Keep on ice, protected from light until processing.
   4. Cut the small intestine just below the pyloric sphincter and above the cecum. Pull the intestine out slowly with the help of forceps, removing the attached mesentery and fat tissue.
   5. Place the small intestine on a paper towel and generously moisten it with PBS. Remove the Peyer's patches from the small intestine with scissors.
      NOTE: To maintain viability, remove the remaining fat tissue, and keep the small intestine moist with PBS during the entire process.
   6. Cut the small intestine longitudinally using scissors, and gently slide the forceps over the open intestine to remove the fecal content and mucus.
   7. Hold the small intestine with forceps, and wash it in 5 mL of PBS on ice by carefully submerging it a few times. Repeat twice.
   8. Cut the small intestine into short pieces (approximately 5 mm), and collect them in a 50 mL conical tube with 10 mL of HBSS¹⁴ with 2% FBS (Table 1). Immediately continue to isolate intraepithelial and lamina propria cells.

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.

1. Pre-warm the shaking incubator, R-20 medium, HBSS, and HBSS-2 mM EDTA (Table 1) at 37 °C.
2. Prepare 10 mL of small intestine digestion medium (Table 1) per sample.
3. Shake the intestine pieces from step 3.3.8 vigorously by hand.
4. Filter each sample through a nylon mesh (approximately 10 cm x 10 cm) over a glass funnel. Wash the sample by adding 10 mL of pre-warmed HBSS over the mesh and discard the flow-through. Repeat one more time.
   NOTE: Use a new mesh for each sample and reuse it throughout the procedure.
5. Remove the mesh from the funnel, and collect the sample from the mesh in a 50 mL conical tube with 10 mL of warm HBSS-2 mM EDTA (step 4.1). Incubate for 10 min at 37 °C, with shaking at 200 rpm.
6. Vortex for 10 s at maximum speed (3,200 rpm) and filter the sample with the mesh over a glass funnel, recovering the flow-through in a 50 mL conical tube.
7. Repeat steps 4.5 and 4.6 twice, recovering the flow-through in the same 50 mL conical tube. The intraepithelial cells are located in this 30 mL fraction. Save the remaining tissue.
8. Single-cell suspension preparation from intraepithelial cells
   1. Centrifuge the 30 mL of cell suspension, recovered in step 4.7, at 450 x g for 5 min at room temperature (RT). Discard the supernatant.
   2. Add 5 mL of PBS and centrifuge at 450 x g for 5 min at RT. Discard the supernatant.
   3. Resuspend the cell pellet in 3 mL of RPMI 10% FBS-20 µg/mL DNase (Table 1). Keep on ice, protected from light until cell staining.
9. Single-cell suspension preparation from lamina propria cells
   1. Wash the remaining small intestine tissue from step 4.7 by pouring over 10 mL of warm HBSS through the mesh over the funnel. Repeat the wash. Collect the tissue from the mesh in a 50 mL conical tube with 10 mL of small intestine digestion medium.
   2. Incubate for 30 min at 37 °C, with shaking at 200 rpm. Vortex at maximum speed for 10 s every 5 min.
   3. Add 10 mL of FACS-EDTA buffer (Table 1) to stop the digestion reaction, and place it on ice.
