Trichuris muris Infection: A Model of Type 2 Immunity and Inflammation in the Gut

1. Propagating Trichuris muris eggs

1. To generate new batches of Trichuris muris eggs, infect 20-30 immunodeficient mice (eg. NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) or 129S6/SvEvTac-Rag2^tm1Fwa (RAG2^-/-)) or genetically susceptible mice (eg. AKR/J) that are 6-8 weeks old with approximately 300 Trichuris muris eggs by oral gavage.
2. After 32–35 days sacrifice the mice by CO₂ asphyxiation.
3. Expose the ventral side of the mouse and wet the abdomen with 70% ethanol.
4. Using forceps grasp the abdominal skin and make a small incision using scissors. Cut the skin to expose the abdominal contents.
5. Identify the cecum and the proximal large intestine, and remove them.
6. Place the cecum and proximal large intestine in a 10 cm petri dish containing Dulbecco’s Modified Eagle Medium (DMEM) with 500 U/ml penicillin and 500 μg/ml streptomycin (P/S). Cut the cecum open to expose the lumen and the worms. Using forceps place cecum in a beaker of phosphate buffered saline (PBS) and shake gently to remove feces.
7. Return cecum to petri dish and using smooth curved forceps (Roboz RS-5047) gently pull out worms and place them in a well of a 6 well plate containing 5 mls of DMEM + P/S.
8. Repeat this process for all of the mice.
9. Put 6 well plate in a Tupperware container with a damp paper towel to maintain humidity and incubate at 37°C for 4 hrs.
10. After 4 hrs remove worms using smooth curved forceps and split them into 2 new wells of the 6 well plate containing 5mL DMEM + P/S. Harvest the contents of the original well and place in a 15 ml falcon tube. Spin tube at 3000 rpm and remove and reserve supernatant as 4 hr antigen (can be used for cell restimulation after PBS dialysis, where typical protein yields are 10-20 mg per 20 mice) and resuspend egg-containing pellet in autoclaved tap water.
11. Put the 6 well plate back at 37°C overnight. Remove and dispose of the worms. Harvest the contents of the wells into a falcon tube and spin at 3000 rpm for 5 min. Remove supernatant and reserve as overnight antigen (can be used for Trichuris Ag specific antibody isotype ELISAs after PBS dialysis, where typical protein yields are 10-20 mg per 20 mice) and resuspend egg-containing pellet in autoclaved tap water.
12. Combine 4 hr and overnight eggs and filter the eggs through the 70μm strainer to remove any leftover worms then transfer into a 75 cm² flask. Keep flask in a dark place covered in foil for 6 weeks at room temperature (RT).
13. Wash eggs in autoclaved tap water and resuspend at 50 viable eggs in 50 μl. To do this, centrifuge eggs at 3000 rpm for 5 min and resuspend in ˜50ml sterile tap water. Remove 50 μl eggs and place on a glass slide. Using the 40x magnification count the number of viable embryonated eggs in the 50 μl. Dilute eggs to give 50 viable eggs in 50 μl. For images of viable eggs please see the study by Kopper and Mansfield¹⁶.

2. Acute infection of experimental mice with Trichuris muris eggs (200 eggs/mouse)

1. Thoroughly resuspend eggs and remove enough volume to treat the number of mice X 2 (eg. for 10 mice take 200 μl x 10 mice x 2 = 4 mls of eggs) and place them in a 14 ml snap cap tube.
2. Invert the 14 ml snap cap tube to mix. Bring up 200 μl of eggs into 1 ml syringe on an appropriate size feeding needle (for mice weighing 25 grams we used 18 gauge 1 1/2″ needles). In one hand scruff the mouse and using the other hand administer eggs by oral gavage .
3. Wait 21 days.