   4. Filter each sample through a 100 µm cell strainer using a serological pipette, recovering the suspension in a 50 mL conical tube placed on ice.
   5. Centrifuge at 600 x g for 6 min at 4 °C.
   6. Discard the supernatant. Wash the cell pellet with 5 mL of PBS and centrifuge at 600 x g for 6 min at 4 °C.
   7. Discard the supernatant, and resuspend the cell pellet in 1 mL of RPMI 10% FBS-20 µg/mL DNase. Keep on ice, protected from light until cell staining.
10. Single-cell suspension preparation from lung
    NOTE: Each lung and small intestine should be processed in parallel by two people to avoid extended handling times, which result in low cell viability.
    1. Prepare 4 mL of lung digestion medium (Table 1) per sample processed.
    2. Remove the RPMI medium from the well containing the lungs from step 3.2.3. Cut the lung into small pieces with fine scissors.
    3. Transfer the lung pieces to a 15 mL conical tube with a spatula. Add 4 mL of lung digestion medium (Table 1).
    4. Incubate for 30 min at 37 °C, with shaking at 250 rpm. When finished, keep on ice until each sample is processed.
    5. Filter each sample through a 100 µm cell strainer, positioned in a single well of a 6-well culture plate. Dissociate the tissue with a syringe plunger.
    6. Recover each sample in a 15 mL conical tube, and add 4 mL of R-2 medium (Table 1). Keep on ice while the other samples are processed.
    7. Centrifuge at 600 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cell pellet in 1 mL of R-5 medium (Table 1).
    8. While centrifuging, prepare 4 mL of 27.5% density-gradient solution (Table 1) per sample to enrich the hematopoietic cell fraction^15,16. This strategy for single-cell separation is more efficient, cost-effective, and less toxic compared to other similar density gradient media¹⁷.
    9. Add 4 mL of 27.5% density-gradient solution to the 1 mL of cell suspension from step 4.10.7, and shake vigorously.
    10. Slowly add 1 mL of R-5 medium (Table 1) on top of the mixed suspension to create two phases.
    11. Centrifuge at 1,500 x g for 20 min at RT, with low acceleration and the brake off. Observe the ring formed between the two phases.
    12. Recover the ring formed between the two phases with a 1 mL micropipette, and resuspend in 4 mL of R-2 medium.
    13. Centrifuge at 450 x g for 5 min at 4 °C. Discard the supernatant. Resuspend the cell pellet in 1 mL of ACK buffer (Table 1), and incubate for 1 min at RT.
    14. Add 4 mL of R-5 medium and centrifuge at 450 x g for 5 min at 4 °C. Discard the supernatant and resuspend the cell pellet in 1 mL of R-10 medium. Keep on ice, protected from light until staining for flow cytometry.
11. Single-cell suspension preparation from lymph nodes
    1. Place the lymph nodes from steps 3.2.2 or 3.3.3 between two pieces of mesh in a well from a 6-well culture plate, and dissociate with a syringe plunger.
    2. Recover the cell suspension in a 1.5 mL conical tube and centrifuge at 450 x g for 5 min at 4 °C.
    3. Discard the supernatant, and resuspend the cell pellet in 1 mL of R-10 medium. Keep on ice, protected from light until staining for flow cytometry.
       NOTE: If clumps are visible, filter the sample through a 100 µm cell strainer.