3. Experimental harvest

1. On day 21 sacrifice the mice using CO₂.
2. Collect blood from mice by cardiac puncture, in a non-heparinized syringe, and store at 4°C. The following day isolate serum and freeze at -20°C. Serum can be used for detection of Trichuris-specific IgGs and IgE by ELISA.
3. Expose the ventral side of the mouse and wet the abdomen with 70% ethanol.
4. Using forceps grasp the abdominal skin and make a small incision using scissors. Cut the skin and underlying membrane to expose the abdominal contents.
5. Identify the small intestine, cecum and colon. Grab the cecum with forceps and pull out of abdominal cavity. With another pair of forceps push small intestine over to the right exposing the mesenteric lymph nodes (mLNs). Remove the mLNs being careful not to cut into the intestines and place in a 15 ml falcon containing 2 mls of media. If desired these can be processed and restimulated in vitro (4×10⁶/ml in 1 ml in 24 well plates with 1 μg/ml αCD3/CD28 or with 50μg/ml 4h Trichuris Ag) for 72 hrs for cytokine analysis by ELISA and intracellular flow cytometry.
6. Cut out the cecum and colon.
7. Using forceps, push out fecal pellets from distal colon and place in a 2 ml Axygen tube. Freeze at -20°C. Fecal pellets can be analyzed by western blot for expression of resistin-like molecule-β (RELM-β).
8. Remove the tip of the cecum (˜0.5 cm). To remove feces hold cecum tip with forceps and flush with PBS using a 3 ml syringe and 22-gauge needle in a petri dish. Place in fresh 4% paraformaldehyde in a 5 ml Falcon snap cap tube. Store at 4°C. These can be paraffin-embedded, sectioned and mounted on slides for histological analysis.
9. Remove a piece of proximal colon (˜ 1 cm) for RNA. Put sample into 2 ml Axygen tube with 500 μl RNAlater. Store at 4°C. These samples can be analyzed for mRNA expression by qPCR.
10. Place the rest of the cecum in a labeled petri dish and freeze at -20°C.

4. Enumeration of Trichuris muris worms in the cecum

1. Remove cecum from freezer and place in a petri dish containing ˜5 ml water.
2. Using scissors, cut open cecum to expose the lumen.
3. Hold the cecum with forceps and vigorously shake the cecum to remove the majority of the feces.
4. Transfer cecum to another petri dish containing water and using smooth curved forceps gently scrape off the IECs the underlying tissue.
5. Transfer cecum to a new petri dish containing water and vigorously scrape remaining tissue with forceps, ripping the tissue into small pieces.
6. Use a dissecting microscope to count all of the worms in all 3 of the petri dishes. As you find a worm remove it from the dish to ensure the same worm isn’t counted twice. If worms are damaged (i.e. broken in half) count only thick or thin ends to get an accurate count.

5. Representative Results

The Trichuris muris infection model allows researchers to quantify parasite burden and systemic immune responses, as well as to histologically examine the inflammatory response within the distal cecum. When the lumen of the harvested cecum is exposed, Trichuris worms can be enumerated using a dissecting microscope (Figure 1A-B). Parameters of immunity to Trichuris also include antibody isotype switching to IgG1 (associated with Th2-type immunity) in resistant animals, and IgG2a (associated with Th1-type immunity) in susceptible animals (Figure 1C). Expression of the goblet cell-specific protein resistin-like molecule-β (RELM-β) is associated with clearance of Trichuris and is detectable by Western blot analysis (Figure 2). The distal portion of the cecum can be stained with Periodic Acid-Schiff (PAS) to assess goblet cell responses and the extent of intestinal inflammation in response to infection (Figure 3).

Figure 1. Quantification of immunity to Trichuris muris. (A) Resistant (B6) mice expel Trichuris muris by day 21 post-infection. By contrast, susceptible (AKR/J) mice become chronically infected with Trichuris, resulting in parasite establishment. Each bar represents the mean ± SEM of 4-8 animals per group. (B) Worms are counted using a dissecting microscope. Top panel shows worms in isolation, bottom panel shows worms in the presence of IECs and feces. (C) Trichuris-specific IgG2a titres are determined by ELISA of diluted serum (1:2 dilution, starting at 1:20 dilution and ending at 1:2560) on plates coated with excretory Trichuris antigen (O/N Ag, 5 μg/ml). Susceptible AKR/J mice have notably higher levels of Trichuris-specific serum IgG2a than resistant B6 mice.

Figure 2. Clearance of Trichuris muris is associated with expression of the goblet cell-specific protein RELM-β⁷. RELM-β production can be detected from fecal pellets from Trichuris-infected resistant (B6), but not susceptible (AKR/J) mice by western blot analysis. Each lane is representative of a single animal, (N)=Naive, (Tm)=21-day Trichuris infected mice.

Figure 3. Histological examination of the distal cecum following Trichuris infection. 5-7 μm sections of the distal cecum stained with PAS. Goblet cell hyperplasia and mucus production are evident in resistant (B6) but not susceptible (AKR/J) mice following Trichuris infection.