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).

1. Cell staining for identification of ILC2s (Figure 3 and Supplementary Figure 2)
   NOTE: This staining procedure is specific for the identification of IL-9-expressing ILC2 cells.
   1. Plate 150 µL per lung sample (approximately 1.8 x 10⁶ cells) from step 4.10.14, and 50 µL per lymph node sample from step 4.11.3 (approximately 0.7 x 10⁶ and 2.2 x 10⁶ cells for mediastinal and mesenteric lymph node samples, respectively) in a 96-well conical bottom culture plate. Add 100 µL of FACS buffer, and centrifuge at 450 x g for 5 min at 4 °C.
   2. Plate 100 µL per small intestine sample (approximately 2.7 x 10⁶ and 0.6 x 10⁶ cells for intraepithelial and lamina propria samples, respectively) from steps 4.8.3 and 4.9.7 in a 96-well conical bottom culture plate. Add 150 µL of FACS buffer and centrifuge at 450 x g for 5 min at 4 °C.
   3. Discard the supernatant, and resuspend each cell pellet in 50 µL of the biotinylated antibody cocktail (Table 2) diluted in FACS buffer. Incubate for 30 min at 4 °C protected from light.
      NOTE: Use FACS buffer with 20 µg/mL DNase for intraepithelial and lamina propria samples.
   4. Add 150 µL of FACS buffer. Centrifuge at 450 x g for 5 min at 4 °C.
   5. Discard the supernatant, and resuspend the cell pellet in 200 µL of FACS buffer. Centrifuge again and discard the supernatant.
   6. Resuspend the cell pellet in 50 µL of the antibody/stain cocktail (Table 2), and incubate for 30 min at 4 °C protected from light.
   7. Add 150 µL of FACS buffer. Centrifuge at 450 x g for 5 min at 4 °C.
   8. Discard the supernatant, and resuspend the cell pellet in 200 µL of FACS buffer. Centrifuge again and discard the supernatant. Repeat the wash (step 5.1.7), and discard the supernatant.
   9. Resuspend the cell pellet in 300 µL of FACS buffer, and analyze by flow cytometry.
   10. For the small intestine and lung samples, use the gating strategy: lymphocytes, single cells, live cells, hematopoietic cells, CD90+Lineage- cells, ST2+ cells, and IL-9+ cells (Figure 3A,B and Supplementary Figure 2A). For the lymph node samples, use the gating strategy: live cells, single cells, CD90+ Lineage- cells, ST2+ cells, and IL-9+ cells (Supplementary Figure 2B,C).
2. Cell staining for identification of IL-9-producing lymphocytes (Figure 2 and Supplementary Figure 3)
   1. Transfer 800 µL per lung sample from step 4.10.14 to a 1.5 mL tube (approximately 14.6 x 10⁶ cells). Centrifuge at 450 x g for 5 min at 4 °C. Discard the supernatant.
   2. For lymph node samples, plate 400 µL of the cell suspension from step 4.11.3 (approximately 5.6 x 10⁶ and 17.5 x 10⁶ cells for mediastinal and mesenteric lymph node samples, respectively) in a 96-well conical bottom plate in two steps.
      1. Transfer 200 µL first, centrifuge at 450 x g for 5 min at 4 °C, and discard the supernatant.
      2. Transfer another 200 µL to the corresponding well. Centrifuge at 450 x g for 5 min at 4 °C, and discard the supernatant.
   3. Resuspend the cell pellet in 50-400 µL of the antibody/stain cocktail (Table 3), and incubate for 30 min at 4 °C protected from light.
      NOTE: Lung samples should be resuspended in 400 µL, mediastinal lymph node samples in 50 µL, and mesenteric lymph node samples in 100 µL of the staining cocktail.
   4. Add 150 µL of FACS buffer to the mediastinal lymph node samples and 100 µL to the mesenteric lymph node samples. Centrifuge at 450 x g for 5 min at 4 °C, and discard the supernatant.
   5. Wash the lung and lymph node samples by resuspending the pellets in 1 mL and 200 µL of FACS buffer, respectively. Centrifuge at 450 xg for 5 min at 4 °C. Discard the supernatant and repeat the wash.
   6. Resuspend the lung samples in 600 µL of FACS buffer, and analyze them by flow cytometry. Use the gating strategy: lymphocytes, single cells, live cells, hematopoietic cells, CD4+TCRβ+ cells, and IL-9+ cells (Figure 2A).
   7. Resuspend the lymph node samples in 300 µL of FACS buffer, and analyze by flow cytometry. Use the gating strategy: lymphocytes, single cells, live cells, CD4+ TCRβ+ cells, and IL-9+ cells (Figure 2B and Supplementary Figure 3A).

6. Determination of absolute numbers of cells in single-cell suspensions

1. Dilute the samples from isolating steps 4.8.3, 4.9.7, 4.10.14, and 4.11.3 with PBS at a 1:20 ratio (10 µL of sample + 190 µL of PBS).
2. Mix 10 µL of each diluted sample from step 6.1 with 10 µL of Trypan Blue. Load 10 µL into a hemocytometer and count the live cells, considering each dilution.
3. Obtain the absolute numbers of cells by multiplying the percentage of the population of interest from live cells determined by flow cytometry (viability dye negative cells) by the total number of live cells in the single-cell suspension after isolation, and dividing this number by 100 (Supplementary Figure 4, Supplementary Figure 5, and Supplementary Figure 6).
   Absolute number = (Percentage of the population of interest from live cells determined by flow cytometry x Total number of live cells in the single-cell suspension)/100

Wyniki

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-...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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